THE DETECTION OF POISONS AND POWERFUL DRUGS AUTENRiETH WARREN LABORATORY MANUAL FOR The Detection of Poisons AND Powerful Drugs BY DR. WILHELM AUTENRIETH PROFESSOR IN THE UNIVERSITY OF FREIBURG i. B. AUTHORIZED TRANSLATION BY WILLIAM H. WARREN, Ph.D. FIFTH AMERICAN EDITION WITH 25 ILLUSTRATIONS PHILADELPHIA P. BLAKISTON'S SON & CO 1012 WALNUT STREET 3 * > COPYRIGHT, 1921, BY P. BLAKISTON'S SON & Co. AUTHOR'S PREFACE Additional matter in "Detection of Poisons" has made the fourth edition considerably larger than the third. The seven chapters now comprised in the book have been entirely revised but the first three chapters remain unchanged in arrangement. Chapter I treats of poisons volatile with steam. Organic poisons, especially the alkaloids, form the subject of Chapter II. Hydrastine and veronal, introduced into this chapter for the first time, have been incorporated into the Stas-Otto process. Chapter III deals with metallic poisons. The toxic substances included in Chapter IV find no place in the three groups just mentioned. As they seldom appear in toxicological examinations, their significance is theoretical rather than practical. The following members of this group have been introduced for the first time, namely, cantharidin, cytisine, ergot, papaverine, pilocarpine, saponin substances, solanine, thebaine, and the toxalbumins, ricin, abrin and crotin. Chapter V- deals with special qualitative and quantitative methods, such as the quantitative estimation of phosphorus in phosphorated oils; the electrolytic detection and estimation of arsenic; the biological test for arsenic; the destruction of organic matter and detection of arsenic by A. Gautier and G. Locke- mann; Karl Th. Morner's estimation of minute quantities of arsenic; methods of estimating alkaloids by H. Matthes, H. Thorns and A. H. Gordin. This chapter also includes A. J. J. Vandevelde's estimation of the toxic action of organic com- pounds by means of blood haemolysis. Chapter VI takes up the estimation of alkaloids and other active principles in raw materials (drugs) and in their prepara- tions. Pharmacopceial as well as other estimations, such as that of nicotine in tobacco, caffeine in tea, coffee, cola prepara- tions, etc., pilocarpine in jaborandum leaves, piperine in pepper, VI AUTHOR S PREFACE solanine in potatoes, and theobromine in cacao and its prepara- tions have been included. The author has endeavored to treat these subjects as thoroughly as possible. Chapter VII describes the methods employed in detecting carbon monoxide -in blood, in recognizing blood itself in stains and in differentiating human from animal blood. The new edition, though more comprehensive in its scope than the last, has lost nothing in clearness because of the rear- rangement of subject-matter. Beginners will probably confine their attention to the first three chapters. Students of phar- macy will undoubtedly add Chapter VI which deals with drug assaying. The other chapters are designed more especially for those who wish to become better acquainted with toxicological procedures. Descriptions of syntheses of organic drugs such as acetanilide, antipyrine, phenacetine, pyramidone, salicylic acid, sulphonal and veronal allow the student to review the methods employed in connection with laboratory work. Structural, formulae of alkaloids and their cleavage-products have been given only when they have been definitely determined or shown to be highly probable. By introducing this specific information the author hopes to stimulate the student's interest in alkaloidal chemistry which has become so important within recent years. More advanced students will find in fine print brief statements about the poisonous action of the better known physiologically active substances as well as their distribution in and elimination from the human organism. Repeated references to larger treatises upon toxicology, especially to R. Robert's " Intoxikationen, " have been made. Numerous citations of literature enable the student to consult original articles for fuller information. The translation of the third and fourth editions into English and Spanish and the proposed translation of the fourth edition into Italian indicate that colleagues in other countries have favorably received this work. WlLHELM AUTENRIETH. FREIBURG IN BADEN. TRANSLATOR'S PREFACE The fourth English edition of Professor Autenrieth's " Auffin- dung der Gifte" was a translation of the fourth completely revised German edition. The present, or fifth, English edition is also a translation of the same work, for a fifth Ger- man edition so far as the writer is aware has not yet appeared. The last English edition, though adhering closely to the text of the German work, included a few subjects not found in the latter. Among these may be mentioned a fuller discussion of the ''normal arsenic" question and the quantitative estimation of arsenic and antimony by the Gutzeit test. These subjects have been retained in the present edition. Owing to the prominence attained of late by wood (methyl) alcohol, due to ignorant or criminally careless substitution of this intoxicant for grain (ethyl) alcohol, this substance has been added to the list of volatile poisons. Aside from minor corrections of the text, the omission and correction of certain tests, the intro- duction of a few new tests of recent appearance in the litera- ture, and the expansion of the index to include authors as well as subjects, no changes of importance have been made in the last edition of this work. WILLIAM H. WARREN. SOMERVILLE, NEW JERSEY. vii CONTENTS PAGE Author's Preface v Translator's Preface vii Introduction i CHAPTER I TESTS FOR PHOSPHORUS AND OTHER POISONS VOLATILE WITH STEAM FROM ACID SOLUTION PHOSPHORUS 5 Scherer's test; Mitscherlich's test; Blondlot and Dusart's test; (a) in the Fresenius-Neubauer apparatus, (b) in the Hilger- Nattermann apparatus; Detection of phosphorous acid; Phosphorus in phosphorated oils; Detection and quantitative estimation by the Mitscherlich-Scherer method; Metabolism in phosphorus poisoning. FURTHER EXAMINATION OF THE DISTILLATE HYDROCYANIC ACID 19 Physiological action; Preliminary test; Detection; Quantitative estimation; Detection in presence of potassium ferrocyanide; Mercuric cyanide; Mercuric cyanide in presence of potassium ferrocyanide. CARBOLIC ACID 26 Action and fate in the organism; Detection; Quantitative estima- tions; i. Gravimetrically; 2. Volumetrically (Beckurts-Koppes- chaar); 3. Volumetrically (J. Messinger-G. Vortmann); Estimation in urine; Carbolic acid in presence of aniline. CHLOROFORM 35 Behavior in the human organism; Distribution in the cadaver; Detection; Quantitative estimation in cadaveric material. CHLORAL HYDRATE 38 Detection; Action and fate in the human organism; Quantitative estimation in blood and tissues. lODOFORM 41 Detection. NITROBENZENE 42 Toxic action; Detection. ANILINE 44 Toxic action; Detection. CARBON DISULPHIDE 46 Toxic action; Detection; Quantitative estimation of carbon disul- phide vapor in air. X CONTENTS PAGE ETHYL ALCOHOL 49 Fate in the human organism; Detection. METHYL ALCOHOL 52 Physiological action; Detection. ACETONE 55 Occurrence in urine; Detection; Acetone in presence of ethyl alcohol; Detection in urine. BITTER ALMOND WATER AND BENZALDEHYDE 57 SYNOPSIS or GROUP I (CHAPTER I) 59 CHAPTER II DETECTION OF THOSE ORGANIC SUBSTANCES WHICH ARE NOT VOLATILE WITH STEAM FEOM ACID SOLUTION STAS-OTTO PROCESS 63 A. EXAMINATION OF ETHER EXTRACT OF THE AQUEOUS TARTARIC ACID SOLUTION 63 PICROTOXIN 66 Detection in beer. COLCHICIN 68 PICRIC Aero 70 Action and elimination; Detection. ACETANILIDE 73 Action; Detection; Examination of acetanilide urine. PHENACETINE ....-." 75 Preparation; Detection. SALICYLIC ACID . . 77 Detection; Quantitative estimation; Detection in urine. VERONAL 79 Preparation; Physiological action; Detection in urine. ANTIPYRINE 82 Preparation; Detection in urine. CAFFEINE 83 Fate in human metabolism; Detection. B. EXAMINATION OF ETHER EXTRACT OF THE AQUEOUS ALKALINE SOLUTION 85 CONHNE . . . ' 89 NICOTINE. ...,.......;,.. 90 Physiological action; Reactions. ANILINE 93 VERATRINE 93 Preparation of crystalline and water soluble veratrine; Constitution; Reactions. STRYCHNINE 96 Physiological action; Detection; Detection in presence of brucine. BRUCINE 100 ATROPINE . IO 2 Constitution; Reactions. CONTENTS XI PAGE HOMATROPINE 105 COCAINE 105 Constitution; Behavior in the animal organism; Detection. PHYSOSTIGMINE no CODEINE in NARCOTINE 113 Constitution; Detection. HYDRASTINE 116 Preparation; Constitution; Reactions. QUININE 119 Constitution; Detection. CAFFEINE 122 ANTIPYRINE 123 Detection in urine. PYRAMIDONE 124 Preparation; Behavior in the organism; Detection. C. EXAMINATION OF ETHER EXTRACT AND OF CHLOROFORM EXTRACT OF THE SOLUTION ALKALINE WITH AMMONIA. a. Ether Extract 126 APOMORPHINE 127 /3. Chloroform Extract 130 Preliminary test for morphine; Purification of crude morphine. MORPHINE 131 Constitution; Detection; Behavior in the animal organism. NARCEINE 138 Constitution; Reactions. SYNOPSIS OF GROUP II (CHAPTER II) 140 CHAPTER III EXAMINATION FOR METALLIC POISONS DESTRUCTION OF ORGANIC MATTER 148 Fresenius-v. Babo Method 148 By free chloric acid 151 C. Mai's Method 152 PRECIPITATION WITH HYDROGEN SULPHIDE 152 METALLIC POISONS I: Examination of that portion of the hydrogen sul- phide precipitate soluble in ammonia-ammonium sulphide. ARSENIC 156 Marsh-Berzelius method; Fresenius-v. Babo method; Bettendorff's test; Gutzeit's test. ANTIMONY, TIN, COPPER 163 METALLIC POISONS II: Examination of that portion of the hydrogen sul- phide precipitate insoluble in ammonium sulphide 165 MERCURY, LEAD, COPPER, BISMUTH, CADMIUM 165 METALLIC POISONS III: Examination for Chromium and Zinc 168 ZINC. . , 168 Xii CONTENTS PAGE CHROMIUM 169 METALLIC POISONS IV: Examination for Barium, Lead and Silver of the insoluble residue left on treatment with potassium chlorate and hydrochloric acid 17 SYNOPSIS OF GROUP III (CHAPTER III) 171 THE ACTION or HEAVY METALS . . 172 FATE, DISTRIBUTION AND ELIMINATION OF METALS IN THE BODY .... 1 73 NORMAL ARSENIC 174 CHAPTER IV 1. EXAMINATION FOR THOSE POISONS WHICH DO NOT BELONG IN THE THREE MAIN GROUPS MINERAL ACIDS HYDROCHLORIC ACID 183 NITRIC ACID '...-. 184 SULPHURIC ACID 186 SULPHUROUS ACID 188 OXALIC ACID 189 Toxic action; Distribution in the organism; Detection. DETECTION OF FREE ALKALIES POTASSIUM HYDROXIDE, SODIUM HYDROXIDE, AMMONIA 192 DETECTION OF POTASSIUM CHLORATE, SANTONIN, SULPHONAL, TRIONAL POTASSIUM CHLORATE 194 Toxic action; Detection; Quantitative estimation; Behavior during putrefaction; Detection in meat. S \NTONIN 198 Constitution; Behavior in the organism; Detection. SULPHONAL 200 Preparation; Detection in urine; Detection of hsematoporphyrin in urine. TRIONAL 203 2. POWERFUL ORGANIC SUBSTANCES SELDOM OCCURRING IN TOXICOLOGICAL EXAMINATIONS CANTHARIDIN .;.'.-". 203 Constitution; Detection. CYTISINE 205 Preparation; Toxic action; Detection. DIGITALIS BODIES . 207 Digitonin, Digitoxin, Digitalinum verum. ERGOT . 209 Alkaloids; Sclererythrin; Detection of ergot in flour; Detection and estimation of the alkaloids. OPIUM 212 Meconic acid; Meconin; Selenious-sulphuric acid reagent for opium alkaloids. CONTENTS Xlll PAGE PAPAVERINE 215 Constitution; Detection. PILOCARPINE 217 PTOMAINES . 219 SAPONINS 220 Physiological action; Detection in foaming beverages, such as beer, etc.; Detection of githagin in flour. HAEMOLYSIS AND PHYSIOLOGICAL SALT SOLUTION 224 SOLANINE 225 Toxic action; Detection. THEBAINE 227 Constitution; Detection. TOXALBUMINS 228 Abrin, Ricin; Crotin; Coagulation of blood and defibrinated blood. CHAPTER V SPECIAL METHODS QUANTITATIVE ESTIMATION OF PHOSPHORUS IN PHOSPHORATED OILS i. W. Straub's method; 2. A. Frankel's and C. Stich's method. . . 231 SPECIAL METHODS FOR DETECTING ARSENIC 233 Separation of arsenic as arsenic trichloride . . t 233 Electrolytic detection of arsenic 233 Destruction of organic matter and detection of arsenic by A. Gautier , and G. Lockemann 234 Electrolytic estimation of minute quantities of arsenic by C. Mai and H. Hurt 237 Quantitative estimation of arsenic and antimony by the Gutzeit method . 240 Biological detection of arsenic by means of penicillium brevicaule 242 Detection of arsenic in organic arsenic compounds, i.e. Cacodylic acid; Arrhenal; Atoxyl. Their detection in urine 245 Quantitative estimation of minute quantities of arsenic by Karl Th. Morner 247 DETECTION OF SALICYLIC ACID IN FOODS AND BEVERAGES; IN WINE, MEAT PRODUCTS, MILK 250 MALTOL 251 CHLORAL HYDRATE IN TOXICOLOGICAL ANALYSIS BY R. MAUCH. ... 251 ALKALOIDAL ESTIMATIONS 253 1. By the picrolonate method of H. Matthes 253 2. By precipitation with potassium bismuthous iodide and decom- position of the precipitate with alkali hydroxide-carbonate by H. Thorns 255 3. By the method of H. M. Gordin 257 Quantitative estimation of strychnine and quinine in presence of each other 258 Estimation of the toxicity of chemical compounds by blood haemo- lysis by A. J. J. Vandervelde 258 xiv CONTENTS CHAPTER VI QUANTITATIVE ESTIMATION OF ALKALOIDS AND OTHER ACTIVE PRINCIPLES IN RAW MATERIALS AND IN THEIR PREPARATIONS PAGE ALKALOIDAL ESTIMATIONS OF DRUGS AND THEIR PHARMACEUTICAL PREPA- RATIONS ACCORDING TO THE GERMAN PHARMACOPCEIA 260 Estimation of alkaloid in aconite root 261 Estimation of cantharidin in Spanish fly 263 Estimation of cinchona alkaloids 264 i. In cinchona bark; 2. In aqueous extract of cinchona and in alcoholic extract of cinchona. Estimation of quinine in mixtures of cinchona alkaloids by the sulphate method 268 i. Cinchona bark; 2. Cinchona extract. Estimation of colchicin in colchicum seeds and in colchicum corms 269 Estimation of alkaloid in pomegranate bark 270 Estimation of caffeine in coffee, tea, cola nuts and Guarana paste 271 i. C. C. Keller's method; 2. A. Hilger-A. Juckenack's method. 3. A. Hilger-H. Gockel's method; 4. Socolof-Trillich-Gockel method; 5. E. Katz's method; 6. K. Dieterich's method. Estimation of alkaloid in ipecacuanha root 277 Estimation of nicotine in tobacco 279 i. R. Kissling's method; 2. C. C. Keller's method; 3. J. Toth's method. Estimation of hydrastine in hydrastis extract 281 Estimation of berberine 282 Estimation of hydrastine by the picrolonate method of H. Matthes and O. Rammstedt 282 i. In fluid extract of hydrastis; 2. In hydrastis root. Estimation of morphine in opium and in its pharmaceutical preparations 283 i. In opium; 2. In extract of opium; 3. In wine of opium and in tincture of opium. Estimation of pilocarpine in jaborandum leaves 286 i. G. Fromme's method; 2. H. Matthes and O. Rammstedt's method. Piperine and its estimation in pepper 287 i. J. Konig's method; 2. Cazeneuve and Caillot's method. Estimation of santonin in wormseed . > 289 i. K. Thaeter's method; 2. J. Katz's method. Estimation of solanine in potatoes 291 i. O. Schmiedeberg and G. Meyer's method; 2. F. v. Morgen- stern's method. Estimation of alkaloid in nux vomica and its preparations .... 293 C. C. Keller's method 293 Method of the German Pharmacopoeia 294 i. In nux vomica; 2. In extract of nux vomica; 3. In tincture of nux vomica. CONTENTS XV PAGE H. Matthes and O. Rammstedt's method. 296 i. In extract of mix vomica; 2. In tincture of nux vomica; 3. In nux vomica. Estimation of strychnine in mixtures of strychnine and brucine by C. C. Keller H. M. Gordin 298 Estimation of theobromine and caffeine in cacao and in chocolate 298 Estimation of alkaloid in the leaves of atropa belladonna, hyo- scyamus niger and datura strammonium 300 Estimation of alkaloid in extract of belladonna, according to the German Pharmacopceia, in extract of hyoscyamus 301 Assay of officinal extracts by E. Merck 302 Extract of belladonna; Extract of cinchona; Extract of strychnine. CHAPTER VII DETECTION OF CARBON MONOXIDE BLOOD, BLOOD STAINS AND HUMAN BLOOD 1. Recognition of carbon monoxide blood 304 2. Detection of blood stains 307 Haematin 308 Spectroscopic detection of blood 310 Other tests for blood 312 Schonbein-van Deen's test; Vitali's procedure in this test; Schaer's procedure; Aloin test. 3. Biological detection of human blood 314 APPENDIX PREPARATION OF REAGENTS A. General alkaloidal reagents 317 B. Special reagents and solutions 320 C. The indicator iodeosine 322 INDEX 323 INTRODUCTION Nearly all the common poisons and drugs may be placed in one of three groups. This classification, based upon the chemical behavior of these substances during isolation from mixtures, is as follows: Group I. The members of this group, when heated, vola- tilize without decomposition and distil with steam from an acid solution. Yellow phosphorus, hydrocyanic acid, carbolic acid, chloroform, chloral hydrate, iodoform, aniline, nitrobenzene, carbon disulphide and alcohol (ethyl and methyl) are the principal substances of this class. Group II. The members of this group are non-volatile, organic substances which do not distil with steam from an acid solution. But hot alcohol containing tartaric acid will extract them from extraneous matter. Alkaloids, many glu- cosides and bitter principles, as well as certain synthetic or- ganic drugs, like acetanilide, phenacetine, antipyrine, pyrami- done, sulphonal and veronal comprise this group. Group III. This group includes all poisonous metals. In toxicological analysis, therefore, poisons are divided into three groups, each of which has its own special methods of pro- cedure. A few poisons like mineral acids, caustic alkalies, oxalic acid and potassium chlorate cannot be conveniently placed in these three groups owing to differences in solubility and other peculiarities. Special tests for such substances must be made with a separate portion of material. Chapter IV contains a description of the methods used in identifying these substances. The material must be thoroughly mixed and divided into three or four approximately equal portions, unless the analysis is to be limited to the detection of a single well-defined sub- stance. One portion is tested for non-volatile, organic sub- stances (Chapter II). The second portion is examined for 1 2 INTRODUCTION volatile poisons (Chapter I) and the residue from this portion is used in testing for poisonous metals (Chapter III). The third portion is tested for substances considered in Chapter IV. The fourth portion is held in reserve in case additional material is needed to verify a doubtful result, or to replace a portion accidentally lost during analysis. Occasionally it is advisable to depart from the general pro- cedure and follow a special method, especially in detecting a single poison, or in estimating it quantitatively. For instance, pure ether would not be the best solvent to use in extracting strychnine quantitatively from an alkaline solution. A mix- ture of ether and chloroform, or better pure chloroform would be preferable, since strychnine is more soluble in the latter solvent than in pure ether. For the same reason chloroform should be used in the quantitative extraction of caffeine or antipyrine. When only a small quantity of material is avail- able for analysis, tests for all three groups of poisons may be made with the same portion. In this case after removal of volatile poisons (Chapter I) the residue should be divided into two unequal portions. The larger portion should be tested for non- volatile, organic poisons (Chapter II). The smaller por- tion together with the residue left after extracting non- volatile, organic poisons should be tested for poisonous metals (Chapter III). It is advisable, however, even in such a case to reserve a portion of material for any contingencies. Organs of the human body like liver, kidneys, spleen, heart, brain, stomach or intestines with contents should be cut into small pieces and then finely chopped before being examined chemically. An organ should first be cut into small pieces with sharp, clean scissors and then minced with a clean chopping knife in a new wooden bowl, or a small meat machine, which has been carefully cleaned, may be used. Material may be held with nickel plated tongs while being cut with scissors. Largely to eliminate the unpleasant odor of viscera and facilitate bringing them by hashing into a condition most favorable to the action of acids or solvents, it has been suggested that they be cooled ( 6 to 10). DETECTION OF POISONS CHAPTER I VOLATILE POISONS Yellow Phosphorus and Other Poisons Volatile with Steam from Acid Solution Scherer's Test. This test should precede the distillation described on page 18. The principle of the test is that moist phosphorus vapor and silver nitrate form black silver phosphide (AgsP), metallic silver, phosphoric and sometimes phosphorous acid. Place the finely divided material in a small flask and cover with water if a sufficient quantity is not present. Cut a V-shaped slit in the cork and place the latter loosely in the mouth of the flask so that the two strips of filter paper are freely suspended (Fig. i). Moisten one strip with silver nitrate and the other with lead acetate solution. 1 Warm gently upon the water-bath (40 to 50). 2 If the silver but not the lead paper is darkened, yellow phosphorus may be present. If both papers are darkened, hydrogen sulphide also is present. In the latter case yellow phosphorus may be pres- ent with hydrogen sulphide. In absence of FIG hydrogen sulphide, darkening of the silver paper is not final proof of yellow phosphorus, for any volatile organic substance having reducing properties, as formalde- 1 A more sensitive "lead paper" may be obtained by using alkaline lead solu- tion prepared by adding excess of sodium hydroxide to the solution of a lead salt whereby Pb(OH)(ONa) and Pb(ONa) 2 are formed. 2 Temperatures in this book are expressed in Centigrade degrees. Tr. 3 4 DETECTION OF POISONS hyde (H.CHO), or formic acid (H.COOH), may give the same result. Scherer's test is of value in proving the absence rather than the presence of yellow phosphorus. It is a good preliminary test, as it excludes phosphorus if the silver paper is unchanged. Distillation. Place a portion of finely divided and thoroughly mixed material in a large round-bottom flask and add enough distilled water for free distillation. Then add tartaric acid solution drop by drop until the mixture is acid after thorough shaking. Practice analyses 1 usually require 20 to 30 drops of 10 per cent, tartaric acid solution. In examining animal material, as the stomach or intestines and contents, or organs, like liver, spleen and kidneys, it is often unnecessary to addmuch water because enough is usually pres- ent. First chop the material in a wooden tray with a steel knife. In a medico-legal analysis the tray should be new. A meat machine which has been carefully cleaned may be used. Thin the material with a little distilled water, acidify with dilute tartaric or sulphuric acid and finally distil. If Scherer's test is positive, begin distilling with the Mit- scherlich apparatus (Fig. 2) ; but if negative, distil in the usual way with the Liebig condenser (see page 18). The distillate may contain : Yellow phosphorus Aniline Hydrocyanic acid Ethyl alcohol Carbolic acid Methyl alcohol Chloroform Acetone Chloral hydrate Carbon disulphide lodoform Benzaldehyde Nitrobenzene Bitter almond water 1 Laboratory practice in detecting poisons may be given by mixing small quan- tities (from 0.03 to 0.05 or o.i gram) of a poison with dry bread or biscuit crumbs, meal or meat. Finely chopped organs (liver, kidney, spleen, etc.), sausage meat, beer, wine or milk may be used. Drugs like morphine, codeine, quinine, acetanilide, phenacetine, antipyrine, caffeine, santonin, sulphonal, veronal, calomel, tartar emetic, subnitrate of bismuth, etc., may be mixed with powdered cane- or milk-sugar. The last kind of practice is especially suitable for students of pharmacy. VOLATILE POISONS YELLOW PHOSPHORUS Mitscherlich Method' of Detecting Yellow Phosphorus The principle of this method is that yellow phosphorus volatilizes with steam and becomes luminous in contact with air. The phosphorescence is best seen in a dark room. FIG. 2. Mitscherlich Apparatus. Procedure. Arrange the apparatus as in Fig. 2. Support the condenser in a vertical position and connect the upper end with the flask by a glass tube about 8 mm. internal diameter. This tube has two right-angle bends and each end passes through 6 DETECTION OF POISONS a cork. Have condenser and tube scrupulously clean to avoid interference with the phosphorescence. Have the flask at most not more than a third full. This pre- caution is necessary because many materials, containing protein substances like albumin,, albumose, etc., and starchy matter, when distilled in aqueous solution, cause more or less foaming which is liable to carry over solid matter into the receiver. Use as the receiver an Erlenmeyer flask containing a little distilled water (3 to 5 cc.) into which the end of the condenser dips. This precaution prevents loss of easily volatile substances like hydrocyanic acid and chloroform. Heat the flask upon a wire gauze of fine mesh, asbestos plate or sand-bath and bring the contents to boiling by raising the temperature gradually. There is some danger of burning or carbonizing organic matter on the bottom of the flask, if heat is applied too strongly or rapidly. When boiling begins, make the room as dark as pos- sible and watch for phosphorescence in the tube and condenser. It usually appears as a luminous ring or band in the upper part of the condenser. When this is distinctly visible, the presence of yellow phosphorus is established. Phosphorescence during distillation with steam is very characteristic of yellow phos- phorus and frequently is the only sure and unquestionable test for this element. Phosphorescence is a process of oxidation by which phos- phorus vapor is changed to phosphorous acid. Should it not appear immediately, distillation must be continued for some time, since certain substances like ethyl or methyl alcohol, ether, turpentine and many other ethereal oils either prevent the phenomenon entirely or seriously retard it. Considerable carbolic acid, creosote, chloroform, chloral hydiate, as well as hydrogen sulphide, may completely prevent phosphorescence. K. Polstorff and J. Mensching 1 have shown that mercuric chloride as well as other mercury compounds may also interfere with phosphorescence. Possibly mercuric chloride carried over by steam is reduced to metallic mercury by phosphorus vapor. In that case the metal should appear in the distillate. The 1 Berichte der Deutschen chemischen Gesellschaft 19, 1763 (1886). VOLATILE POISONS 7 fact that both metallic mercury and phosphoric acid can be detected in the distillate favors the supposition that action takes place between phosphorus vapor and mercuric chloride. Phosphorescence, however, often appears when these sub- stances have passed over. But even when prolonged dis- tillation fails to give a positive result, this must not be accepted as final proof of the absence of phosphorus until other tests have been made. Whatever the result, evaporate a portion of the distillate to dryness on the water-bath with excess of saturated chlorine water, or with a little fuming nitric acid. Phosphorus always imparts a strong odor to the distillate. Small drops of phosphorus appear if the quantity is large, and the solution contains phosphorous acid. Dissolve the residue from evaporation in 2 to 3 cc. of water and test in two separate portions for phosphoric acid. 1. Ammonium Molybdate Test. Acidify the solution with a few drops of concentrated nitric acid. Add an equal volume of ammonium molybdate solution and warm to about 40. Phos- phoric acid precipitates yellow ammonium phospho-molybdate. 2. Ammonium Magnesium Phosphate Test. Add magnesia mixture 1 to the second portion. Phosphoric acid gives a white crystalline precipitate of ammo- nium magnesium phosphate (H 4 N)- MgP0 4 .6H ! 0. Vigorous shaking FlG ' favors precipitation. When only traces of phosphoric acid are present, long standing is necessary before the precipitate appears. Always examine the precipi- tate with the microscope. It should consist of well-formed 1 Magnesia mixture is a clear solution prepared by mixing equal volumes of magnesium chloride, ammonium chloride and ammonium hydroxide (about 10 per cent.) solutions. It contains the readily soluble double chloride of ammo- nium and magnesium which is not decomposed by ammonium hydroxide. This reagent is prepared as needed and should be perfectly clear and colorless. 8 DETECTION OF POISONS crystals or at least should be crystalline. These crystals are transparent, acicular prisms (Fig. 3) . Notes. A. Fischer 1 states that substances interfering more or less with the detection- of phosphorus by the Mitscherlich method are usually less troublesome if Hilger and Nattermann's procedure is used (see page 15). The essential feature of the latter process consists in allowing steam charged with phosphorus to pass into the air, or in admitting air into the apparatus. Detection of Phosphorus and Phosphorous Acid (Blondlot 2 -Dusart 3 ) When the Mitscherlich method fails to show phosphorus, it is often necessary to test for phosphorous acid. This is the first product in the oxidation of phosphorus and is easily formed. The Blondlot-Dusart method shows the slightest trace of phos- phorous (H 3 PO 3 ) and hypophosphorous (HsPC^) acids as well as yellow phosphorus. The method consists in converting yellow phosphorus into phosphine (PH 3 ) by nascent hydrogen. The lower oxidation products of phosphorus, namely, hypo- phosphorous (H 3 PO 2 ) and phosphorous (H 3 PO 3 ) acids, 4 when warmed with zinc and dilute sulphuric acid are reduced to phosphine by nascent hydrogen: H 3 P0 2 + 4 H = PH 3 + 2H 2 O, H 3 PO 3 + 6H = PHj. + 3 H 2 0. . Phosphine, or hydrogen charged with phosphorus vapor, burns with a characteristic green flame (Dusart's reaction) : 2 PH 3 + 4 2 = P 2 6 + 3 H 2 0. The green flame is easily recognized by depressing a cold porcelain dish or plate upon the flame. Detection of phosphorus by the Blondlot-Dusart method depends upon these two facts. A toxicological analysis usually deals with the detection of traces of yellow phosphorus. Hydrogen after acting in the nascent state upon the material is not directly examined for 1 Pflueger's Archiv, 97, 578 (1903). 2 Journal de Pharmacie et de Chimie (3), 40, 25. 8 Comptes rendus de 1'Academie des Sciences, 43, 1126. 4 Nascent hydrogen will not reduce ordinary, ortho-phosphoric acid (H 3 PO 4 ), and its derivatives, pyrophosphoric (H 4 P 2 O 7 ) and meta-phosphoric (HPO 3 ) acids, to phosphine. VOLATILE POISONS 9 phosphorus but is first passed in to dilute silver nitrate solution. Phosphorus and phosphine precipitate black silver phosphide 1 (Ag 3 P): PH 3 + sAgNOs = Ag 3 P + 3 HN0 3 . Thus traces of yellow phosphorus may be concentrated in the silver precipitate from which nascent hydrogen will liberate phosphine : Ag 3 P + 3H = PH 3 + 3Ag. If hydrogen produces a black or gray precipitate in the silver solution, phosphorus is not necessarily present, as hydrogen sulphide, arsine, stibine and reducing organic compounds be- have similarly with silver nitrate. A black precipitate there- fore should always be examined for phosphorus by the Dusart reaction. In the detection of yellow phosphorus, the Blondlot-Dusart method combines two distinct operations, namely: 1. Preparation of the silver phosphide precipitate. 2. Examination of this precipitate in the Dusart apparatus. Procedure, i. Preparation of Silver Phosphide. Thin the finely divided material with water in a capacious flask where hydrogen is being evolved from phosphorus-free zinc and pure dilute sulphuric acid (1:5). In testing for phosphorous acid alone (see page 14) use the filtrate from an aqueous extract of the material, or the filtrate from the residue left after the Mitscherlich distillation (see page 5). Nascent hydrogen should act for 1.5 to 2 hours, or even longer, and pass through neutral silver nitrate solution in the receiver at the end of the apparatus. If yellow phosphorus is present, the hydrogen will contain phosphorus and phosphine and cause a black precipi- tate of silver phosphide in the silver nitrate solution. Collect the precipitate upon a small ash-free paper, wash with a little cold water and examine in the Dusart apparatus as described elsewhere. 1 Phosphorus cannot be determined quantitatively as silver phosphide because this compound is partially decomposed by water. Phosphoric and phosphorous acids pass into solution: (a) 2 A g3 P + sO + 3H 2 O = 6Ag + 2 H 3 PO 4 , (b) 2 Ag 3 P + 3 + 3 H 2 = 6Ag + 2 H 3 P0 3 . 10 DETECTION OF POISONS If there is silver phosphide in the precipitate, the filtrate will contain phosphoric or phosphorous acid (see Note, page 9). To detect phosphoric acid, first add hydrochloric acid to remove excess of silver from this filtrate. Filter through paper pre- viously well washed with acid and water and completely expel hydrochloric acid from the filtrate by evaporation upon the water-bath with concentrated nitric acid. Dissolve the resi- due in a little warm water and test for phosphoric acid with ammonium molybdate or magnesia mixture. 2. Examination of the Silver Precipitate (AgsP) for Phos- phorus. Two forms of apparatus may be used for this purpose, namely: (a) Fresenius-Neubauer 1 Apparatus. Generate hydrogen in flask A (Fig. 4) from pure phosphorus-free zinc and dilute FIG. 4. Fresenius-Neubaxier Apparatus. sulphuric acid. Fill U-tube C with pieces of pumice stone saturated with concentrated potassium hydroxide solution to absorb any hydrogen sulphide. Use hard glass for tube D and 1 C. R. Fresenius, Qualitative chemische Analyse, XVI edition, page 521. VOLATILE POISONS 11 have the tip F of platinum. The part marked E 1 is a glass stop-cock or screw-tap. Reservoir B serves to hold liquid from A when cock E is closed. A platinum tip is essential, other- wise the flame instead of being colorless will always be yellow from sodium in the glass. The place where the platinum tip is fused into the glass should be cooled by wrapping cotton around the glass and keeping it moist. Procedure. Open E and let hydrogen from A pass for some time through the apparatus to expel air. Then close E and liquid in A will rise into B. Now open E just enough to allow hydrogen to burn with a small flame which should be colorless in the dark. If there is no trace of green in the inner cone and a porcelain dish depressed upon the flame does not show an emer- ald-green coloration, hydrogen is phosphorus-free. It is well to repeat this test. To test the silver precipitate for phosphorus, wash it with the paper into B with a little water. When the entire precipitate is in A, close E until all the liquid has risen from A into B. Then open E, light the hydrogen and examine the flame in the dark. If the precipitate contains a trace of silver phosphide, the inner cone will be green and a porcelain dish depressed upon the flame will show an emerald- green coloration. Have the hydrogen flame small so that its color may be observed for some time. (b) Hilger-Nattermann 2 Apparatus. Reduction takes place in a 100 cc. flask closed by a rubber stopper with three holes, two of which are for right-angle tubes just passing through the stopper and the third for a thistle-tube going to the bottom of the flask (Fig. 5). Hydrogen from a Kipp generator enters the flask by one tube and leaves by the other. Attach to the latter a U-tube filled with pieces of pumice stone saturated with concentrated potassium hydroxide solution to absorb hydrogen sulphide. Connect the other end of the U-tube with a hard 1 Fresenius and Neubauer use a screw pinch-cock instead of a gas-cock but by means of a short rubber connector they interpose an ordinary cock between the gas-flask A and the U-tube C. 2 Forschungsbericht iiber Lebensmittel und ihre Beziehungen zur Hygiene, etc., 4, 241-258 (1897). 12 DETECTION OF POISONS glass tube tipped with platinum. 1 Cut the paper containing the precipitate into small pieces and place in the flask which contains in addition a few pieces of phosphorus-free zinc and enough water to seal the thistle-tube. Light the hydrogen after it has passed through the apparatus for some time and been found free from air by the usual test. Seen in the dark the flame should be entirely colorless and burn without a green cone or a greenish glow. 2 Hilger and Nattermann advise a spectro- scopic examination of the flame to determine the purity of the FIG. 5. Hilger- Nattermann Apparatus. zinc. Pure zinc gives a hydrogen spectrum which shows only an orange colored line in place of the yellow sodium line. The minutest trace of phosphorus will give three green lines lying to the right of the line D . The color of two of these lines is more pronounced than that of the third. Having thus tested the purity of zinc and sulphuric acid, pour a few cc. of dilute sul- phuric acid (i : 5) through the thistle-tube into the flask con- taining zinc and the silver precipitate. If the latter contains phosphorus, the flame will show, though not always at once, a green coloration which should be examined with the spectroscope. 1 Hilger and Nattermann use a platinum tipped blow-pipe instead of a glass tube tipped with the same metal. Cotton, which is kept moist and acts as a cooler, is wrapped around the blow-pipe below the tip. 2 Zinc entirely free from phosphorus which will stand this test is difficult to obtain. VOLATILE POISONS 13 The Mitscherlich method affords a distillate especially suit- able for the Blondlot-Dusart test. If this imparts a green color to the hydrogen flame, there can be no question about the pres- ence of phosphorus. Although the Blondlot-Dusart test is very delicate, many chemists refuse to accept it as a substitute for the Mitscherlich test. Selmi states that animal material like brain, which con- tains organic phosphorus compounds, yields after putrefaction a distillate that often gives a black precipitate with silver ni- trate solution. This will impart a green tinge to the hydrogen flame in the Blondlot-Dusart test. Z. Halasz,' however, has failed to confirm Selmi's results. He examined two kinds of animal material by the Blondlot-Dusart method. First, he tested nor- mal brains (man, calf, hog); second, the brain and other organs of rabbits that had been given poisonous doses of phosphorus by the mouth and subcutaneously. He examined these organs when fresh and also from week to week after more or less pronounced putrefaction had set in, but could not detect phosphorus in the brain in a single instance. These experiments disprove the earlier idea that phosphorus normally present in the brain is so changed during putrefaction that it can be detected by the Blondlot-Dusart reaction. He also failed to detect phosphorus in the brain of rabbits poisoned by this element, though he found it in other organs, as stomach and intestines, and in those rich in blood, as liver, lungs and kidneys. He could always detect small or large quantities of phos- phorus in any organ which this element had directly reached, or by which it had been indirectly absorbed. If any compound containing phosphorus is really formed in the brain during putrefaction, Halasz concluded that it is not volatile with steam and does not give the Blondlot-Dusart reaction. On the basis of these experiments Halasz holds that the Blondlot-Dusart method of detecting phosphorus is just as reliable for forensic purposes as that of Mitscherlich. Procedure of Halasz in the Blondlot-Dusart Method Make a thin mixture of very finely divided material and boiled water in a flat- bottom flask where hydrogen is being generated from phosphorus-free zinc and dilute sulphuric acid. Warm upon the water-bath and pass the gas through an absorption-tube provided with several bulbs and containing neutral silver nitrate solution. Concentrated sulphuric acid and a little platinic chloride may be added toward the end to hasten the evolution of gas. Nascent hydrogen thus acts upon phosphorus in the animal material for 2-2.5 hours. Finally wash the silver precipitate carefully with water and transfer it with the paper to the Blondlot- Dusart apparatus. 1 Zeitschrift fur anorganische Chemie 26, 438 (1901). 14 DETECTION OF POISONS Detection of phosphorous Acid The reduction of phosphorous acid to phosphine by zinc and dilute sulphuric acid takes place very slowly. Hilger and Nattermann state that even a few- milligrams require the action of nascent hydrogen for 10 to 14 days. Moreover careful manipulation is necessary because silver phosphide is quite unstable. Water decomposes this substance into metallic silver and phosphorous acid and the nitric acid present oxidizes the latter to phosphoric acid. Therefore when special attention must be given to phosphorous acid, Hilger and Nattermann recommend examining the silver precipitate (presumably Ag 3 P) after 2 days, or at most 3, for phosphorus by the Blondlot-Dusart method and the nitrate for phosphoric acid (see page 10). Detection of Phosphorus in Phosphorated Oils 1. Straub's 1 Test. If a phosphorated oil is placed on the surface of copper sulphate solution, phosphorus will gradually pass from the oil to the aqueous solution and first form black copper phosphide. The latter, acting as a carrier of oxygen, oxidizes phosphorus still in the oil to phosphoric acid which dissolves in the water. Shake 10 cc. of phosphorated oil in a test-tube with 5 cc. of i per cent, copper sulphate solution. According to the amount of phosphorus a black or light brown coloration will appear in the emulsion at once, or in a few minutes, and at most after 2 hours. Phosphoric acid in the aqueous solution may be recog- nized by ammonium molybdate. At least 0.0025 per cent, of phosphorus may be detected in this way. A practical, therapeutic application of this reaction may be made in acute phos- phorus poisoning. Administration of copper sulphate solution may prevent absorption of free phosphorus still in the gastro-intestinal tract. 2. The Mitscherlich test is also applicable to phosphorated oils, even though the oil may contain only 0.0002 gram of phosphorus in 100 grams. But phosphorescence will not appear unless air is admitted into the tube from time to time. Phosphorus in oils' cannot be determined quantitatively by the distillation method, for not more than 36 to 41 per cent, of 1 W. Straub, Miinchener medizinische Wochenschrift 50, 1145; Archiv der Pharmazie 241, 335 (1903); and Zeitschrift fiir anorganische Chemie 35, 460 (1903)- VOLATILE POISONS 15 phosphorus will distil over. The quantitative method recom- mended by Straub (see page 231) may be used in that case. Detection and Quantitative Estimation of Phosphorus (Mitscherlich-Scherer) Acidify a weighed portion of material with dilute sulphuric acid and add a little ferrous sulphate. Distil in a gentle stream of carbon dioxide, using a large flask fitted with a two-hole stopper. Expel air completely from the apparatus by carbon dioxide before heating. Use as receiver a flask fitted with a PIG. 6. Hilger-Nattermann Apparatus for Detecting and Quantitatively Esti- mating Phosphorus. two-hole stopper. Pass the end of the condenser through one hole and connect the other with a U-tube containing silver nitrate solution. Evaporate the distillate upon the water- bath with strong bromine water, or with concentrated nitric acid, to oxidize phosphorus or any phosphorous acid formed. Dissolve the residue in a little water and precipitate phos- 16 DETECTION OF POISONS phoric acid with magnesia mixture. Weigh the precipitate as magnesium pyrophosphate, Mg 2 P207. Heat the contents of the U-tube with concentrated nitric acid. Precipitate silver as silver chloride and filter. Concentrate the filtrate by evapo- ration and precipitate phosphoric acid with magnesia mixture as before. Combine this precipitate with the other. In dis- tillation some phosphorus separates as globules in the first receiver and any carried over as vapor by carbon dioxide is re- tained by silver nitrate solution. As the steam distillation of phosphorus is very slow, the process should be carried on for at least 3 hours. Hilger and Nattermann recommend the ap- paratus in Fig. 6 not only for detecting phosphorus but for estimating it quantitatively. Air may be mixed with phos- phorus vapor by means of stop-cock K and the characteristic phosphorescence will appear. Remarks Upon the Mitscherlich Test. Hilger and Nattermann state that 0.00006 gram of yellow phosphorus is the smallest quantity that can be detected by the Mitscherlich method. When 200 cc. of water containing 0.0003 gram of phosphorus were distilled, there was brilliant phosphorescence for 5 minutes. The degree of dilution seems to have no effect upon the result, at least not within limits occurring in practice. Hydrogen sulphide, always present in putrefying animal matter, has no apparent effect upon phosphorescence. Free phosphorus can be detected in putrid organic matter even after the lapse of considerable time. Putrefactive and digestive processes appear to prevent oxidation of phosphorus. Dragendorff detected phosphorus in an exhumed body several weeks after death. Neumann found free phosphorus in a human body fourteen days and Elvers eight weeks after death. When an acid aqueous solution is distilled in the Mitscherlich apparatus, the flask residue always contains phosphoric (H 3 PO 4 ), phosphorous (H 3 PO 3 ) and hypophosphorous (H 3 PO 2 ) acids and red phosphorus. Distillation of a solution of 0.0644 gram of phosphorus gave only 71.33 per cent, in the distillate. The residue contained: Phosphorus as phosphoric acid (H 3 PC>4) 18.93 per cent. Phosphorus as phosphorous acid (H 3 PO 3 ) 2.15 per cent. Phosphorus as hypophosphorous acid (HsPC^) 4-27 per cent. Phosphorus as red phosphorus 2 . 98 per cent. 28.33 Oxidation of phosphorus may be prevented by distilling in a current of carbon dioxide as in the Mitscherlich-Scherer method (see page 15). Metabolism in Phosphorus Poisoning. Phosphorus has a very poisonous action upon the processes of metabolism. Present as a vapor in the blood and tissue fluids, it retards normal oxidative processes occurring in the animal organ- VOLATILE POISONS 17 ism'during metabolism. In phosphorus poisoning the usual course of chemical metabolism is wholly changed. Fat instead of being oxidized is deposited in the organs in large quantity (fatty degeneration of the liver). Different observers believe there is formation of fat from protein. During phosphorus poisoning the quantity of protein broken down is greatly increased. In human metabolism this applies to protein in both food and tissues. Yet the needs of the organism are not satisfied and the conclusion is that the changes are not as complete as in normal protein metabolism. This increase in the breaking down of tissues in phosphorus poisoning recalls similar changes which take place during respiration in insufficient oxygen. Accompanying these abnormal processes are certain nitrogenous and non-nitrogenous products of metabolism which either are not normally formed in the organism or appear merely as intermediate steps in the formation of the oxidative products of metabolism. Decomposition of the protein molecule goes in part only as far as the amino-acids. Consequently in phosphorus poisoning the urine almost always contains CH 3 \ >CH - CH 2 -- CH(NH 2 ) - COOH Leucine (a-amino-isobutyl-acetic acid), CH 3 / /OH (i) Tyrosine (p-oxyphenyl-a-aminopropionic C 6 H 4 < acid) X CH 2 - CH(NH 2 ) - COOH (4) CH 3 - CH(OH) - COOH Sarcolactic acid (dextro-lactic acid). In acute phosphorus poisoning the following acids can be detected in the urine in greatly increased quantity: /OH (i) CeH^ Para-oxyphenyl-acetic acid. X CH 2 - COOH (4) xOH (i) Para-oxyphenyl-propionic acid (hydro- CeH^ para-cumaric acid). X CH 2 - CH 2 - COOH (4) S - CH 2 - CH(NH 2 ) - COOH Cystine, | , has also been detected in phosphorus S - CH 2 - CH(NH 2 ) - COOH urine. In phosphorus poisoning there is a marked decrease in the urea-content of the urine but a decided increase in total nitrogen. A considerable part of the nitrogen, that is to say, 25 per cent, or more of the total nitrogen, appears to leave the body as ammonia. The urine of adults usually contains from 2 to 5 per cent, of the total nitrogen as ammonia. The increase of ammonia may have some con- nection with the increase in formation of acid during phosphorus poisoning. Peptone-like substances, the presence of which is attributed to profound dis- turbance of metabolism, frequently appear in the urine in phosphorus poisoning. Various observers believe there is no longer any doubt as to the appearance of geniune peptonuria. A glycosuria may also appear, or be easily induced by a diet rich in sugar. In accord with this observation is the fact that the liver of an animal poisoned by phosphorus is without the power to change glucose of the blood into glycogen and store up the latter. In phosphorus poisoning the alkalinity of the blood rapidly diminishes owing to the increased formation of acid. Since persons poisoned by phosphorus have icterus (jaundice), bile-pig- ment, or at least urobilin, can be readily detected in the urine. 2 18 DETECTION OF POISONS The amounts of oxygen and carbon dioxide, which the organism respectively takes up and gives off, show a marked diminution during phosphorus poisoning. Only 48 per cent, of carbon dioxide, as compared with 100 per cent, under normal conditions, may be eliminated. In brief the chief characteristics of phosphorus urine are a strong acid reac- tion; presence of protein (peptone-like substances) ; and frequently occurrence of the amino-acids mentioned above, as well as fat cylinders, cell detritus, free fat globules and blood-corpuscles. Further Examination of the Distillate When phosphorescence has been distinctly observed in the Mitscherlich apparatus, it is advisable to stop distillation and change the Liebig condenser to its customary position. This simpler method of distilling is shown in Fig. 7 and should al- ways be used in toxicological analysis when there is no occasion to test for phosphorus. PIG. 7. Distillation with Liebig Condenser. Since the several poisons appearing here are not equally volatile with steam, it is best to collect the distillate in two or three fractions. The first will contain most of the easily volatile substances like hydrocyanic acid, chloroform, ethyl and methyl alcohol, acetone, iodoform and nitrobenzene. The others (second and third) will contain substances less easily volatile with steam like carbolic acid, aniline, chloral hydrate and carbon disulphide. This must not be understood to mean that the first part of the distillate will be free from substances that volatilize VOLATILE POISONS with difficulty, and the latter part free from those that volatilize easily. In the main such will be the separation, but either part of the distillate may contain traces of substances which will appear in larger quantity in the other part. The proper procedure is to distil until 5 to 10 cc. of liquid have been collected. Divide the distillate into several portions and test for hydrocyanic acid, chloroform, ethyl and methyl alcohol, acetone, and, if necessary, also for iodoform and nitro- benzene. Use the second and third portions (10 to 20 cc.) to test for carbolic acid, aniline, chloral hydrate and carbon disulphide. Several of these volatile substances have a characteristic odor, which makes it possible to recognize them with great certainty in the original material and especially in the distil- late. First, test the distillate for each individual substance by its most characteristic reaction. Test for hydrocyanic acid by the Prussian blue or sulphocyanate reaction; for ethyl alcohol, acetone and acetaldehyde by Lieben's reaction; for methyl alcohol by one of the oxidation tests; for carbolic acid and aniline by Millon's reaction; for chloroform, chloral hydrate and iodoform by the phenylisocyanide reaction; for aniline with calcium hypochlorite solution; and finally, for carbon disulphide with lead acetate and potassium hydroxide solutions. When there is reason to believe that a certain substance is present, confirm the result by making other characteristic tests. It is seldom necessary to examine the distillate for all the members of the group. HYDROCYANIC ACID, HCN Physiological Action. In whatever way applied, hydrocyanic acid is absorbed, even from the skin. So rapid is the absorption of this poison that there is evidence of an intoxication after a few seconds, or a few minutes at most. Part of the poison thus absorbed passes from the body unchanged by way of the lungs. Another part, usually much less, is eliminated by the kidneys and passes into the urine. Sweat also is said to contain hydrocyanic acid. Most of the absorbed hydrocyanic acid, though variable in quantity, undergoes chemical change within the organism whatever be the form of its chemical com- bination. Hydrocyanic acid is supposed to combine with loosely bound sulphur of proteins and form sulphocyanic acid (HSCN) which is not nearly as toxic as hydrocyanic acid. (Antidote for hydrocyanic acid.) Hydrocyanic acid after the 20 DETECTION OF POISONS manner of the cyanohydrin reaction 1 might combine chemically with carbohy- drates of the blood and tissues. Finally, putrefactive changes as well as ferment action within the cadaver might convert hydrocyanic acid into ammonium for- mate. 2 The last statement may explain the disappearance of hydrocyanic acid until only traces remain in the cadaver. Thus the possibility of making more than an approximately quantitative determination of hydrocyanic acid taken internally is precluded from the beginning. Yet there are instances where the poison has been found in the human cadaver after 14 days, and even after 100 and 180 days. After 48 days the author obtained enough hydrocyanic acid in the distillate from stomach and intestinal contents of a child 4^ years old to give the Prussian blue test in three different portions of the distillate after 3 to 4 hours. Undoubtedly hydrocyanic acid has a very poisonous effect upon ferments for it kills certain vegetable and animal enzymes, or at least strongly retards their action. This acid interferes particularly with the action of that enzyme which causes transfer of oxygen from blood-corpuscles and thereby gives rise to oxida- tive processes (oxidation ferment, "respiration ferment"). Careful experiments in metabolism have shown that warm-blooded animals under the influence of hydrocyanic acid take up less than the normal amount of oxygen and con- sequently give off less carbon dioxide, even though relatively large quantities of oxygen are administered artificially. R. Kobert (Intoxikationen) regards hydro- cyanic acid poisoning as an internal asphyxiation of the organs in presence of an excess of oxygen. The oxidative processes of the blood are checked and so little oxygen is used that the venous blood becomes arterial, that is to say, contains a large quantity of oxyhaemoglobin. As a result the color of the venous blood is bright red. This change of venous to arterial blood seems to be permanent in cold-blooded but usually only transitory in warm-blooded animals. The appearance of lactic acid in the blood and urine is due to the disturbing influence of the poison up'on the oxidative processes of the organism. The processes of normal metabolism in warm-blooded animals finally oxidize lactic acid to car- bon dioxide and water. Consequently the appearance of lactic acid in the blood is very transitory and it is not found in the urine at all. The occurrence of lactic acid in the blood and a decrease in its alkalinity are concurrent. As a result of very deficient oxidation during hydrocyanic acid poisoning, dextrose not infre- quently appears in the urine. The blood therefore in hydrocyanic acid poisoning is characteristically changed. Venous blood becomes bright red. And moreover blood which contains this acid cannot liberate oxygen from hydrogen peroxide, that is to say it has lost its catalytic power. 3 Such a compound as cyano-hajmoglobin appears to exist and * R ~ \) + HCN = R ~ C ^ OH (R denotes an y radical) OH 2 ,O 'H-CN =H-C/ O H 2 \Q - NH 4 J Hydrocyanic acid poisons platinum black just as it does blood ferments. Put about 5 cc. of 3 per cent, hydrogen peroxide solution in each of two test-tubes. Add to one i or 2 drops of hydrocyanic acid (about i per cent, solution) and to both a trace of platinum black. Pure hydrogen peroxide at once gives off oxygen vigorously, whereas that containing hydrocyanic acid does not VOLATILE POISONS 21 its formation in the blood of a person poisoned by hydrocyanic acid would seem probable, yet for some unknown reason the union of this acid with haemoglobin takes place either not at all or only with great difficulty. In a chemical examination for hydrocyanic acid and potas- sium cyanide the contents of the stomach and intestines, or- gans rich in blood as liver, brain and heart, the blood itself and sometimes the urine are most important. Examine such material at once for hydrocyanic acid which may be recognized by its characteristic odor, provided putrefaction has not gone too far. Preliminary Test. A special test (Schonbein-Pagenstecher reaction) for hydrocyanic acid should precede distillation. Acidify a portion of the original material in a small flask with tartaric acid solution. Then suspend in the flask (see Fig. i) a strip of " guaiac-copper " paper 1 without letting it touch the liquid. Gently warm the contents of the flask upon the water- bath. Neither hydrocyanic acid nor potassium cyanide is present, unless the paper is turned blue or bluish green. But the only conclusion to be drawn from a positive test is that hydrocyanic acid, or an easily decomposable cyanide, may be present. Further conclusions should not be drawn from a positive result, since other substances like ammonia, volatile ammonium compounds, hydrochloric acid and especially oxi- dizing agents like ozone, hydrogen dioxide, nitric acid and chlorine will turn the paper blue. Consequently, though very delicate, this test cannot be accepted as conclusive proof of the presence of hydrocyanic acid. Mechanism of the Reaction. Hydrocyanic acid has nothing directly to do with this reaction. But it forms ozone with copper sulphate and that turns the guaiaconic acid of guaiac resin blue. Cupric cyanide (a) is an intermediate product which furnishes ozonized oxygen as shown in (/3) : (a) CuSO 4 + 2HCN = Cu(CN) 2 + H 2 SO 4 , (0) 6Cu(CN) 2 + 3H 2 = 6CuCN + 6HCN + O 3 . 1 Prepare "guaiac-copper" paper by saturating strips of filter paper with freshly prepared, 10 per cent, alcoholic tincture of resin of guaiac. Dry these strips in air and moisten before using with very dilute aqueous copper sulphate solution (1:1000). 22 DETECTION OF POISONS The actual chemical examination for hydrocyanic acid is made by adding tartaric acid solution to the finely divided material and distilling as described (see page 18). This acid volatilizes easily with steam and most of it will appear in the first part of the distillate. Therefore use the first 5 or 10 cc. of distillate for the tests. Note cautiously the odor of the distil- late, which is characteristic, and then proceed as follows: 1. Prussian Blue Test. Add to the solution (distillate) a little potassmm hydroxide solution; then i or 2 drops of fer- rous sulphate solution and i drop of ferric chloride solution. Shake well and warm gently. Finally acidify with dilute hy- drochloric acid. If much hydrocyanic acid is present, a pre- cipitate of Prussian blue will appear immediately. But if the quantity is small, the solution will have merely a blue or bluish green color. After a long time (10 to 12 hours) a flocculent precipitate of Prussian blue will settle to the bottom of the test tube. The limit of delicacy is 1:5, 000,000. 1 Mechanism of the Reaction. 2 Hydrocyanic acid and potassium hydroxide form potassium cyanide which with ferrous sulphate produces ferrous cyanide (a). The latter combines with more potassium cyanide, forming potassium ferrocyanide (0) which with ferric chloride precipitates Prussian blue (y), the ferric salt of hydroferrocyanic acid (H 4 Fe(CN 6 ). () FeS0 4 + aKCN = Fe(droquinol (CeH^OHMi^))- These enter into synthesis with sulphuric acid and appear in urine as ethereal salts of sulphuric acid. The dark color of "carbolic urine" is largely due to further oxidation of hydroquinol, whereby colored products (quinone?) are formed. In carbolic acid poisoning, urine often has a pronounced dark color (greenish to black). Urine in other cases is amber-yellow at first, but standing in air gives it a deeper color. When carbolic acid poisoning is suspected, the urine should be examined chemically. "Carbolic urine" differs from normal human urine in being nearly free from sulphuric acid, 1 the so-called "preformed sul- phuric acid." Consequently barium chloride solution, in presence of excess of acetic acid, gives only a slight precipitate of barium sulphate or none at all. Filter when there is a precipitate and warm the clear filtrate with a few cc. of concentrated hydrochloric acid. An abundant precipitate of barium sulphate will usually appear. The mineral acid decomposes phenyl-sulphuric acid into phenol and sulphuric acid which is then precipitated. Normal human urine 1 This is sulphuric acid present in urine as sulphates. It is also termed "pre- formed sulphuric acid," by which is meant that it enters the body as such. In this respect it differs from "ethereal," or "conjugate" sulphuric acids, which result from syntheses within the body. VOLATILE POISONS 27 contains considerably more "sulphate sulphuric acid" (A sulphuric acid) than "ethereal sulphuric acid " (B sulphuric acid). The average proportion between the two being: A SO^B S(>4 = 10:1. Barium chloride solution, added to normal urine in presence of acetic acid, produces a heavy precipitate of barium sulphate. Distribution of Carbolic Acid in the Human Body After Poisoning C. Bischoff 1 examined organs, removed from a man who died 15 minutes after taking 15 cc. of liquid carbolic acid, and found the poison distributed as stated in the table below. The organs in this case were perfectly fresh. Only a small por- tion of the stomach was received. Weight Organ Phenol 242 grams Contents of stomach and intestine o. 171 gram 112 grams Blood 0.028 gram 1480 grams Liver 0.63 7 gram 322 grams Kidney 0.201 gram 1445 grams Brain 0.314 gram Bischoff distilled with steam until the distillate gave no further precipitate with bromine water. The results show how rapidly carbolic acid is absorbed, and how soon it is distributed throughout the body. E. Baumann 2 has published certain facts relating to the quantity of carbolic acid formed during putrefaction of protein substances. Baumann states that he . obtained from 100 grams of fresh pancreas and 100 grams of moist fibrin, mixed with 250 cc. of water, after 6 days of putrefaction 0.073 to 0.078 gram of tri- bromophenol, corresponding to 0.0208 to 0.022 gram of phenol. Urine gives a distinct test for carbolic acid 15 minutes after the poison has been taken by the mouth, or hypodermically. This shows how rapidly carbolic acid is absorbed. Most of the carbolic acid absorbed is eliminated in 4 or 5 hours. Schaffer 3 found the quantity of conjugate sulphuric acid in urine to increase in exact proportion to the quantity of carbolic acid taken. Tests for the Detection of Carbolic Acid Carbolic acid distils quite easily with steam. Yet, to remove the last traces, long-continued distillation is necessary. If fractional distillation is made, when carbolic acid is present, this substance will appear in the first and second fractions and even in the third. Usually carbolic acid can be recognized by its peculiar odor. When much carbolic acid is present, the distillate is milky. Colorless or reddish globules may be seen 1 Berichte der Deutschen chemischen Gesellschaft 16, 1337 (1883;. 2 Berichte der Deutschen chemischen Gesellschaft 10, 685 (1877) and Zeit- schrift fur physiologische Chemie i, 61 (1877-78). 3 Journal fur praktische Chemie, Neue Folge 18, 282 (1878). 28 DETECTION OF POISONS floating in the liquid. Excess of potassium or sodium hydroxide solution will dissolve carbolic acid and render the distillate perfectly clear. Pure, anhydrous carbolic acid melts at 40 to 42 and distils at 178 to 182. Decomposition of protein substances produces phenol and especially para-cresol in small quantity. Traces of phenols can almost always be detected in distillates from animal matter in an advanced stage of decomposition. Millon's reagent, and usually bromine water, will give positive tests with such distillates. I. Millon's Test. Millon's reagent, 1 heated with a solution containing only a trace of carbolic acid, produces a red color. An aqueous solution containing only 20 mg. of carbolic acid, diluted i : 100,000, will give a distinct red color. If the phenol solution is not very dilute, the color will appear even in the cold. Though a very delicate test, it is not characteristic of carbolic acid, because several other aromatic compounds behave simi- larly. This is true of derivatives of mon-acid phenols like the three cresols, salicylic acid, 2 para- hydroxy-benzoic acid, para-hydroxy- phenyl-acetic acid, para-hydroxy- phenyl-propionic acid (hydro-para- cumaric acid 3 ) and tyrosine. Aniline heated with Millon's reagent also gives a dark red color. 2. Bromine Water Test. Excess of bromine water produces a yellow- ish white, crystalline precipitate, even Wlth V ^ dllute C ^ O ]ic add SOlu r of 1:20,000. tions. It is a very delicate test for carbolic acid. Phenol diluted i : 50,000 yields, after some time, a precipitate made up in part of well-formed crystals (Fig. 8). 1 For the preparation of this reagent see page 321. Traces of salicylic acid volatilize with steam, at least in such quantity that it can be detected with Millon's reagent. 3 Para-hydroxy-phenyl-acetic acid and hydro-para-curnaric acid are formed in the putrefaction of proteins but are not volatile with steam. VOLATILE POISONS- 29 If there is a sufficient excess of bromine water to give the supernatant liquid a brownish red color, the precipitate consists only of tribromophenyl hypobromite, C 6 H 2 Br 4 O. R. Benedikt 1 regards this compound as a brom-phenoxy-tribromo- benzene with the structure OBr , whereas Thiele and Eichwede 2 have ascribed to it the structure I O A I BrC CBr /\ BrC CBr HC CH \/- HC CH C \/ Br C Br 2 This reaction takes place so easily that carbolic acid may even be determined quantitatively as this tetrabromo-derivative (see page 31). It melts at 132- OH I34 with evolution of bromine and crystallizes as lemon-yellow J, leaflets from alcohol-free chloroform or ligroin. Heated with / /?\ alcohol, acetone, xylene, or aqueous sulphurous acid, this com- BrCe 1 2CBr pound loses bromine and changes at once to 2,4,6-tribromo- I II phenol, melting at 93-94. Salicylic alcohol (saligenin), sali- ^/ CH Cylic aldeh y de > salic ytic acid and para-hydroxy-benzoic acid C are converted quantitatively by an excess of saturated bromine Br water even in the cold into tribromo-phenyl hypobromite. 3. Ferric Chloride Test. Very dilute ferric chloride solution, added drop by drop, imparts a blue-violet color to aqueous carbolic acid solutions. Addition of dilute hydrochloric or sulphuric acid changes this color to yellow. This test is not as delicate as i and 2. It is entirely negative in presence of min- eral acids. The limit of delicacy is about i : 1000. 4. Hypochlorite Test. Add a few cc. of ammonium hydroxide solution to a dilute, aqueous carbolic acid solution, and then 2 or 3 drops of freshly prepared calcium or sodium hypochlorite solution. Gentle warming will produce a blue color. Very dilute carbolic acid solutions after some time give only a green to blue-green color. F. A. Fliickiger 3 allows bromine vapor to come into contact with the phenol solution which has been mixed with a little ammonium hydroxide solution in a porcelain dish. 1 Annalen der Chemie und Pharmazie 199, 127 (1879). 2 Berichte der Deutschen chemischen Gesellschaft 33, 637 (1900). 3 Pharmaceutische Chemie, page 287 (1879). 30 DETECTION OF POISONS 5. Nitrite Test Mix a carbolic acid solution with a dilute alcoholic solution of ethyl nitrite, C 2 H 6 -O-N = O, 1 or iso- amylnitrite, C 5 Hn-0-N = O, 2 and add concentrated sulphuric acid from a pipette so that it forms a distinct under-layer. A red zone will appear at the contact surface of the two liquids. This is a very delicate test. This test may also be made by adding the liquid under ex- amination as an upper layer upon concentrated sulphuric acid containing a trace of red fuming nitric acid. 6. H. Melzer's Benzaldehyde Test. 3 Add 2 cc. of concen- trated sulphuric acid to i cc. of the solution (distillate) to be tested for carbolic acid, then i or 2 drops of benzaldehyde and heat. The mixture, at first yellowish brown, will become dark red. At the same time a red resinous substance will appear, unless the solution is too dilute. When cold add 10 cc. of water and enough potassium hydroxide solution to give a distinct alkaline reaction. If carbolic acid is present, a violet-blue color will appear. To obtain this coloring-matter, acidify the solution, extract with ether and evaporate the solvent. Alka- lies, added to alcoholic solutions of the coloring-matter, produce a blue color which acids discharge. This is a very delicate test. One cubic centimeter of 0.05 per cent, carbolic' acid solu- tion (= 0.0005 gram of carbolic acid), will still give the blue color very distinctly. Note. In absence of phenol concentrated sulphuric acid produces a dark brown color with benzaldehyde. According to A. Russanow 4 the first condensa- tion product between phenol and benzaldehyde in presence of concentrated sulphuric acid is para-dihydroxy-triphenyl-methane which crystallizes in yellow- ish needles: C ' H6 \ ; HjC 6 H 4 -OH C 6 H 8V /C 6 H 4 -OH w >C = i O + i = H 2 + >C< (i, 4). H|C 6 H 4 -OH H / \C 6 H 4 -OH Benzaldehyde " Phenol P-Dihydroxy-triphenyl-methane Alkalies dissolve the pure crystals without color but, if these solutions are exposed to air, oxidation takes place and a red or red-violet color appears. Prob- l The officinal preparation is called "Spiritus Aetheris Nitrosi." 2 Amylium nitrosum of pharmacists. 3 Zeitschrift fur analytische Chemie 37, 345 (1898). 4 Berichte der Deutschen chemischen Gesellschaft 22, 1943 (1889). VOLATILE POISONS 31 ably benzaurine, dihydroxy-triphenyl-carbinol, is first formed. This compound is a brick-red crystalline powder soluble in alkalies with a violet color. QUANTITATIVE METHODS OF ESTIMATING PHENOL i. Gravimetric Estimation as Tribromophenol The principle of this method is based on the complete pre- cipitation of phenol from aqueous solution as tribromophenyl hypobromite by an excess of saturated bromine water (see Test 2) . The precipitate is practically insoluble in cold bromine water and the results are very satisfactory. 1 Procedure. Place the aqueous phenol solution in a large glass-stoppered flask. Add gradually, while shaking, saturated bromine water until the supernatant liquid has a red-brown color and bromine vapor is visible above the solution. Let stand 2-4 hours and shake frequently. Then collect the pre- cipitate in a weighed Gooch crucible and dry in a vacuum des- iccator over sulphuric acid to constant weight. On the basis of the following proportion calculate the weight of phenol corre- sponding to the weight of the precipitate: C 6 H 2 Br 4 O : C 6 H 5 -OH = Wt. of Ppt. found : x 409.86 94-05 Since the ratio -~r- = 0.2295, the weight of phenol may be 409.00 found by multiplying the weight of the precipitate by 0.2295. 2. Beckurts-Koppeschaar 2 Volumetric Method Dilute sulphuric acid liberates hydrobromic acid from potas- sium bromide (a) and bromic acid from potassium bromate (/3). These two acids react according to (7) : (a) KBr + H 2 SO 4 = KHSO 4 + HBr, (ffl KBrO 3 + H 2 SO 4 = KHSO 4 + HBrO 3 , (7) S HBr + HBrOs = 3Br 2 + 3H 2 O. 1 The following results were obtained by F. Beuttel: Phenol taken CeH 2 Br 4 O Phenol found Per cent, found 1. 0.103 grm. 0.4538 grm. 0.0997 g rn i. 96.2 2. 0.2072 grm. 0.8806 grm. 0.2014 grm. 98.6 3. 0.2072 grm. 0.8708 grm. 0.2006 grm. 98.6 2 Archiv der Pharmazie 24, 570 (1886). 32 DETECTION OF POISONS Therefore addition of dilute sulphuric acid to a mixture of potassium bromide and bromate solutions liberates bromine which will convert phenol into a mixture of tribromophenol and tribromophenyl hypobromite. The excess of free bromine and also the loosely bound bromine atom of tribromophenyl hypobromite will displace iodine from potassium iodide and finally all the phenol will be present as tribromophenol: C 6 H 2 Br 3 OBr + 2 KI = C 6 H 2 Br 3 OK + KBr + I 2 One molecule of phenol requires 6 atoms of bromine, as shown by the equation: 5 KBr + KBrOs + 6H 2 SO 4 + C 6 H 8 OH = C 6 H 2 Br 3 OH + 3 HBr + 6KHSO 4 + 3H 2 0. The following standard solutions are required: 1. o.oi n-potassium bromide solution, containing - 595.6 grams = = 5.956 grams KBr in 1000 cc. 2. o.oi n-potassium bromate solution, containing - 167.17 grams = - = 1.6717 grams KBr0 3 in 1000 cc. 3. o.i n-sodium thiosulphate solution, containing o.i Na2S 2 O3.5H 2 O grams = 24.83 grams in 1000 cc. 4- Potassium iodide solution, containing 125 grams of KI in 1000 cc. Procedure. Put about 25 cc. of aqueous phenol solution (distillate) into a flask having a tight glass stopper. Add 50 cc. each, of o.oi n-potassium bromide and o.oi n-potassium bro- mate solutions, then 5 cc. of pure concentrated sulphuric acid and shake vigorously for several minutes. The gradually increasing opalescence of the solution becomes more and more marked, as tribromophenol and tribromophenyl hypobromite are precipitated. The yellow color which soon appears shows excess of bromine. Open the flask in 15 minutes, add 10 cc. of potassium iodide solution, shake and titrate free iodine in 5 minutes with o.i n-sodium thiosulphate solution. VOLATILE POISONS 33 6 gram-atoms Br 6 X 79.96 Calculation. --- = - --^- = 4.7976 grams of bromine are set free from a mixture of 1000 cc. of o.oi n-potassium bromide solution and 1000 cc. of o.oi n-potasssium bromate solution. A mixture therefore of 50 cc. of each of the two solutions will give 0.2399 gram of bromine. This quantity of bro- mine can convert 0.04704 gram of phenol into tribromophenol : 6Br : C 6 H 6 OH 479.76 94.05 = 0.2399 : x (x = 0.04704) i cc. of o.i n-sodium thiosulphate solution corresponds to 0.012697 gram of iodine and this quantity of iodine to 0.007996 gram of bromine. But 0.007996 gram of bromine will convert 0.00157 gram of phenol into tribromophenol: 6Br : C 6 H 5 OH 479.76 94.05 = 0.007996 : x (x = 0.00157) Consequently, for each cc. of o.i n-sodium thiosulphate solution used, subtract 0.00157 from 0.04704 gram of phenol. This determines the quantity of car- bolic acid in the 25 cc. of distillate taken. 3. Messinger-Vortmann 1 Volumetric Method Excess of iodine (8 atoms of iodine to i molecule of phenol dissolved in 4 molecules of potassium hydroxide) , added to an alkaline phenol solution at 50-60, will produce a dark red, non- crystalline precipitate. One molecule of phenol requires 6 atoms of iodine. 1. CH 6 OH + 3 I, = C 6 H 2 I 3 OH + 3 HI, 2. 3HI + 3 KOH = 3 H 2 + 3 KI. This red precipitate dissolves in hot potassium hydroxide solution with a red-brown color and appears as white 2, 4, 6- tri-iodophenol, melting at 154-156, on addition of an excess of dilute sulphuric acid. Messinger and Vortmann regard the red compound as di-iodophenyl hypoiodite (C 6 H 3 I 2 OI) which potassium hydroxide converts into the more stable isomeric tri- iodophenol: 01 OH #\ is converted by 1C CI potassium hy- 1C CI droxide into HC CH HC CH H I Red di-iodo- White 2. 4, 6-tri- phenyl hypoiodite iodophenol 1 Berichte der Deutschen chemischen Gesellschaft 22, 2312 (1889); and 23, 2 753 (1890). See also Kossler and Penny, Zeitschrift fiir physiologische Chemie 17, 117 (1892). 34 DETECTION OF POISONS Procedure. 1 The reaction between the alkaline phenol solu- tion and iodine is rather slow in the cold but is hastened at 50 to 60. Place a measured volume of aqueous phenol solution (5 to 10 cc.) in a small flask and add a measured volume of o.i n-potassium hydroxide solution until the mixture is strongly alkaline. Warm gently by dipping the flask in water at 60 and add 10-15 cc. more of o.i n-iodine solution than the volume of o.i n-potassium hydroxide solution used, or until the excess of iodine produces a strong yellow color. Agitation will cause a deep red precipitate to appear. Cool the solution, acidify with dilute sulphuric acid and dilute to a definite volume (250 to 500 cc.). Filter an aliquot portion (100 cc.) rapidly and determine excess of iodine with o.i n-sodium thiosulphate solu- tion. Calculation. Each molecule of phenol requires 6 atoms of iodine. Therefore i atom of iodine = -~ = = 6 6 1 5-^75 phenol. 1000 cc. of o.i n-iodine solution, containing o.i gram-atom of iodine, correspond therefore to 1.5675 grams of phenol. Note. This method will not give satisfactory results, unless at least 3 molecules of sodium or potassium hydroxide are taken for i molecule of phenol. Estimation of Phenol in Urine In determining carbolic acid in urine, the regular occurrence of phenols must not be overlooked. After a mixed diet, the quantity of normal human urine passed in 24 hours will yield approximately 0.03 gram of phenols (phenol and more especially para-cresol) . In certain diseases where there is excessive bacterial decomposition within the organism, in the intestines for example, urine contains more of these phenols and, consequently, more conjugate sulphuric acids. Even external application of carbolic acid, for instance the use of carbolic acid water as a lotion, is sufficient to i ncrease the quantity of phenyl-sulphuric acid in urine. Detection of Carbolic Acid in Presence of Aniline Aniline closely resembles carbolic acid in behavior toward Millon's reagent and ne water. But the two substances can be easily separated. Add potas- 1 Use 0.5 to i per cent, carbolic acid solution for laboratory experiments. VOLATILE POISONS 35 sium hydroxide solution in large excess and distil. The distillate will contain aniline alone. Or make the solution strongly acid with dilute sulphuric acid, and extract with ether which will dissolve only carbolic acid. Evaporate the ether extract at a moderate temperature and examine the residue. CHLOROFORM Behavior in the Human Organism. When inhaled, chloroform first passes from the air into the blood-plasma which then transmits it to the red blood-corpuscles JT where it may accumulate in relatively large quantity. Air passed through blood will remove chloroform completely. Pohl (see Cl C Cl Robert's " Intoxikationen ") states that blood may contain 0.62 I per cent, of chloroform, three-fourths of which will be in the red blood-corpuscles. At the height of a harmless narcosis the blood contained only 0.035 per cent, of chloroform. Absorption of chloroform is rapid from all parts of the body. The stimulative action of chloroform on the mucous membranes of the respiratory passages explains such disturbances as coughing, secretion of saliva and reflex slowing of respiration and heart-beat, occurring at the beginning of narcosis. Dilatation of the blood-vessels of organs living after death is due to paralysis caused by even small doses of chloroform. A drop in blood-pressure accompanies paralysis of the brain and the heart's action is feebler and slower. Several researches regarding the effect of inhaled chloroform upon human and animal metabolism have shown an increase in the quantity of nitrogen in the urine after prolonged narcosis because more protein is decomposed. The amount of neutral sulphur and chlorine in the urine also increases. The increase of the latter is due in part at least to the conversion of chloroform into chloride. The acidity of the urine is also much higher. A final characteristic of chloroform urine is the high content of reducing substances. The increased protein decom- position in chloroform narcosis affects both reserve protein and that of the tissues. This may explain degeneration in red blood-qorpuscles, glandular organs, the heart, etc., after frequent narcoses or one of long duration. The temporary or permanent paralysis of isolated animal or vegetable cells, such as leucocytes, ciliated cells, yeast cells, algae and spores, is evidence of the antiseptic action of chloroform when present in proper concentration in air or in a liquid. This explains the use of i per cent, aqueous chloroform solution as an antiseptic. Added to urine it acts as a preservative. Therefore it may be used in the study of the action of enzymes but not of bacteria, though all micro-organ- isms are not paralyzed or killed by chloroform water. Pohl and Hans Meyer have studied the distribution of chloroform in the body and found that the red blood-corpuscles and the brain are most likely to show this poison. After chloroform has been inhaled, some will appear in the gastric juice but at most only traces in the urine. In but two out of 15 cases of chloro- form narcosis was this poison found in the urine and then only in traces. Robert states that as a rule it is the exception to find chloroform itself in the cadaver, because part of the poison is converted into chloride in the human organ- ism and part is quickly exhaled during respiration. Usually it is possible to detect chloroform in the breath of patients even 24 hours after narcosis. Bii- dinger states that the mucus of the respiratory passages retains chloroform. 36 DETECTION OF POISONS Tests for the Detection of Chloroform Chloroform distils easily with steam and appears in the first fraction in largest quantity. When much chloroform is present , it will separate from the distillate as heavy, colorless globules, whereas a small quantity will remain in solution. This solution usually has the characteristic odor and sweetish taste of chlo- roform. The following tests should be applied to the first fraction. 1. Phenylisocyanide Test. Add i or 2 drops of aniline to the chloroform solution (distillate), and then a few cc. of aqueous, or alcoholic potassium hydroxide solution. Gentle heat will produce phenylisocyanide (CeHsNC). The penetrating and very repulsive odor of this compound is easily recognized. CHCls + C 6 H 5 .NH 2 + 3KOH = C 6 H S .NC + aKCl + 3H 2 O. A. W. Hofmann states that this test will show with certainty i part of chloroform in 5000 to 6000 parts of alcohol. Note. Chloral, chloral hydrate, bromoform, iodoform and tetrachloro- methane also give this test. The fact that aniline boiled with potassium hydroxide solution gives a peculiar, faintly ammoniacal odor, even when chloroform is absent, must not be over- looked. There is small chance, however, of confusing this odor with the repulsive smell of phenylisocyanide. In doubtful cases warm some water, containing a drop of aniline and a trace of chloroform, with potassium hydroxide solution and compare the odor with that in question. 2. Schwarz's Resorcinol Test. 1 Dissolve about o.i gram of resorcinol ( C H dW)j|?y m 2 cc - of water > add a few dr P s of sodium hydroxide solution and finally the liquid containing chloroform. This mixture heated to boiling will develop even in very dilute solution a yellowish red color attended by a beau- tiful yellowish green fluorescence. Chloral, bromal, bromoform and iodoform also give this test. 3- Lustgarten's 2 Naphthol Test. Dissolve a few centigrams of a- or /3-naphthol hi i or 2 cc. of 33 per cent, aqueous potas- sium hydroxide solution. Warm to 50 and add the solution to 1 Zeitschrift fur analytische Chemie, 27, 668. 2 Monatshefte fur Chemie, 3, 715 (1882). VOLATILE POISONS 37 be tested. Chloroform will produce an evanescent blue color which in contact with air will change to green and then to brown. This color is less stable when /3-naphthol is used. Acidification of the blue solution will precipitate naphthol col- ored by a red dye-stuff. This precipitate is usually brick-red. Chloral, bromal, bromoform and iodoform also give this test. 4. Fujiwara's Pyridine 1 Test. Mix 2 cc. of pyridine with 3 cc. of 10 per cent, sodium hydroxide solution, heat to boiling and add i cc. of the liquid to be tested. Even a trace of chloro- form will produce a bright, blue-red color. Chloral, bromoform, iodoform and several other similar compounds also respond to this test. One part of chloroform in 1,000,000 parts of water, 500,000 parts of ether, or 300,000 parts of ethyl alcohol can be detected by this test. It is equally sen- sitive toward the other substances mentioned. 5. Cyanide Test. Seal the liquid to be tested for chloroform in a glass tube (pressure- tube) 2 with a little solid ammonium chloride and alcoholic potassium hydroxide solution. Heat for several hours in a boiling water-bath. Cool the tube, re- move the solution and test for hydrocyanic acid by the Prussian blue reaction. A positive test means that the distillate con- tained chloroform. The following reactions take place: (a) CHC1 3 + H 3 N + 3KOH = HCN + 3 KC1 + 3H 2 O, (0) HCN + KOH = KCN + H 2 O. 6. Reduction Tests, (a) With Fehling's Solution. Warm the liquid containing chloroform with Fehling's solution. A red precipitate of cuprous oxide will appear. (b) With Ammoniacal Silver Nitrate Solution. Add excess of ammonium hydroxide to silver nitrate solution and then the liquid containing chloroform. Heat will produce a black precipitate of metallic silver. These reactions are not characteristic of chloroform, because many volatile organic substances, as formic acid and aldehydes 1 Chemical Abstracts n, 3201 (1917). 2 An ordinary citrate of magnesium bottle is a convenient apparatus for this test. Wrap a towel around the bottle, place it in the water-bath and gradually raise the temperature to boiling. Do not remove the bottle until it is cold. Tr. 38 DETECTION OF POISONS which may occur in distillates from animal material, reduce Fehling's and ammoniacal silver nitrate solutions. Quantitative Estimation of Chloroform in Cadavers (Ludwig-Fischer 1 ) Mix a weighed portion of material with water and distil as long as there is any chloroform. To tell when this point is Breached, apply the phenylisocyanide test to a few cc. of liquid collected at the end of distillation. Add some calcium car- bonate to combine with free hydrochloric acid. Warm the distillate to about 60 and draw washed air through it by suc- tion. Pass this air through a combustion-tube heated to high temperature and then into silver nitrate solution acidified with nitric acid. Weigh the precipitated AgCl (N). Calculation : 3 AgCl : CHC1 3 = N : x. This method is based upon the fact that chloroform heated with steam above 200 is decomposed into carbon monoxide, hydrochloric and formic acids: (a) CHCl, + H 2 O = CO + 3 HC1. (0) CHCU + 2H 2 O = H.COOH + 3 HC1. In a series of blank experiments B. Fischer has shown that the stomach, stomach contents and blood, of a person who has not taken chloroform, give no volatile chlorine compounds under these conditions. By this method B. Fischer found in the cadaver of a laborer, who had died during chloroform narcosis, the following quantities of chloroform: Weight Organ Chloroform 985 grams Stomach and contents and parts of the intestine o.i gram 780 grams Lungs and blood from the heart o-55 gram 445 grams Portions of spleen, kidneys and liver traces 480 grams Brain 0.07 gram From these results it appears that most of the chloroform was in the brain and blood. CHLORAL HYDRATE Cl C1_C_Q Chloral hydrate distils very slowly with steam from an acid I solution. Therefore the complete distillation of a large quantity H C OH of chloral hydrate requires considerable time. Chloral hydrate I appears as such in the distillate. 1 Jahresbericht des chemischen Untersuchungsamtes der Stadt Breslau fur die Zeit vom i April ^94 bis 31 Marz 1895. VOLATILE POISONS 39 Tests for the Detection of Chloral Hydrate Chloral hydrate like chloroform will give the phenyliso- cyanide, resorcinol, naphthol and pyridine tests. But the distillate containing chloral hydrate does not have the charac- teristic chloroform odor which is also scarcely perceptible in very dilute aqueous chloroform solutions. Jaworowski 1 suggests the following tests to differentiate chloral hydrate from chloroform: 1. Test with Nessler's Solution. Add a few drops of this re- agent to an aqueous chloral hydrate solution and shake. It will produce a yellowish red precipitate, the color of which will change after a while to a dirty yellowish green. This is an aldehyde reaction. 2. Test with Sodium Thiosulphate. Boil a few cc. of chloral hydrate solution with 0.2-0.3 gram of solid sodium thiosulphate. This will give a turbid liquid of brick-red color. A few drops of potassium hydroxide solution will remove the turbidity and change the color to brownish red. When the quantity of chloral hydrate is not too small, it may also be detected by the following procedure: Decomposition of Chloral Hydrate. Heat a portion of the distillate for 30 minutes under a reflux condenser with calcined magnesium oxide (MgO) upon a boiling water-bath. Magne- sium formate and chloroform are produced by decomposition of chloral hydrate. 2 CC1 3 .CH(OH) 2 + MgO = aCHCl, + Mg(OOCH) 2 + H 2 O. Proceed as follows to detect these products : Chloroform. Distil a few cc. from the solution in the flask and test for chloroform by the phenylisocyanide, resorcinol, a-naphthol and pyridine tests. Formic Acid. Filter the icsidue from the distillation, con- centrate the nitrate to a few cc. by evaporation and divide into two parts for the following reduction tests: (a) Reduction of Mercuric to Mercurous Chloride. Add a few drops of mercuric chloride solution and warm. Formic 1 Pharmaceutische Zeitung fiir Russland 33, 373, und Zeitschrift fur analytische Chemie, 37, 60 (1898). 40. DETECTION OF POISONS acid, if present, will produce a white precipitate of mercurous chloride (calomel) : Mg(OOCH) 2 + 4HgCl 2 = 2'Hg 2 Cl 2 + MgCl 2 + 2HC1 + 2CO 2 . (b) Reduction of Silver Nitrate. Warmed with silver nitrate solution, formic acid and its salts produce a black precipitate of metallic silver: Mg(OOCH) 2 + 4AgN0 3 = 4Ag + Mg(NO 3 ) 2 + 2HNO 3 + 2CO 2 . Detection of Chloral Hydrate in Powders or Solutions Extract a powder with cold water containing sulphuric acid, filter, extract the filtrate several times with ether and spon- taneously evaporate the ether extracts in a shallow dish or on a clock-glass. Chloral hydrate imparts to the residue its char- acteristic pungent odor. The odor of chloroform is easily recognized by warming the residue with sodium hydroxide solution: CC1 3 -CH(OH) 2 + KOH = CHC1 3 + H.COOK + H 2 O The phenylisocyanide, resorcinol, naphthol and pyridine tests, as well as that with Nessler's reagent, should be applied to the residue. In the case of an aqueous solution of chloral hydrate, first acidify with dilute sulphuric acid and repeatedly extract with ether. Evaporate the ether extracts and examine the residue as already described. Note. Pure chloral hydrate forms transparent crystals which are dry, perma- nent and colorless. This compound has a pungent odor, its taste being caustic and faintly bitter. It dissolves with ease in water, alcohol and ether; and in 5 parts of chloroform. It melts at 58. Action and Fate of Chloral Hydrate in the Human Organism Applied locally chloral hydrate acts as a strong stimulant. Taken internally it frequently stimulates the stomach. When it reaches the blood, it acts like chloroform in paralyzing the brain, spinal cord and heart but usually no previous stimulation is noticeable. There is marked decrease in blood-pressure due to paralysis of the blood-vessels. Death from chloral hydrate poisoning is occa- sioned by impaired circulation and respiration, in consequence of which the quan- tity of oxygen taken in and of carbon dioxide given off is considerably diminished. H. Meyer has shown that the narcotic action of chloral hydrate depends, as does that of all compounds of the alcohol and chloroform group, upon the affinity of the poison for lipoids, the fatty constituents of the nervous system. It is also VOLATILE POISONS 41 held by the blood, especially by the red blood-corpuscles. Later it appears unchanged, most abundantly in the cells of the brain and spinal cord (Kobert, ' ' Intoxikationen ") . Only very little chloral hydrate taken internally passes as such into the urine. As shown by v. Mering and Musculus, 1 the greater part by conjugation with gly- curonic acid forms urochloralic acid (CsHnClsO?) which is eliminated as such in the urine. This conjugated acid undergoes hydrolysis, when boiled with dilute acids, and gives trichlor-ethyl alcohol and free dextro-rotatory glycuronic acid: CsHnCUpT + H 2 = CC1 3 -CH 2 OH + HOOC-(CH.OH) 4 -CHO Urochloralic Trichlor- Glycuronic acid. ethyl alcohol. acid. Urochloralic acid is therefore trichlor-ethyl glycuronic acid. It is crystalline and with heat reduces silver solution as well as alkaline copper and bismuth so- lutions. Consequently chloral urine behaves much like sugar urine but differs from the latter in being strongly laevo-rotatory. The reduction of the aldehyde chloral, to its corresponding primary alcohol, trichlor-ethyl alcohol, is especially noteworthy as regards the behavior of chloral hydrate in the human organism. Quantitative Estimation of Chloral Hydrate in Blood and Tissues (Archangelsky 2 ) Distil the material for 12-20 hours with its own weight of 20 per cent, phos- phoric acid, repeating the process if the distillate is turbid or yellow. To com- plete the decomposition of chloral hydrate into chloroform and formic acid, add 50 cc. of sodium hydroxide solution to the distillate and concentrate on the water- bath to about 20 cc. Neutralize the solution exactly and heat for 6 hours on the water-bath with an excess of mercuric chloride solution. Finally weigh the precipitated mercurous chloride. Satisfactory results were obtained by this method when known quantities of chloral hydrate were added to blood and organs. Using this method Archangelsky has shown that chloral hydrate is not uniformly distributed in the blood but is contained especially in the blood-cor- puscles. When narcosis begins there is less chloral hydrate in the brain than in the blood. But later the percentage of the poison in the brain is higher than in the blood. Archangelsky has further shown how much chloral hydrate the blood must contain before narcosis can appear. A dog's blood must contain 0.03-0.05 per cent. When the blood contains 0.12 per cent., respiration ceases. IODOFORM lodoform crystallizes in shining hexagonal leaflets or plates. It may also T appear as a rather fine crystalline powder, lemon-yellow in color and having a penetrating odor somewhat like saffron. The melting-point I C I of iodoform is approximately 120. It is nearly insoluble in water; I soluble in 50 parts of cold and in about 10 parts of boiling alcohol; and soluble in 6 parts of ether. It is also freely soluble in chloroform. 1 Berichte der Deutschen chemischen Gesellschaft 8, 662 (1875); and v. Mer- ing, Ibid., 15, 1019 (1882). 2 Archiv fur experimentelle Pathologic und Pharmakologie, 46, 347 (1901). 42 DETECTION OF POISONS Detection of lodoform lodoform distils quite easily with steam and gives a milky distillate having a characteristic odor. Extract this distillate with ether and carefully test the residue left by the spontaneous evaporation of the solvent. If much iodoform is present, it will form yellow hexagonal plates. Dissolve the ether residue in a little alcohol, and use this solution for the following tests: i. Lustgarten's 1 Test. Gently warm a few drops of alcoholic iodoform solution in a test-tube with a little sodium phenolate (CeHs.ONa) solution. 2 If iodoform is present, a red substance will be deposited on the bottom of the tube. A few drops of dilute alcohol will dissolve this precipitate with a carmine-red color. Also make the resorcinol, pyridine and phenylisocyanide tests (see pages 36 and 37). NITROBENZENE Nitrobenzene has a strong poisonous action. Administration of very small quantities of this compound has produced death in human beings. There are J^Q^ records in the literature of several cases where 20 drops, and even j 7 to 8 drops, have caused fatal results. But on the other hand complete recovery has followed poisoning by much larger doses. Fatal poisonings have come also from inhaling nitrobenzene vapor. Within recent years nitrobenzene has been used to some extent as HC CH an abortifacient. Nitrobenzene poisons the blood and changes its \/ appearance. The blood has a chocolate color and at the same time the red blood-corpuscles change their shape and go into solution. As a result the blood is incapable of uniting with oxygen. The blood of persons poisoned by nitrobenzene is said to contain less than i per cent, of oxygen so that death is caused by asphyxiation. Healthy blood contains about 17 per cent, of oxygen by volume. There seems to be no methaemoglobin in blood containing nitrobenzene. Such blood examined spectroscopically shows the two oxyruemoglobin bands and also a special absorption-band between C and D (Fihlene's nitrobenzene band). It is proba- ble that the slight solubility of this poison necessitates a definite incubation period, for 2 to 3 hours usually elapse after nitrobenzene has been taken before 1 Monatshefte fur Chemie, 3, 715 ( Z 882). 2 Prepare sodium phenolate solution by mixing 20 grams of phenol with 40 grams of sodium hydroxide and 70 grams of water. VOLATILE POISONS 43 signs of intoxication appear. A woman, who had taken 10 drops of mirbane oil as an abortifacient, gave no indication of intoxication, that is to say, uncon- sciousness and cyanosis, for 8 hours after taking the poison. Nitrobenzene not only profoundly changes the blood but it irritates and paralyzes the central nervous system (see R. Kobert, "Intoxikationen")- Some nitrobenzene passes into the urine. Although it has been stated that the organism does not convert nitrobenzene into aniline, Rossi 1 found in the viscera of a person, who had died from supposed nitrobenzene poisoning, aniline which evidently had been formed as a result of putrefaction. Conse- quently in cases of fatal poisoning, tests for nitrobenzene should be made im- mediately after death. In nitrobenzene poisoning human urine contains a brown pigment but only rarely haemoglobin or methaemoglobin. Urine contain- ing nitrobenzene will reduce Fehling's solution. It is also unfermentable and distinctly laevo-rotatory. A conjugated glycuronic acid is possibly concerned in these reactions. Detection of Nitrobenzene In nitrobenzene poisoning the urine and all the organs have the odor of this compound. For the chemical tests the material should first be distilled with water. Nitrobenzene distils quite easily with steam and appears in the distillate as yellowish globules. These are heavier than water and have a character- istic odor. Vigorously agitate the globules, when separated as completely as possible from water, with granulated tin and a few cc. of concentrated hydrochloric acid, until there is no odor of nitrobenzene. Pour the acid solution from undissolved tin, and add an excess of potassium hydroxide solution to decompose the double chloride of aniline and tin. Extract free aniline with ether. Withdraw the aqueous liquid from the separating funnel, and evaporate the ether extract spontaneously in a small glass dish. Aniline, formed by reducing nitrobenzene, will remain as globules which usually have a red or brown color. Dissolve these globules by agitation with water, and use this solution for the hypochlorite and phenylisocyanide tests (see pages 45 and 36). Mechanism of the Reaction. Nitrobenzene is reduced by nascent hydrogen to aniline (a) which combines with the excess of hydrochloric acid forming aniline hydrochloride (/3). From the latter compound potassium hydroxide liberates aniline (y) : 1 Chemical Abstracts 9, 1341 (1915). 44 DETECTION OF POISONS (a) C 6 H 6 -N0 2 + 6H = C 6 H 5 -NH 2 + 2 H 2 O, 03) C 6 H 6 -NH 2 + HC1 = C6H5-NH2.HC1 1 , M C 6 H 6 NH 2 .HC1 + KOH = C 6 H 5 -NH 2 + H 2 O + KC1. ANILINE Toxic Action. Aniline is moderately toxic in its action. Doses of 1.5 to 2 grams, administered in the course of a day, have proved fatal to small dogs. It is not possible to state definitely the average lethal dose for human beings. Very ^r-rr serious results are said to have followed a dose of 3 or 4 grams of aniline. The lethal dose is certainly less than 25 grams, for that C quantity of aniline was sufficient to kill a healthy man. Even ^\ inhalation of aniline vapor may cause severe or fatal intoxications. H 9 |T H Aniline produces methaemoglobin and therefore poisons the HC CH blood. The conversion of oxyhaemoglobin into methaemoglobin \/ by aniline may be demonstrated by adding an aqueous aniline solution to blood in a test-tube. Aniline changes their form and partially decomposes red blood-corpuscles. Thereby the quan- tity of available oxygen in the blood is so diminished that it amounts to only 5 to 10 volumes instead of 15 to 20, the normal quantity. The number of red blood-corpuscles is diminished in aniline poisoning but not that of the white blood-cells. R. v. Engelhardt has shown that aniline is partly changed in the human organ- ism into aniline black, or into a similar compound insoluble in water. At the climax of aniline poisoning blue-black granules may be seen in every drop of blood and also in the urine. Aniline is oxidized in the system to para-aminophenol (C6H4.OH,NH 2 (i,4)). Like all phenols this compound forms an ethereal sul- phate with sulphuric acid, 2 namely, para-aminophenyl-sulphuric acid (HO.SO 2 .- O.C 6 H 4 .NH 2 (i,4). This acid is eliminated through the kidnej^s as an alkali salt and then appears in the urine. A part of the para-aminophenol is also elim- inated as a conjugate of glycuronic acid. 3 The reduction of Fehling's solution by urine containing aniline is due to this conjugated acid. In severe cases of poisoning unchanged aniline has also been found in the urine. Usually urine that contains aniline has a very dark color. Besides the substances mentioned, a dark pigment has been detected 1 Organic ammonium bases resemble ammonia in combining with acids to form salts. Trivalent nitrogen of the free base is changed to pentavalent nitrogen in the salt: III /H C 6 H 6 - N< + HC1 = C 6 H 6 - N~ \TT \~ \C1 Aniline Aniline hydrochloride 2 This conjugation takes place with elimination of water: H 2 N.C 6 H 4 .OH + HO.S0 2 .OH = H 2 O + H 2 N.C 6 H 4 .O.SO 2 .OH (i, 4 ) 3 Glycuronic acid, C 6 Hi O 7 = ^)C-(CH.OH) 4 -COOH, is closely related to glucose. It is an uncrystallizable syrup. If its aqueous solution is boiled, the acid is partly converted into the internal anhydride, glycurone (C 6 H S O 6 ), which crystallizes well. VOLATILE POISONS 45 in urine in aniline poisoning as well as haemoglobin, methaemoglobin and an abundance of urobilin (R. Robert, "Intoxikationen"). Detection of Aniline Aniline is a rather feebly base and part of it will pass over with steam, when the acid solution is distilled. There will be enough in the distillate for detection by the tests described below. In estimating aniline quantitatively in any kind of mate- rial the distillation must be as complete as possible. Mix the substance with water, make strongly alkaline with sodium hydroxide or carbonate solution and distil in a current of steam. Since 30 parts of water at 15 dissolve i part of aniline, the distillate may contain considerable of this amine. When the quantity is large, oil-drops will appear. An aqueous aniline solution (aniline water) colors pine wood and elder pith in- tensely yellow. The following tests should be used for aniline : 1. Hypochlorite Test. Add a few drops of aqueous calcium or sodium hypochlorite solution drop by drop to a portion of the distillate. A violet-blue or purple- violet color, gradually chang- ing to a dirty red, will appear if aniline is present. Addition of a little dilute aqueous phenol solution containing some ammonia will produce a blue color which is quite stable. This test is sensitive i : 66,ooo. r 2. Phenylisocyanide Test. Heat a portion of the distillate with a few drops of chloroform and potassium hydroxide solu- tion. The repulsive odor of phenylisocyanide will show the presence of aniline. 3. Bromine Water Test. Bromine water added to a solution containing aniline will produce a flesh-colored precipitate. This test is sensitive i : 66,000. 4. Chromic Acid Test. 2 Mix a trace of pure aniline with 4 to 5 drops of concentrated sulphuric acid in a porcelain dish and add a drop of aqueous potassium dichromate solution. After a few minutes the mixture beginning at the edge will take on a 1 Test this experimentally with very little aniline. For example, dissolve a small drop in 30 cc. of water and take only 2-3 cc. of this dilute solution for the test. 2 Beissenhirtz reaction, Annalen der Chemie und Pharmazie, 87, 376 (1853). 46 DETECTION OF POISONS pure blue color. Addition of 1-2 drops of water produces at once a deep blue color. To apply this test to the distillate, first extract with ether, evaporate the ether solution and test an oily residue as described. CARBON DISULPHIDE Carben disulphide, CS 2 , is a colorless liquid having a characteristic odor and a high index of refraction. It is only slightly soluble in water. There is some difference of opinion as regards the solubility of carbon disulphide in water. 1000 cc. of water dissolve 13-14 2 . 03 parts (Page) 15-16 i. 8 r. parts (Chancel; Par mentier) 15-16 2-3 ' parts (Ckindi) 15-16 3-S-4-S2 parts (Peligot) Carbon disulphide is miscible in all proportions with absolute alcohol, ether, ethereal and fatty oils. Toxic Action. Carbon disulphide administered internally has a very poisonous action upon the blood causing especially decomposition of red blood-corpuscles. Even inhalation of carbon disulphide vapor frequently occasions severe poi- soning. Carbon disulphide was formerly considered a typical producer of methaemoglobin but recent investigations have not confirmed this opinion. It has a very injurious action upon the red blood-corpuscles and causes haemolysis. R. Robert (Intoxikationen) states that its power of dissolving lipoids is respon- sible for its injurious action upon the blood and the central nervous system. E. Harmsen 1 has recently come to practically the same conclusion. He considers carbon disulphide a powerful blood poison because it decreases the number of red blood-corpuscles and the quantity of haemoglobin and brings about a leuco- cytosis. 2 Detection of Carbon Disulphide Carbon disulphide distils very slowly with steam. Con- sequently the second or third fraction of the distillate should be used in testing for this substance. If 40 cc. are distilled from 100 cc. of water containing 2 drops of carbon disulphide, the following 10 cc. will give a distinct test. If the quantity of carbon disulphide is small, it will remain in solution. Such a solution does not have a strong odor. Carbon disulphide may be recognized by the following tests : 1 Vierteljahrsschrift fur gerichtliche Medizin, 30, 442 (1905). 2 Leucocytosis means a temporary increase in the number of white blood- corpuscles (leucocytes) as compared with the number of red blood-corpuscles. Normally there are about 350 red to i white blood-corpuscle, whereas in leucocy- tosis the proportion is 20 : i. VOLATILE POISONS 47 1. Lead Acetate Test. Add a few drops of lead acetate solution to the liquid containing carbon disulphide. It will cause neither a precipitate (distinction between 82 and H^S) nor a color. Add excess of potassium hydroxide solution and boil. A black precipitate (PbS) will appear. This is a very delicate test. 2. Sulphocyanate Test. Heat an aqueous solution of carbon disulphide for a few minutes with concentrated ammonium hydroxide solution and alcohol. Ammonium sulphocyanate (H 4 NSCN) is formed together with ammonium sulphide. Concentrate this solution upon the water-bath to about i cc. and acidify with dilute hydrochloric acid. Add a drop of ferric chloride solution and a deep red color will appear. This test will show even traces of carbon disulphide, for example 0.05 gram in i cc. of water. Mechanism of the Reaction : (a) 4NH 3 + CS 2 . = (H 4 N)SCN + (H 4 N) 2 S, (0) FeCl 3 + 3 (H 4 N)SCN = Fe(SCN) 3 + 3(H 4 N)C1. 3. Xanthogenate Test. Shake a few cc. of distillate for several minutes with 3 or 4 times its volume of saturated solu- tion of potassium hydroxide in absolute alcohol. Faintly acidify the solution with acetic acid and add i or 2 drops of copper sulphate solution. If carbon disulphide is present, a brownish black precipitate of cupric xanthogenate will appear. This will soon change to a yellow, flocculent precipitate of cuprous xanthogenate, CS(SCu) (OC 2 H 5 ). Vitali's procedure is somewhat different and consists in adding solid ammonium molybdate to the alkaline reaction-product and then in acidify- ing with dilute sulphuric acid. The appearance of a red color indicates carbon disulphide. Mechanism of the Reaction. Alcoholic potassium hydroxide acts like potas- sium alcoholate (C 2 H 5 -OK) and converts carbon disulphide into potassium xanthogenate /SK CS 2 + C 2 H 5 -OK = C=S \OC 2 H 5 This compound treated with cupric salts gives first a brownish black precipitate of cupric xanthogenate: /SK ' /S 2C==S + CuS0 4 = (S = C( ) 2 Cu + K 2 SO 4 \OC 2 H 5 X OC 2 H 5 4g DETECTION OF POISONS The cupric salt then forms cuprous xanthogenate andethyl xanthogen disulphide: /OC 2 H 6 /OC 2 H 5 s = c s = c< s = c\ X)C 2 H B X OC 2 H 6 X OC 2 H 6 Cupric Ethyl xanthogen Cuprous xanthogenate disulphide xanthogenate Quantitative Estimation of Carbon Disulphide in Air Inhalation of air containing carbon disulphide has frequently given rise to chronic poisoning. Persons thus affected have usually been laborers in rubber fac- tories. Consequently experiments have been made to determine the maximum quantity of carbon disulphide air may contain without injury to health. The results of these investigations may be summarized as follows: CS 2 in mgrs. per liter of air 1. 0.5-0.8 No injurious effect. 2. 1.3 Slight uneasiness after several hours. 3. - 3.4 Uneasiness in 30 minutes. 4. 6.0 Uneasiness in 20 minutes. 5. 10.0 Paralysis attended by after-effects last- ing several days. The exact danger limit for persons obliged to live for weeks at a time in an atmo- sphere containing carbon disulphide should be placed below 3 mg. per liter of air. Air in factories, where operatives work in presence of carbon disulphide vapor, should never exceed this limit. In rubber factories the air is said fre- quently to contain 2.5 to 3 mg. per liter. Since experiments have shown that 93 to 96 per cent, of the carbon disulphide breathed was exhaled unchanged, an exceedingly small quantity is capable of producing toxic symptoms. Procedure. Place a saturated alcoholic solution of potassium hydroxide in a Peligot absorption-tube and draw through this solution 10 to 20 liters of air con- taining carbon disulphide vapor. A quantitative formation of potassium xan- thogenate (see above) will take place. Dilute the contents of the receiver at the. end of the experiment with 96 per cent, alcohol and bring the volume to 50 cc. Measure an aliquot portion of this solution and dilute with water. Faintly acidify the solution with acetic acid and remove excess- of acid with acid sodium carbonate. Add freshly prepared starch solution and o.i n-iodine solution until there is a permanent blue color. Iodine converts potassium xanthogenate according to equation (I) into ethyl xanthogen-disulphide : KS.CS.OC 2 H 6 S.CS.OC 2 H 5 I. Ii + = 2KI + | KS.CS.OC 2 H 2 S.CS.OC 2 H 5 VOLATILE POISONS 49 E. Rupp and L. Krauss 1 think the action of iodine upon potassium xanthogen- ate is expressed by equation (II) : II. 2 KS.CS.OC 2 H 6 + H 2 + 2! = KS.CS.SK + 2 C Z H 5 .OH + aHI + S. Both equations require the same quantity of iodine, namely, 2 atoms for 2 molecules of xanthogenate. A difference therefore in the mechanism of the reaction has no influence on the combining relations of the iodine and the method is applicable to the quantitative determination of xanthogenate. 1000 cc. of o.i n-iodine solution, containing o.i gram-atom of iodine, corre- spond to o.i gram-molecule of CSz = 7.6 grams. ETHYL ALCOHOL 2 Fate in the Human Organism. Ethyl alcohol brought in contact with many different parts of the organism is very rapidly absorbed, but especially easily from an empty stomach. Although there is practically no absorption of non- volatile aqueous liquids from the stomach, ethyl alcohol, is freely absorbed. After absorption it passes into the blood and is then jj C H distributed to all organs (see chloral hydrate). Experiments upon dogs, colts and adult horses (see Kobert, " Intoxikationen ") have ^ shown that blood at the climax of narcosis contains 0.72 per cent, jj of ethyl alcohol . There is stupor even when o. 1 2 per cent, is present. There is difference of opinion among toxicologists regarding alcoholic intoxication, as to whether the poison is distributed uniformly through- out the body, or accumulated in the brain in larger quantity than in other organs. The following percentages of ethyl alcohol, found in the organs of a man, who had died at the climax of severe acute ethyl alcohol poisoning, lend support to the latter view: liver 0.21, brain 0.47 and blood 0.33 per cent. It appears from these results that the brain takes up an especially large quantity of ethyl alcohol. Uncertainty concerning the subsequent fate of ethyl alcohol in the organism has finally been removed. Experiments have shown that ethyl alcohol is never eliminated unchanged through the skin. At most only 1-1.5 per cent, passes off through the kidneys and only 1.6-2 per cent, through the lungs. Strass- mann 3 found the quantity eliminated by the lungs somewhat higher (5-6 per cent.) and by the kidneys 1-2.5 P er cent. The remainder is completely oxidized in the human organism to carbon dioxide and water. B. Fischer found the following quantities of ethyl alcohol in organs removed from a man who had probably died from drinking too much brandy: Weight Organ Ethyl Alcohol 2720 grams Stomach and intestines 30.6 grams 2070 grams Heart, lungs and blood 10.85 grams 1820 grams Kidneys and liver 7.8 grams 1365 grams Brain 4.8 grams 1 Berichte der Deutschen chemischen Gesellschaft 35, 4257 (1902). 2 The word "alcohol," unqualified by an adjective, i.e., methyl, amyl, etc., means ethyl alcohol. Tr. 3 Pfliiger's Archiv, 49, 315 (1891). 50 DETECTION OF POISONS Detection of Ethyl Alcohol Ethyl alcohol distils easily with steam and consequently most of it will be in the first fraction. If present in sufficient quantity, it can be recognized in the distillate by its odor. The following tests should be made: 1. Lieben's lodoform Test. 1 Gently warm the liquid (40- 50), add a few cc. of aqueous iodo-potassium iodide solution, or a small crystal of iodine, and enough potassium hydroxide solution to give the liquid a distinct yellow to brownish color. If, ethyl alcohol is present, a yellowish white to lemon-yellow precipitate of iodo- form will appear immediately, or as the solution cools. If the quantity of ethyl alcohol is very small, a precipitate will form on long stand- ing. When iodoform is deposited slowly, the crystals are very perfect PIG. 9 .-Iodof orm Crystals. hexagonal plates and sta rs (see Fig. 9) . Note. This iodoform test is very delicate but not characteristic of ethyl alcohol, since other primary alcohols, except methyl alcohol, and many secondary alcohols, as well as their oxidation products, aldehydes and ketones, give iodo- form under the same conditions. Acetic ether, aceto-acetic ether, lactic acid, etc., also give iodoform. The correct explanation of the iodoform reaction is probably the following: Iodine and potassium hydroxide form potassium hypo-iodite (KOI) by reaction (a). This compound brings about the oxidation of ethyl alcohol to acetic aldehyde (/3) and at the same time substitutes iodine for hydrogen in the latter (y). Finally tri-iodo-acetic aldehyde is decomposed by the excess of potassium hydroxide into iodoform and potassium formate (6) : (a) 2KOH + I, = KI + H 2 O + KOI, . (/3) CH 3 .CH 2 .OH + KOI = CH 3 .CHO + H 2 O + KI, (7) CH 3 .CHO + 3KOI = 3 KOH + CI 3 .CHO, (5) CI 3 .CHO + KOH = CHI 3 + H.COOK. 2. Berthelot's Test. Shake the liquid containing ethyl alcohol with a few drops of benzoyl chloride and about 5 cc. of sodium hydroxide solution (10 per cent.), until the irritating 1 Annalen der Chemie und Pharmazie, Supplement Band, 7, 218. VOLATILE POISONS 51 odor of benzoyl chloride has gone. The aromatic odor of ethyl benzoate will appear. C 6 H 6 .COC1 + C 2 H 5 .OH + KOH = C 6 H 5 .CO.OC 2 H 5 + KC1 + H 2 O Ten cc. of 0.5 per cent, ethyl alcohol will give a distinct odor of this ester. 3. Chromic Acid Test. Warm the liquid containing ethyl alcohol with dilute sulphuric, or hydrochloric acid, and add i or 2 drops of very dilute potassium dichromate solution. The color of the liquid will change from red to green, and at the same time the odor of acetaldehyde will be recognized. This test is not characteristic of ethyl alcohol because many other volatile organic compounds behave similarly. Mechanism of the Reaction (a) K 2 Cr 2 O 7 + H 2 S0 4 = K 2 SO 4 + H 2 Cr 2 O7(H 2 O + sCrO,), (0) 3 C 2 H 6 .OH + 2 Cr0 3 + 3H 2 S0 4 = 3 CH 3 .CHO + Cr 2 (SO 4 ) 3 + 6H 2 O. % Acetaldehyde 4. Ethyl Acetate Test. Mix the liquid containing ethyl alcohol with the same volume of concentrated sulphuric acid. Add a very small quantity of anhydrous sodium acetate and heat. Ethyl acetate will be recognized by its odor. () C 2 H 6 .OH + H 2 SO 4 = CjHsO.SOa.OH 1 + H 2 O, (/3) CH 3 .CO.ONa + C 2 H 6 O.SO 2 .OH = CH 3 .CO.OC 2 H 5 + NaHSO 4 . 5. Vitali's Test. Thoroughly mix a few cc. of distillate in a glass dish with a small piece of solid potassium hydroxide and 2 or 3 drops of carbon disulphide. Let this mixture stand for a short time without warming. When most of the carbon disul- phide has evaporated, add a drop of ammonium molybdate solution and then an excess of dilute sulphuric acid. If the dis- tillate contains ethyl alcohol, a red color will appear. Potas- sium xanthogenate (CS(OC 2 H 5 )(SK)) is first formed. This compound gives a red color with ammonium molybdate. Acetone and acetaldehyde produce a similar color. This test is given distinctly by 5 per cent, aqueous ethyl alcohol solution. . Dissolve 0.2 gram of rosaniline base in 20 cc. of cold saturated sulphurous acid solution. If the color is not discharged in 24 hours, add 10 cc. more of sul- phurous acid. Repeat, if necessary, until there is no color and dilute to 200 cc. 6 Analyst 33, 417 (1908). VOLATILE POISONS 55 monium persulphate (H 4 N.SO 4 ) and 3 cc. of dilute sulphuric acid d : 5). Dilute the mixture to 20 cc. with water and distil, collecting in test-tubes 5 separate 2 cc. portions. Reject the first two portions, which contain any acetic aldehyde, and add to the last three a few drops of 0.5 per cent, morphine hydro- chloride solution. Finally run into each test-tube 3 cc. of concentrated sulphuric acid as an under layer. A violet ring will appear at the contact-surface of the two liquids, if formal- dehyde is present. This test will not show with certainty less than 5 per cent, of methyl alcohol in ethyl alcohol. Quantitative Estimation of Methyl Alcohol The method of Leach and Lythgoe, 1 requiring the use of the Zeiss immersion refractometer, furnishes a rapid means of estimating methyl alcohol in presence of ethyl alcohol. This instrument gives a reading of 14.5 for distilled water at 20, a maximum reading of 101 for 75 per cent, ethyl alcohol and 91 for 100 per cent.; and a maximum reading of 39.8 for 50 per cent, methyl alcohol, 14.9 for 91 per cent, and 2 for 100 per cent. 2 ACETONE Human urine almost always contains a very small quantity of acetone as a physiological constituent. Under pathological conditions, especially in diabetes jj mellitus (diabetic acetonuria), urine contains much more. It is also present in urine in protracted high fever, digestive disturb- H C H ances, severe forms of carcinoma (carcinomatous acetonuria), etc. I Finally, acetone has been found in urine in considerable quantity | ~ in various intoxications (toxic acetonuria), for example, in poison- H C H ing by phosphorus, carbon monoxide, atropine, curare, antipyrine, pyrodine, sulphuric acid, extract of male fern; in chronic lead poisoning; and in chronic morphinism after discontinuance of the drug (see R. Robert, "Intoxikationen"). Acetone is not poisonous nor in the least corrosive. Man and animals can tolerate considerable quantities of acetone taken internally. It seems to produce no effect, though it may possibly possess very feeble narcotic properties. Arch- angelsky found dogs to show signs of narcosis when the blood contained 0.5 per cent, of acetone. Even smaller doses produce narcosis in rabbits and have an injurious action upon the blood and kidneys. 1 Journal of the American Chemical Society 27, 964 (1905). 2 Tables giving the percentages of methyl alcohol corresponding to the dif- ferent readings will be found in the original communication of the authors. 56 DETECTION OF POISONS Distillates from human urine, as well as from blood and various organs, as liver, spleen, kidneys, brain, etc., often contain acetone, or more correctly per- haps, substances like acetone. This is especially the case when cadaveric mate- rial has begun to putrefy. Acetone is a clear, colorless liquid boiling at 56. It has a peculiar, fruity odor and is neutral in reaction. It is miscible in all proportions with water, ethyl alcohol and ether. It distils easily with steam. Detection of Acetone i. Lieben's lodoform Test. Add a few cc. of aqueous iodo- potassium iodide solution, or a small crystal of iodine, to an aqueous solution of acetone and then potassium hydroxide solution drop by drop until the color is yellow. lodoform immediately separates, even in the cold, as a yellowish white precipitate which is usually amorphous. Acetone differs from ethyl alcohol in giving iodoform, when ammonium hydroxide solution is substituted for potassium or sodium hydroxide solution (Gunning's acetone test). Acetaldehyde resembles acetone in giving iodoform in the cold and under conditions the same as those stated above. Note. Potassium hypo-iodite (a) probably converts acetone into tri-iodo- acetone (CHj.CO.CIs) (/3) and this compound is then decomposed by potassium hydroxide into iodoform and potassium acetate (y) : (a) 6KOH + 3 I 2 = 3 KI + 3 KOI (0) CH,.CO.CH 3 + 3 KOI = CH 3 .CO.CI 3 (7) CHs.CO.CIs 4- KOH = CHI 3 + CH 3 .CO.OK. 2. Legal's Test. Add a few drops of freshly prepared sodium nitroprusside solution to a liquid containing acetone, and then potassium hydroxide solution. A red or reddish yellow color will appear. This color soon changes to yellow. Add an excess of acetic acid to the solution. The solution will now have a carmine to purplish red color, according to the quantity of acetone present. Heat will change this color to violet. Ethyl alcohol does not give Legal's test, though acetaldehyde does. The red color caused by aldehyde fades upon addition of acetic acid, and changes to green with heat. Le Nobel states that ammonium hydroxide, or ammonium carbonate solution, may be substituted for potassium hydroxide solution in Legal's test, but under these conditions the red color is very slow to appear. Le Nobel's modification, however, eliminates the possibility of confusing acetone with acet- aldehyde. VOLATILE POISONS 57 3. Penzoldt's Test. Prepare a hot, saturated, aqueous ortho-nitro-benzaldehyde (C 6 H 4 .NO 2 .CHO(i,2)) solution and allow it to cool. Add this solution to the liquid containing acetone, and also some sodium hydroxide solution. At first the color of the mixture is yellow. It then becomes green, and a blue precipitate of indigotin is formed in 10 to 15 minutes. When indigotin is present in traces only, shake the solution with chloroform. This solvent will dissolve the coloring matter and become blue. 4. Reynolds' Test. Acetone will dissolve freshly precipi- tated mercuric oxide, and this test is based upon this property. Add mercuric chloride solution to the distillate, and an alcoholic potassium hydroxide solution. Shake thoroughly and filter. Add ammonium sulphide solution to the clear filtrate as an upper layer. If acetone is present, there will be a black zone (HgS) where the two solutions meet. Detection of Acetone in Urine. Acidify 200 to 500 cc. of urine with a few drops of sulphuric acid and distil. Collect 20 to 30 cc. of distillate. This will contain the entire quantity of acetone in the urine. Acetone thus obtained may pos- sibly be derived from aceto-acetic acid which is often present in human urine, especially in a severe case of diabetes mellitus. Distillation decomposes this acid into acetone and carbon dioxide. CH 3 .CO.CH 2 .CO.OH = CH 3 .CO.CH 3 + CO 2 . Detection of Ethyl Alcohol and Acetone in Mixtures. Ethyl alcohol may be detected in presence of acetone by Berthelot's test. On the other hand, ace- tone may be distinguished from ethyl alcohol by Legal's or Penzoldt's test. BITTER ALMOND WATER AND BENZALDEHYDE Bitter almond water (Aqua Amygdalee Amarae of the Phar- macopoeia) contains hydrocyanic acid. Only a small portion of this acid, however, is free so that it can be precipitated by silver nitrate solution. The greater part is combined as the / H cyanohydrin of benzaldehyde, C 6 H 5 .C^OH, which does not re- act with silver nitrate. But potassium hydroxide solution will decompose this compound. C 6 H 5 CH(OH)CN + KOH = KCN + H 2 O + C 6 H 5 .CHO. 58 DETECTION OF POISONS Pure benzaldehyde, also called hydrocyanic acid-free oil of bitter almonds, is not poisonous. It is oxidized to benzoic acid in the body and eliminated in the urine partly as that acid and. partly as hippuric acid after conjugation with glycocoll (amino-acetic acid) : (a) C 6 H 5 -CHO + O = C 6 H 6 -COOH, (/3) CeHs-COOH + H 2 N-CH 2 -COOH = C 6 H 6 -CO-NH-CH 2 -COOH. Benzoic acid Glycocoll furnished Hippuric acid by the organism Ordinary commercial oil of bitter almonds contains hydro- cyanic acid and is poisonous in proportion to the quantity of this acid present. Test for hydrocyanic acid by shaking about 2 cc. of oil of bitter almonds with 20 cc. of potassium hydroxide solution and making the Prussian blue test. When oil of bitter almonds is mixed with other material, distil with steam from a solution acidified with tartaric, or dilute sulphuric acid, and test the first part of the distillate for hydrocyanic acid. If benzaldehyde is present, the distillate at the same time will be milky and have the characteristic odor of that compound. Distil until the drops of water are perfectly clear. Benzaldehyde may be detected with certainty, and at the same time distinguished from nitro- benzene which has a somewhat similar odor, by adding a few drops of potassium hydroxide solution to the milky distillate, to combine with any hydrocyanic acid, and extracting with ether. The ether upon evaporation will deposit benzaldehyde as globules, which can be positively identified by conversion into benzoic acid. Heat the globules for a few minutes in a small flask, attached to a reflux condenser, with about 10 cc. of potassium dichromate solution and a little dilute sulphuric acid. Cool, extract with ether and evaporate the ether solution in a glass dish. When the material contains benzaldehyde, this residue will consist of benzoic acid. This substance may be further identified by its melting-point (120-121), its property of subliming and the test with ferric chloride solution. 1 1 Dissolve the residue in a small quantity of water, and neutralize benzoic acid by heating the solution to boiling with excess of calcium carbonate. Filter and add a few drops of ferric chloride solution. If benzoic acid is present, a flesh- colored precipitate of basic ferric benzoate will appear. Tr. VOLATILE POISONS 59 SYNOPSIS OF GROUP I Scherer's Test for Phosphorus Precedes Distillation The material to be examined must, first be rendered uniform by grinding or chopping. Add sufficient water to thin the mass, acidify with tartaric acid and distil. If the preliminary test for phosphorus (Scherer's) is positive, distil in the Mitscherlich apparatus; otherwise distil as usual with a Liebig condenser. It is advisable to collect the distillate in two or three fractions. Test the first 5 to 10 cc. of distillate for hydrocyanic acid, chloroform, ethyl and methyl alcohol, acetone and possibly also for nitre-benzene and iodoform. Use the remainder of the distillate in testing for carbolic acid, chloral hydrate and carbon disulphide. Phosphorus. Phosphorescence in Mitscherlich apparatus during distillation in a dark room. Evaporate distillate with strong chlorine water, or a little fuming nitric acid, and test the residue for phosphoric acid. As an alternative procedure, examine the original material, or at least the Mitscherlich dis- tillate, for phosphorus by the Blondlot-Dusart method. Hydrocyanic Acid. Odor. Schonbein's preliminary test. Prussian blue test. Sulphocyanate test. Nitroprusside test. Silver nitrate test. Alkaline phenolphthalin test. Carbolic Acid. Odor. Red color with MilloH's reagent. Yellowish white precipitate with bromine water. Violet color with ferric chloride solution. Chloroform. Separation of colorless globules, when the quantity is large. Odor. Phenylisocyanide test, when heated with aniline and potassium hydroxide solution. Reduces silver nitrate and Fehling's solutions with heat. Red color with resorcinol and potassium hydroxide solution. Blue color with naphthol and potassium hydroxide solution. Blue-red color with pyridine and sodium hydroxide solution. Chloral Hydrate. Gives chloroform reactions. Brick-red precipitate with Nessler's solution which in time becomes yellowish green. Gives chloroform and magnesium formate, 60 DETECTION OF POISONS when heated with magnesium oxide and water. Test for formate with silver nitrate or mercuric chloride solution. lodoform. Odor. Distillate milky and yellowish white. Ether extract of distillate leaves crystals upon evaporation, gives chloroform reactions. Nitrobenzene. Yellowish globules with characteristic odor. Reduced to aniline, when shaken with tin and hydrochloric acid. Test for aniline. Aniline. Violet color with, calcium hypochlorite solution. Phenylisocyanide test, when heated with chloroform and potassium hydroxide solution. Flesh-colored precipitate with bromine water. Dark red color on warming with Millon's reagent. Carbon Bisulphide. Black precipitate, or only black colora- tion (PbS), when heated with lead acetate and potassium hydroxide solutions. Formation of ammonium sulphocyanate by evaporation with concentrated ammonium hydroxide solu- tion and detection with ferric chloride solution. Formation of potassium xanthogenate, when shaken with alcoholic solu- tion of potassium hydroxide and detection with copper sulphate solution. Ethyl Alcohol. lodoform test. Odor of ethyl benzoate, when shaken with benzoyl chloride and sodium hydroxide solu- tion. Green color and aldehyde odor, when heated with potas- sium dichromate and hydrochloric acid- Vitali's test. Methyl Alcohol. Oxidation tests with copper, potassium permanganate and ammonium persulphate. Acetone. Gives iodoform, even in the cold, with iodine and potassium hydroxide or ammonium hydroxide solution. Legal's test. Indigotin test. Reynolds' test. CHAPTER II NON-VOLATILE POISONS 1 Alkaloids, Glucosides and Synthetic Compounds Non-volatile with Steam from Acid Solution Put a portion of finely chopped material into a large flask, and thoroughly mix with two or three times as much absolute ethyl alcohol. 2 Add enough tartaric acid solution to give the mixture a distinct acid reaction after shaking. Laboratory experiments usually require 20 to 30 drops of 10 per cent, tartaric acid solution. Avoid a large excess of tartaric acid, since it may act as an objectionable impurity in the ether extract, owing to its solubility in that solvent. Connect the flask with a glass tube (80 to 100 cm. long) serving as a reflux cooler. Frequently shake and heat 10 to 15 minutes upon the water-bath. In the exti action of a large quantity of material from a cadaver, con- nect the flask with an upright Liebig condenser used as a reflux cooler (Fig. -10). Cool the flask contents and filter to remove fat and other insoluble matter. Wash the residue with ethyl alcohol. Evaporate the filtrate, which must have an acid reac- tion, to a thin syrup in a glass dish upon the water-bath. Thor- oughly mix this residue with 100 cc. of cold water. Usually this causes an abundant separation of fat and resinous matter, especially when parts of a cadaver are examined. Filter and evaporate the nitrate to dryness, or to a syrup, upon the water- 1 The isolation of these toxic substances from cadaveric material, food, etc., is necessary before tests establishing their presence can be made. Mixtures used for laboratory practice, consisting of dog biscuit, meat, comminuted organs (liver, kidneys, spleen), sausage meat, etc., with any of the poisons of this group, should be examined according to the method outlined above. 2 Commercial ethyl alcohol usually contains basic compounds, the presence of which is objectionable. They should be removed by adding tartaric acid to the ethyl alcohol and distilling. Ethyl alcohol should not be used in toxicologi- cal analysis, unless an actual test has shown it to be free from such impurities. Tr. 61 62 DETECTION OF POISONS c bath. Thoroughly mix this residue with absolute ethyl alcohol. As a result of this treatment, a whitish substance, which is more or less viscous or slimy, usually remains undissolved. This residue, which consists chiefly of protein substances (albumin, albumoses and peptones), dextrin-like compounds and in part also of inorganic salts, frequently becomes granular upon standing. Tartrates of the alkaloids and other organic poisons are dissolved. The larger the quantity of ab- solute ethyl alcohol used, the more com- plete the precipitation of those substances ., which interfere more or less with the t^^r** ^" detection of organic poisons. Again evap- orate the filtered alcoholic solution upon the water-bath, and dissolve the residue in about 50 cc. of water. If the solution is not perfectly clear, filter through a moistened paper. The result of this procedure is a solution containing alkaloidal tartrates and other organic substances belonging to this group. This solution should have an acid reaction and be practically free from protein substances, fat, resinous bodies and coloring matter. If the solution fulfils these requirements, it is ready to be examined for organic poisons accord- ing to the "Stas-Otto" method. The utmost care must be taken in preparing this solution, because definite conclusions cannot be drawn from the uncertain tests given by impure material. For the purpose of isolating alkaloids from viscera free from ptomaines and other impurities, Magnin and Zappi 1 suggest the following procedure. Finely comminute the material and macerate in water acidulated with a few drops of dilute sul- 1 Chemical Abstracts 9, 2748 (1915). NON-VOLATILE POISONS 63 phuric acid (i :4). Warm at 40 for 1-2 hours to facilitate extraction and set aside for 18-20 hours. Filter, add an equal volume of 90-92 per cent, ethyl alcohol to the filtrate, then 10 cc. of 30 per cent, aluminium sulphate solution and finally 10 cc. of 15 per cent, potassium hydroxide solution. After 2-3 hours filter and concentrate the filtrate in vacuo to a sirupy consistency at a temperature not exceeding 45-50. Treat the residue with 20 times its volume of 90-92 per cent, ethyl alcohol, set aside for 18-20 hours, filter and again concentrate in vacuo as above. Dissolve the residue in water and use the solution for the extraction of alkaloids. Ptomaines and other impurities are almost completely eliminated by this procedure. When the material is a powder mixed with cane- or milk- sugar, it is usually possible, after the aqueous solution has been acidified with tartaric acid, to extract diiectly with ether and continue according to the Stas-Otto method. Frequently in suspected poisoning an examination of beer, wine, black coffee, infusion of tea, food, etc., is necessary. In such cases the process outlined above may often be considerably shortened. Acidify the material with aqueous tartaric acid solution, if necessary, and evaporate in a glass dish upon the water-bath. Treat the residue thoroughly with absolute ethyl alcohol and filter. Evaporate the filtrate uporr the water- bath and dissolve the residue in tepid water. Filter this solution, if necessary, and then examine according to the Stas-Otto process. STAS-OTTO PROCESS A. Examination of Ether Extract of Tartaric Acid Solution Thoroughly extract the acid aqueous solution (see process of preparation described above) two or three times with ether, using each time about the same quantity of solvent. Employ a separating funnel for this purpose (Fig. n). Pour the com- bined ether extracts into a dry flask loosely stoppered. If the solution stands for i or 2 hours at rest, a few drops of water usually settle out. Decant the ether solution and pour through 64 DETECTION OF POISONS a dry filter. Slowly evaporate this solution in a small glass dish upon a water-bath previously heated slightly above 35. Do not have gas burning during this operation! Examine the residue as described below. An excellent method of evaporat- ing ether consists in setting a small glass dish (8 to 10 cm. in PlG. II. Separating Funnels and Glass Crystallizing Dishes. diameter) upon a hot water-bath and dropping the filtered ether extract into it as fast as the solvent evaporates. Thus a large quantity of extract may be evaporated in a small dish. The advantage of this method is the ease with which the residue can be removed for the various tests. The residue is usually quite small and it is not advisable to have it distributed over too large a surface. NON-VOLATILE POISONS 65 Examine the residue from the ether extract for the following substances : Picrotoxin Caffeine Antipyrine Colchicin Acetanilide Salicylic Acid Picric Acid Phenacetine Veronal Evaporation of the ether extract, even in the absence of members of the group, usually leaves a more or less viscous residue, containing tartaric and lactic acids as well as fatty, resinous and colored substances. This is especially so in analyses of cadaveric material. Moreover ether extracts certain metallic salts from aqueous solutions, for example, mercuric cyanide 1 and chloride. First, note the general appearance and taste of the residue. Then examine it with a microscope. Very definite conclusions as to the presence or absence of certain substances can fre- quently be drawn. A very bitter residue should be examined carefully for picrotoxin and colchicin. If there is a pronounced yellow color, the examination should include picric acid also. Veronal is colorless and has a very bitter taste. A tasteless, or only faintly bitter, residue probably does not contain these sub- stances and should be examined for acetanilide, antipyrine, caffeine, phenacetine and salicylic acid. The residue from evaporation of the ether extract may con- tain the following substances: Picrotoxin. Usually a thick syrup which gradually solidifies and becomes crystalline. Tastes intensely bitter. Colchicin. Yellowish, amorphous residue which does not become crystalline. Tastes intensely bitter. Dissolves in water with a yellowish color, which increases in intensity on addition of a few drops of dilute hydrochloric acid. 1 Ether to some extent will extract mercuric cyanide from a tartaric acid solu- tion which is not too dilute. For instance, it will remove appreciable quantities from 100 cc. of o.i per cent, mercuric cyanide solution, but the extraction will not be complete. The solution after five extractions will still give a distinct test for mercury. Ether will not remove even a trace of mercuric cyanide from o.oi per cent, solution. To test for cyanide, add ammonium sulphide solution to the ether residue. This will precipitate mercuric sulphide and the nitrate will con- tain ammonium sulphocyanate (see hydrocyanic acid, page 22). 5 QQ DETECTION OF POISONS Picric Acid. Usually appears as a syrup which gradually solidifies and becomes crystalline. Tastes very bitter. Resi- due intensely yellow, giving yellow aqueous solutions not in- tensified by hydrochloric or sulphuric acid. Acetanilide. Leaflets or flattened needles. Has a faint, burning taste but is not bitter. Phenacetine. Inodorous and tasteless leaflets and small needles. Antipyrine. Residue a syrup which is rarely crystalline. Tastes mildly bitter. Very easily soluble in water. Caffeine. Residue composed of shining needles frequently in radiating clusters. Tastes mildly bitter. Salicylic Acid. Crystallizes frequently in long needles. Tastes harsh and at the same time sweet and acid. Veronal. Crystalline needles having an agreeable, bitter taste. PICROTOXIN Picrotoxin, CsoI^Ois, the poisonous principle of Cocculus indicus, the fruit of Menispermum Cocculus, crystallizes from hot water in long colorless needles melting at 190-200. It dissolves with difficulty in cold water but more readily in hot water or ethyl alcohol. It is slightly soluble in ether but freely soluble in chloroform, amyl alcohol and glacial acetic acid. Its alcoholic solution is neu- tral and laevo-rotatory. Picrotoxin has a very bitter taste. It is not as readily soluble in acids as in pure water, but is soluble in caustic alkalies and aqueous ammonia, forming unstable, salt-like compounds which do not crystallize. Pi- crotoxin behaves toward strong bases as if it were a weak acid. Heated to boiling with twenty times its volume of benzene, it is decomposed into picro- toxinin and picrotin. The former passes into solution but picrotin is almost completely insoluble: Cl 6 Hl8p7 Picrotoxin Picrotoxinin Picrotin Chloroform brings about this cleavage even more easily. On the other hand, if picrotoxinin and picrotin in molecular proportions are dissolved in hot water, picrotoxin crystallizes out as the solution cools. Treated with bromine direct or dissolved in water or ether, picrotoxin is first split into picrotoxinin and picrotin. The former is immediately converted into monobromo-picrotoxinin, Ci 6 Hi B BrO 6 , but picrotin remains almost unchanged. Monobromo-picrotoxinin is soluble with difficulty in water but is reduced by zinc dust and acetic acid to picrotoxinin. Picrotin is almost non-toxic, whereas picrotoxinin has a very poisonous action. Picrotoxin is a powerful convulsive poison, standing in its action between cicu- toxin and strychnine. NON-VOLATILE POISONS 67 R. Meyer and P. Bruger 1 regard picrotoxin as a complex of the two compounds, picrotin and picrotoxinin, crystallizing together in definite but not molecular proportion, and not as a molecularly constituted chemical compound. Detection of Picrotoxin 1. Fehling's Test. Dissolve picrotoxin in a small test-tube, using 10-20 drops of very dilute sodium hydroxide solution. Add a few drops of Fehling's solution 2 and warm but do not shake. A red or yellowish red precipitate forms and settles to the bottom. If the ether residue, not too little of which should be taken, fails to give a clear solution in very dilute sodium hydroxide solution, filter through moistened paper and examine the filtrate with Fehling's solution. 2. Anunoniacal Silver Test. Warm picrotoxin with aqueous silver nitrate solution containing a slight excess of ammonium hydroxide solution. The reducing action' of picrotoxin will produce a black precipitate of metallic silver, or a dark brown color when only traces are present. 3. Oxidation Test. Picrotoxin, treated with a little con- centrated sulphuric acid in a porcelain dish, first becomes orange-red and then dissolves when stirred forming a reddish yellow solution. A drop of potassium dichromate solution will produce a red-brown color around the margin of the drop. If the two liquids are thoroughly mixed, there is an immediate dirty brown color which passes into green on long 'standing. A green color alone is without significance, since many organic substances capable of reducing chromic acid to chromic oxide produce the same result. 4. H. Melzer's Test. 3 Put some picrotoxin upon a watch- glass and add i or 2 drops of a mixture of benzaldehyde and absolute ethyl alcohol. Careful addition of a drop of concen- trated sulphuric acid will produce a distinct red color. If the watch-glass is tilted, red streaks will run from the substance through the liquid. Use a freshly prepared, 20 per cent, solution of benzaldehyde in absolute ethyl alcohol. Benzaldehyde alone gives a yellowish brown color with concentrated 1 Berichte der Deutschen chemischen Gesellschaft 31, 2958 (1898;. 2 Fehling's solution heated by itself should not give a precipitate of cuprous oxide. 3 Zeitschrift fur analytische Chemie 37, 351 and 747 (1898). 68 DETECTION OF POISONS sulphuric acid. Ethyl alcohol is added as a diluent to diminish this color as much as possible. Under these conditions the solution has a light yellow color, and the dark red tint caused by picrotoxin is very clearly denned. This red color is unstable and, beginning at the margin, gradually fades into a pale pink or violet. H. Kreis 1 has found that cholesterine and phytosterine 2 give similar colors with Melzer's reagent. 5. Langley's Test. Mix picrotoxin with about 3 times the quantity of potassium nitrate, and moisten the mixture with the smallest possible quantity of concentrated sulphuric acid. Then add strong sodium hydroxide solution in excess and an intense red color will appear. Detection of Picrotoxin in Beer First, neutralize the beer with magnesium oxide. Then evaporate 500 cc. or more to a syrup upon the water-bath. Digest this residue with 4 or 5 times its volume of ethyl alcohol and evaporate the alcoholic extract. Dissolve the residue in hot water and filter the solution through a moistened paper. Acidify the fil- trate with dilute sulphuric acid and extract repeatedly with ether, or better with chloroform. Evaporate these extracts and test the residue for picrotoxin. Should the residue from the ether or chloroform be too impure, dissolve it again in hot water, filter, evaporate and extract with ether or chloroform. To purify picrotoxin further, precipitate colored substances from its aqueous solution with lead acetate, filter and remove lead from the filtrate by hydrogen sulphide. The filtrate from lead sulphide upon evaporation, or extraction with ether or chloro- form, will give nearly pure picrotoxin. The very bitter taste of picrotoxin as well as its strong tendency to crystallize are additional characteristics of this substance. COLCHICIN Colchicin, C22H 2 5NOe, an alkaloid occurring in all parts of the meadow saffron, Colchicum autumnale, is a yellowish, amorphous powder which is poisonous and very bitter to the taste. It is freely soluble in water, ethyl alcohol, and chloro- form, less so in ether and benzene, and almost insoluble in petroleum ether. Solutions of colchicin have a more or less yellowish color which becomes more pronounced upon addition of acids or alkalies. These solutions have very faint basic properties. Consequently ether or chloroform, but not benzene nor petro- leum ether, will extract colchicin from an acid, aqueous solution. Upon evaporation of the solvent, colchicin will appear as a yellowish, sticky residue resembling a resin or varnish. Heated with water containing sulphuric acid, colchicin splits into colchicein and methyl alcohol. Boiling the alkaloid 1.5-2 hours with 60 parts of i per cent, hydrochloric acid will produce the same result: C22H25N0 8 + H 2 = C 21 H 23 N06 + CH 3 .OH Oolchicm Colchicein Methyl Alcohol 1 Chemiker-Zeitung 33, 21 (1899). 2 A substance very similar to cholesterine, and named paracholesterine or phytosterine, is found in the seeds of certain plants. (Perkin and Kipping, Organic Chemistry, page 608.) NON-VOLATILE POISONS 69 On the other hand, colchicin is formed when colchicein is heated to 100 with sodium methylate (CH 3 .ONa) and methyl iodide (CHa.I). Since colchicein on treatment with hydriodic acid yields three molecules of methyl iodide, colchicein as well as colchicin contains three methoxyl groups. Heated with strong hydro- chloric acid, colchicein loses acetic acid and passes into trimethyl-colchicinic acid. Consequently colchicein and colchicin contain an acetyl group ( CHs. CO ) . The formula of colchicin, that is to say, of methyl-colchicein, may be written as follows: CH 3 0\ /NH.CO.CHs CHsO^C 15 H 9 < CH 3 O/ X CO.OCH 3 Detection of Colchicin Aqueous colchicin solutions, especially in presence of dilute mineral acids, have a yellow color. Unless the ether residue has this characteristic, colchicin is absent. 1. Tannic Acid Test. This reagent will precipitate colchicin from aqueous solution, if not too dilute, as white flocks. This test, however, is not characteristic of colchicin. 2. Nitric Acid Test. Nitric acid (Sp. gr. 1.4 = 66 per cent.) dissolves colchicin with a dirty violet color which soon changes, when stirred, to brownish red and finally to yellow. Addition of dilute sodium or potassium hydroxide solution, until the reaction is alkaline, produces an orange-yellow or orange-red color. 3. Sulphuric Acid Test. Concentrated sulphuric acid dis- solves colchicin with an intense yellow color. A drop of nitric acid added to such a solution produces a green, blue, violet and finally a pale yellow tone. Excess of potassium hydroxide solu- tion will now bring out an orange-red color. Erdmann's reagent (see page 3 20) dissolves colchicin with a blue to violet color. 4. Hydrochloric Acid Test. Concentrated hydrochloric acid dissolves colchicin with an intense yellow color. Add two drops of ferric chloride solution and heat the mixture 2-3 minutes in a test-tube. The color deepens and the solution on cooling, especially if diluted with the same volume of water, becomes green or olive-green. Finally shake the solution with a few drops of chloroform. This solvent becomes yellowish brown, or garnet-red, and the aqueous solution retains its green color. Zeisel's reaction. 70 DETECTION OF POISONS Purification of the Residue Containing Colchicin To isolate as pure colchicin as possible from the yellow residue, extract with warm water. Filter the solution and, when cold, extract it first with petroleum ether. This will remove fatty, resinous and colored impurities but not colchicin. Then extract with chloroform. Or precipitate colchicin from aqueous solution, which must not be too dilute, with tannic acid. Collect this precipitate upon a filter and wash with cold water. Mix the moist precipitate with freshly precipitated, washed lead hydroxide. Dry the mixture, grind to a powder and extract with chloroform. Evaporation of the solvent will leave nearly pure colchicin. PICRIC ACID Picric acid, or 2,4,6-trinitrophenol, crystallizes from water in light yellow leaflets and from ether in lemon-yellow, rhombic prisms. It melts at 122.5. Though soluble in cold water with difficulty, picric acid dissolves freely in hot water, as well as in ethyl alcohol, ether and benzene. Aqueous solutions have an acid reaction, a very bitter taste and dye animal fibers fast yellow. Material containing picric acid has a yellow or C yellowish green color. Physiological Action and Elimination. Picric acid is OaN Y |j- NO2 q u it e an active poison. Taken internally it produces a HC CH striking yellow pigmentation first of the conjunctiva and then of the entire skin, usually designated as "picric acid C icterus." Picric acid and its salts like most nitro-com- J,~ pounds decompose the red blood-corpuscles forming met- haemoglobin. Consequently it is a blood-poison. At the same time it irritates the central nervous system and causes convulsions. Finally it exercises its power of precipitating proteins in acid solution. This is especially noticeable in those organs of the body, for example, the stomach and QTT kidneys, which, owing to necrotic tissue changes, have an iacid or only a faintly alkaline reaction. The organism re- duces picric to picramic acid which does not so readily o T_^\, ^TTT P re cipitate protein. By thus changing picric acid the 2 organism rids itself of the poison. In picric acid poisoning HC CH tne urine has a marked red color owing to formation of picramic acid. Some picric acid passes into the urine unchanged. Elimination is slow. In one case (see R. NO 2 Kobert, "Intoxikationen"), after administration of a single Picramic acid dose of i gram of picric acid, its presence in the urine could be recognized for 6 days. The urine was ruby-red, clear, acid and free from albumin and bile-constituents. Picric acid was also easily detected in the feces. NON-VOLATILE POISONS 71 Detection of Picric Acid Material containing picric acid has a more or less yellow or yellowish green color. Aqueous, alcoholic and ethereal solu- tions show the same color. Finely divided animal material should be extracted several hours under a return-condenser with ethyl alcohol containing hydrochloric acid to decompose com- pounds of picric acid with albumins and thus bring the acid into solution. Filter and evaporate such an alcoholic extract upon the water-bath. Treat the residue, which is yellow, yellowish green, or frequently yellowish red or reddish brown, with warm water and filter the extract. The filtrate itself may be tested directly for picric acid, or it may first be extracted as usual with considerable ether. The following tests may then be applied to the residue left on evaporating the ether extract : i. Isopurpuric Acid Test. Gently heat (50-60) an aqueous solution of picric acid with a few drops of saturated, aqueous potassium cyanide solution (i : 2). The solution will become red owing to formation of potassium isopurpurate. One milli- gram of picric acid, dissolved in 5 cc. of water, will give a distinct test. Isopurpuric acid does not exist in the free state but is present in this test as the potassium salt. Nietzki and Petri 1 regard isopurpuric acid (CgHaOjNs) as a dicyano-picramic acid = 5-oxy-6-amino-2,4-dinitro-isophthalic nitrile; whereas Borsche 2 considers it a dicyano-dinitro-oxy-/3-phenyl hydroxylamine: OH OH I II c c s\ /\ O 2 N C C NH 2 10 2 N C C NH.OH I II II NC C C CN NC C C CNj N0 2 N0 2 Xietzki-Petri Borsche 1 Berichte der Deutschen chemischen Gesellschaft 33, 1788 (1900). 2 Ibid., 33, 2718 and 2995 (1900). 72 DETECTION OF POISONS 2. Picramic Acid Test. (a) Heat picric acid solution with a few drops of sodium hydroxide solution and glucose. Picra- mic acid, formed by reduction of picric acid, colors the solu- tion deep red. Avoid excess of sodium hydroxide solution, otherwise there will be a red color due solely to the action of the alkali upon glucose. (|8) The test may also be made by warming picric acid solution with a few drops of sodium hydroxide and ammonium sulphide solutions. This will reduce picric acid and produce a red color. In both reactions (a and 0) picric acid is reduced to picramic acid, 2-amino-4,6-dinitro-phenol : OH OH c c s\ /\ O 2 N C 6 2 C NO 2 2 N C 6 2 C NH 2 | || +6H= | || + 2 H 2 0. HC CH HC CH \4/ . \4/ C C NO 2 NO 2 Picric acid Picramic acid The presence of fat and other impurities materially influence this test. 3. Dyeing Test. Dissolve the substance containing picric acid in hot water and put white threads of wool, silk and cotton in the solution. In a few hours (12 to 24) remove the threads and thoroughly rinse in pure water. If picric acid is present, the wool and silk will be dyed yellow but not the cotton. In other words, picric acid is not fast upon vegetable fibers like cotton. Picric acid, diluted i : 100,000, will still produce a yellow color upon wool. 4. Ammoniacal Copper Test. Add a few drops of ammonia- cal copper sulphate solution (copper sulphate solution and an excess of ammonia) to an aqueous picric acid solution. A yel- lowish green precipitate, consisting of hexagonal needles with a polarizing action upon light, will appear. Picric acid, diluted i : 80,000, will give this test. NON-VOLATILE POISONS 73 ACETANILIDE Acetanilide crystallizes in colorless and inodorous, shin- ing leaflets. It has a faint, burning taste; melts at 113 to 114; is soluble'in 230 parts of cold water, in about 22 parts r of boiling water and in 3.5 parts of ethyl alcohol; and is I I j freely soluble in ether and still more so in chloroform. All HC CH acetanilide solutions are neutral. Heated to boiling with \/ potassium hydroxide solution (I) and also with fuming hydrochloric acid (II), acetanilide is decomposed into its constituents: I. CeHs.NH.CO.CHs + KOH = C 6 H 5 .NH 2 + CH 3 .CO.OK. II. C 6 H 6 .NH.CO.CH 3 + HC1 + H 2 O = C 6 H 5 .NH 2 .HC1 + CH 3 .COOH. Physiological Action. Being an aniline derivative, acetanilide has the poison- ous properties of that amine though in less degree. R. Robert (" Intoxikatio- nen") refers to several instances of acetanilide poisoning which did not terminate fatally. In one case a student took a teaspoonful of the drug. There was stupor, uneasiness, marked cyanosis and lowering of the pulse. A purgative and restorative (stimulant) were used but there was considerable exhaustion for several days. The picture was nearly the same in the case of a man who took 2 grams of antifebrine daily for 2 days in succession. Preparation. Boil aniline and glacial acetic acid together for several hours under a return-condenser: C 6 H 6 -NH 2 + CHs-COOH = C 6 H 5 -NH-CO-CH 3 + H 2 O. Detection of Acetanilide Ether or chloroform will extract acetanilide completely from an acid aqueous solution. 1. Indophenol Test. Boil acetanilide with about 4 cc. of fuming hydrochloric acid and evaporate to a few drops (about 10). Cool and add 4 cc. of saturated, aqueous carbolic acid solution. A few drops of calcium hypochlorite solution will produce a violet-red color. In time the color will become deeper , especially if the mixture is shaken. Then carefully add ammon- ium hydroxide solution as a surface-layer which will take on a permanent indigo-blue color. The indigo-blue color is characteristic of acetanilide only when preceded by the red-violet color, since a mixture of aque- ous phenol and hypochlorite solution gives a blue color with ammonia (see carbolic acid). Phenacetine also gives the indophenol test. 2. Phenylisocyanide Test. Boil acetanilide with 5-6 cc. of alcoholic potassium hydroxide solution. Cool, add 2 or 3 74 DETECTION OF POISONS drops of choloroform and again heat. The offensive odor of phenylisocyanide will be developed. Potassium hydroxide decomposes acetanilide into aniline and potassium acetate (see Reaction I above). The former with chloroform gives phenylisocyanide. 3. Calcium Hypochlorite Test. Boil acetanilide a few min- utes with alcoholic potassium hydroxide solution as in test 2. Dilute with water and extract aniline with ether. This sol- vent upon evaporation will deposit aniline as an oily liquid. Dissolve the latter in water and test with calcium hypochlorite. Examination of Acetanilide Urine 1 Scarcely more than traces of unaltered acetanilide appear in urine even after large doses. The most essential change occurring in the body is oxidation of the benzene ring which produces aceto-para-aminophenol. This like most phenols forms a conjugate sulphuric acid and appears in the urine as a salt of aceto-para- aminophenyl sulphuric acid: H OH O SO, OH A A A HC CH HC CH HO\ HC CH + 0=||| + >SOo = I || HC CH oxidation HC CH HO/ HC CH \/ \/ conjugation \/ c c c NH.CO.CHj NH.CO.CHs NH.CO.CH 3 Acetanilide Aceto-p-aminophenol Aceto-p-aminophenyl sulphuric acid To some extent also, a conjugate glycuronic acid of aceto-para-aminophenol is formed. These compounds, heated with concentrated hydrochloric acid, give para-aminophenol which can be detected by the indophenol test previously described. O.S0 2 .OH OH c c H/\H H /\H J- JL + 2H 2 = H 2 S0 4 + CH 3 .COOH + | || HC . CH HC CH V v ? c NH.CO.CH 3 NH 2 p-aminophenol Such urine, boiled a few minutes with concentrated hydrochloric acid, will usually give the indophenol test. But the test will be more certain, if para- * To study the behavior of acetanilide in the body, take at night 0.3 gram of s substance at a dose twice in the course of 3 hours and examine the urine passed in the next 12 hours. NON-VOLATILE POISONS 75 aminophenol is first isolated. Boil a larger quantity of urine (300 to 500 ccj a few minutes with about 10 cc. of concentrated hydrochloric acid. Then add an excess of sodium carbonate and repeatedly extract the cool urine with large quantities of ether. Distil or evaporate the ether. Para-aminophenol usually appears as a reddish or brownish oil. An aqueous solution of this substance will give the indophenol test. PHENACETINE Phenacetine, or p-aceto-phenetidine, crystallizes in shining leaflets, Which are without color, odor or taste, and melts at 134 to 135. Phenacetine is soluble in about 1400 parts of cold water, 70 parts of boiling water, i6 parts of ethyl alcohol and freely soluble in ether and chloroform. Its solutions are neutral. Concentrated sul- phuric acid dissolves it without color. Phenacetine is very closely related to acetanilide but does not give the phenylisocyanide test. Preparation. The gradual addition of crystallized phenol to cold dilute nitric acid (sp. gr. i.n = 17.5 per cent.) results in the formation of a mixture of o- and p-nitro-phenol. Since the ortho-compound is volatile with steam, complete separation of the two products is possible by steam distilla- tion. The residual p-nitro-phenol is converted into its sodium salt which is heated in sealed tube with ethyl bromide and thus changed to p-nitro-phenetol. The latter is reduced by means of nascent hydrogen from tin and hydrochloric acid to p-amino-phenetol, or p-phenetidine, which is then boiled with glacial acetic acid and converted into aceto-p-phenetidine, or phenacetine: HC Hi CH L OC 2 H 6 OH JOH JSJPNa O^a Br:.C 2 H 6 OC 2 H 5 A A r 1 C HC CH HC CH HC CH HC CH 1 II - 1 II - HC CH HC CH I !l HC CH - 1 II HC CH \/ \/ C C C Y __. 1 | HO XO 2 N 2 NO 2 NiO, j 2R 4 H: Phenol p-Nitro- phenol Na salt of p- nitro-phenol p-Nitro- phenetol OC 2 H 6 OC 2 H 5 i i /\ ^\ HC CH HC CH 1 II HC CH / C C i HNiH : NH.CO.CHs iHOLCO.CHs p-Phenetidine Phenacetine 76 DETECTION OF POISONS Detection of Phenacetine The extraction of phenacetine by ether or chloroform from an aqueous tartaric acid solution is complete. 1. Oxidation Test. Boil phenacetine for several minutes with 3 cc. of concentrated hydrochloric acid. Dilute with 10 cc. of water and filter when cold. A few drops of chromic acid solution added to the filtrate will gradually produce a ruby-red color. Strong chlorine water may be substituted for chromic acid. 2. Indophenol Test. Boil phenacetine i or 2 minutes with about 2 cc. of concentrated hydrochloric acid. Dilute with water and add a few cc. of aqueous carbolic acid solution. Filter the solution when cold. If a few drops of freshly pre- pared calcium hypochlorite solution are added, the filtrate will have a fine carmine-red color. Addition of ammonium hydrox- ide solution in excess will change this color to violet-blue. Freshly prepared chlorine water, or 3 per cent, chromic acid solution, may be substituted for hypochlorite solution as an oxidizing agent. 3. Autenrieth-Hinsberg Test. 1 (a) With Dilute Nitric Acid. Heat phenacetine to boiling with a few cc. of dilute nitric acid (10 to 12 per cent.). It is soluble and gives an intense yellow to orange-red color. As the solution cools, if sufficiently concentrated, nitro-phenacetine 2 will crystallize in long, yellow needles which melt at 103. This test is delicate, and char- acteristic of phenacetine, especially when nitro-phenacetine can be obtained in crystals and its melting-point determined. It serves to distinguish phenacetine from acetanilide and anti- pyrine, both of which give colorless solutions when warmed with dilute nitric acid. (6) With Concentrated Nitric Acid. A few drops of con- centrated nitric acid poured upon phenacetine produce a yellow 1 Archiv der Pharmacie 229, 456 (1891). 1 The structural formula of mono-nitro-phenacetine is as follows: /OC 2 H 6 i 3 \NH(C 2 H 3 0) 4 NON-VOLATILE POISONS 77 to orange-red color. Part of the phenacetine is dissolved with the same color and heat completes the solution. Nitro-phen- acetine crystallizes as the solution cools. SALICYLIC ACID COOH Salicylic acid, or ortho-oxy-benzoic acid, crystallizes in long, | white needles soluble in about 500 parts of cold and in 15 C parts of boiling water; and freely soluble in ethyl alcohol, ether, C<^ yCO = 2HC1 + / C CH I I I CH 3 N C ; N^ CH 3 N CO NH 2 Caffeine Dimethyl- Monomethyl- alloxan urea Fate of Caffeine in Human Metabolism. Only a very small part of the caffeine taken into the body passes through unchanged and appears in the urine. About 10 per cent, appears in the urine as decomposition products. The remainder may be changed into normal end-products of human metabolism. Most of the nitrogen of caffeine is eliminated as urea. A very important fact is the cleavage of methyl groups with formation of the first decomposition products of caffeine, namely, dimethyl- and monomethyl-xanthines. Of the mono methyl xanthines, 7-monomethyl-xanthine is formed especially. Of the dimethyl-xanthines, paraxanthine = i,7-dimethyl-xan thine is found. Both of these compounds appear in urine after administration of caffeine. Paraxanthine is isomeric with theophylline, or i,3-dimethyl-xanthine, and with theobromine, or 3 , 7-dimethyl-xanthine. The structural formulae of these cleavage-products of caffeine in animal metabolism are as follows: HN CO (i) CH 3 .N CO OC C N/ OC C NH | II \r> CH (3 ) CH..N-C-N* 7-Methyl-xanthine Theophylline (i) CH 3 .N CO HN CO II II OC C-N.CH 3 ( 7 ) OC C N.CH 3 ( 7 ) >CH \CH HN C N^ ( 3 ) CH 3 .N C N^ Paraxanthine Theobromine Detection of Caffeine Ether will extract more caffeine from an aqueous alkaline solution than from an aqueous tartaric acid solution. Since NON-VOLATILE POISONS 85 caffeine dissolves with some difficulty in ether, but more easily in chloroform, the latter solvent is usually employed after the solution has been made alkaline with ammonia. After dis- tillation of solvent, caffeine appears in concentric clusters of long shining needles. In an analysis by the Stas-Otto method caffeine will appear in all three extracts. 1. Oxidation Test. Pour a few cc. of saturated chlorine water 1 over caffeine and evaporate the solution to dryness upon the water-bath. A reddish brown residue will remain. If a few drops of ammonium hydroxide solution are added, a fine purple-red color will immediately appear. This test may be made by covering the dish containing the residue with a glass plate moistened with a drop of strong ammonia. Or two matched watch-glasses may be used, the material containing caffeine being evaporated to dryness with chlorine water upon one glass which is then placed for a short time upon the other glass containing a drop of strong ammonia. This test, known as the murexide reaction, is also given by xan thine, theobromine, i- and y-monomethyl-zanthine and paraxanthine, especially if made as described by E. Fischer. 2 Heat the material to boiling in a test-tube with strong chlor- ine water, or with hydrochloric acid and a little potassium chlorate, evaporate the liquid to dryness in a dish and moisten the residue with ammonia. 2. Tannic Acid Test. This reagent, added to an aqueous caffeine solution, causes a heavy white precipitate which is soluble in an excess of the acid. This test is not characteristic of caffeine. B. Examination of Ether Extract of Alkaline Solution (Most of the alkaloids appear here) Add enough sodium hydroxide solution to the acid solution separated fiom ether to make it strongly alkaline. The alkali 1 A convenient method of preparing a saturated, aqueous chlorine solution is to heat potassium chlorate with hydrochloric acid and pass the chlorine into a small quantity of water. 2 Berichte der Deutschen chemischen Gesellschaft 30, 2236 (1897). 86 DETECTION OF POISONS will liberate alkaloids from their salts and combine with mor- phine and apomorphine, if present. Thoroughly extract this alkaline solution with about the same quantity of ether. This solvent will dissolve all alkaloids except morphine, apomorphine and narceine. Separate the ether from the aqueous solution and again extract with a fresh quantity of ether. In certain cases 3 or 4 such extractions may be required. Pour the ether extracts into a dry flask, stopper loosely and set aside for i or 2 hours. A few drops of water always settle to the bottom of the flask. Carefully decant the ether and pour through a dry filter. Evaporate the nitrate with gentle heat in a glass dish (8 to 10 cm. in diameter). Let the last part of the ether solution evaporate spontaneously. If small globules having a strong odor appear, the residue must be examined for coniine and nicotine. If there is no trace of these volatile alkaloids, gently heat the residue upon the water-bath to expel water left by evaporation of the ether. Remove the dish from the water- bath as soon as this has been accomplished. It is not advisable to heat the residue too long, as it tends to become viscous. This residue, obtained by extracting the alkaline solution with ether, may contain any alkaloid except morphine, apomorphine and narceine. It should be examined for Coniine Atropine Hydrastine Nicotine Scopolamine Pilocarpine Aniline Cocaine Quinine Veratrine ' Physostigmine Caffeine Strychnine Codeine Antipyrine Brucine Narcotine Pyramidone. First, note the general appearance of the residue and then examine with the microscope. Taste it cautiously. Certain alkaloids may be recognized beforehand by this test. Special tests should then be made at once. The various alkaloids appear in the residue as follows : Strychnine. Fine needles having an exceedingly bitter taste. Brucine. Usually a white, amorphous powder having a very bitter taste. NON-VOLATILE POISONS 87 Veratrine. Usually an amorphous powder having a sharp, burning taste. Atr opine and Quinine. A varnish which is resinous and sticky. Rarely crystalline or in the form of a powder. Codeine. A thick, viscous syrup which after a time becomes solid, especially if stirred with a glass rod, and frequently crystalline. Caffeine. Long, silky needles having a faintly bitter taste. These are frequently concentrically arranged. Antipyrine. A syrup which gradually becomes crystalline, especially if stirred. It has a mild, bitter taste and dissolves very easily in water. 1 Pyramidone. Usually as fine needles which have a faintly bitter taste. It is easily soluble in water. Frequently ether leaves only a slight, tasteless residue. In that case alkaloids are absent. Such residues often consist of fat, resinous matter, or traces of nitrogenous substances (Peptones and their cleavage-products? Creatinine?). Parts of a cadaver, even when quite fresh, usually give small residues at this point. Alkaloids may be absent and every step in the process may have been performed with the greatest care. To be quite sure that alkaloids are absent, dissolve a portion of the residue in water containing a drop of dilute hydrochloric acid. Filter, if necessary, and distribute this solution upon several watch-glasses. Test with the following alkaloidal reagents: Mercuric Chloride, Picric Acid, lodo-Potassium Iodide, Tannic Acid, Potassium Mercuric Iodide, Phospho-Molybdic Acid, Potassium Bismuthous Iodide, Phospho-Tungstic Acid. . Unless these reagents give distinct and characteristic pre- cipitates, alkaloids are absent. It is advisable in every instance 1 Most of the alkaloids are only slightly soluble in cold water. Some cannot be detected satisfactorily by purely chemical means. Others have no characteristic tests. Such substances should not be selected for laboratory practice. They may cause beginners to think that the experienced toxicologist relies upon similar uncertain methods when he seeks to identify an alkaloid in an actual analysis. 88 DETECTION OF POISONS to make this preliminary test for alkaloids. Only a small por- tion of material is required and these general reagents show even traces of alkaloids. To exclude mistakes and oversights in toxicological analysis, dissolve the ether residue, should it be very small, in a few cc. of very dilute hydrochloric acid (about i per cent, of HC1). Evaporate this solution upon the water-bath and dissolve the residue in a little water. Inject this solution from a hypodermic syringe into the lymph-sac on the back of a small but lively frog. If the frog shows no sign of poisoning in the course of several hours, it is quite likely that the residue does not contain any very poisonous alkaloid.' In making special tests for alkaloids, distribute the residue upon several watch-glasses, using a platinum or nickel spatula or a small penknife. Or dissolve the residue in a little hot ethyl alcohol, filter the solution, distribute upon watch-glasses and evaporate at a gentle heat. R. Mauch 1 dissolves the residue in 75 per cent, aqueous chloral hydrate solution and uses this solution in testing for alkaloids. (The details of this method will be found on page 251.) . Purification of the Alkaloidal Residue If alkaloids are contaminated with greasy, resinous or fatty substances, many of the tests will either fail entirely or give uncertain results. In this case the residue must be purified in one of two ways. 1. Thoroughly mix the residue with cold water containing hydrochloric acid. Filter to remove insoluble matter (fatty or resinous substances), add sodium hydroxide solution to the filtrate until alkaline and extract with ether. The alkaloids obtained by evaporating the solvent are usually quite pure. 2. Or dissolve the residue in hot amyl alcohol, extract this solution with a few cc. of very dilute sulphuric acid and with- draw the acid solution from the separating funnel. Amyl alcohol will retain greasy and colored impurities, and the alka- loids will be in the aqueous solution as sulphates. Add sodium 'Richard Mauch (Mittheilungen aus dem Institut des Herrn Prof. Dr. E. Schaer i i Strassburg), "Festgabe des Deutschen Apotheker-Vereins," Strassburg, NON-VOLATILE POISONS 89 hydroxide solution in excess and extract with ether. This method of purifying the alkaloidal residue is especially recom- mended, when there is considerable coloring matter. W. H. Warren and R. S. Weiss 1 have suggested picrolonic acid 2 as a means of purifying alkaloids. An alkaloid like strychnine, whose picrolonate is very in- soluble, may be precipitated from aqueous solution and thus separated from other substances which prevent purification. The precipitated picrolonate may be collected on a filter, washed with water and then warmed with dilute sulphuric acid which discharges the bright yellow color of the picrolonate causing the alka- loid to pass into solution and precipitating pale yellow picrolonic acid. By extracting with acetic ether, in which picrolonic acid is especially soluble, the aqueous solution of the alkaloid is left colorless. Neutralization with sodium hydroxide solution and extraction with ether will give a very pure alkaloid. CONIINE Coniine, a-normal-propyl-piperidine, CgHnN, occurs in all parts of spotted hemlock (Conium maculatum) together with n-methyl-coniine, conhydrine, pj 2 7-coniceine and pseudo-conhydrine. It is a color- C less, oily, very poisonous liquid which becomes yellowish or brown in contact with air and is par- dally resinified. It is slightly soluble in cold but *CH CH CH CH even ^ ess soluble in hot water. Coniine is miscible \/ with ethyl alcohol, ether, chloroform and benzene in all proportions. The unpleasant, narcotic odor of this alkaloid, sometimes said to resemble that of mouse urine, is more intense than the odor of nicotine. Coniine as it occurs in nature is dextro-rotatory, 3 ()D = +18.3, and rather a strong base. Heated with acetic anhydride, it forms acetyl-coniine : H C * C 8 H 16 N.CO.CH 3 + CH,COOH; Shaken with benzoyl chloride and sodium hydroxide solution, it forms benzoyl- coniine: cS*CoS + NaOH = C8H 16 N.CO.C 6 H 8 + NaCl + H 2 O; and with nitrous acid nitroso-coniine: CsH 16 N.NO + H 2 O. All these reactions show that coniine is a secondary base. 1 The Journal of Biological Chemistry, 3, 330 (1907). 2 For the preparation of this reagent, see page 320. 3 The optical activity of this alkaloid is occasioned by the presence of the asym- metric carbon atom marked with an asterisk in the structural formula. 90 DETECTION OF POISONS Detection of Coniine The alkaloidal reagents especially delicate with coniine are: iodo-potassium iodide (1:8000), phospho-molybdic acid (1:5000), potassium mercuric iodide (1:8000) and potassium bismuthous iodide (1:5000). Gold and platinum chlorides fail to precipitate coniine when the concentration is less than i : 100; whereas they will precipitate nicotine when the concen- tration of the solution is as low as i: 10,000 and 1:5000. When coniine is present, the residue left by the ether solution has the characteristic odor of this alkaloid. The two following tests should then be made: 1. Solubility Test. Dissolve a drop of coniine in just enough cold water to give a clear solution. Gently heat the solution and it will become milky, because coniine is more easily soluble in cold than in hot water. A coniine solution which is milky when hot becomes clear on cooling. Aqueous coniine solutions have an alkaline reaction. Test the solution with red litmus paper. 2. Crystallization Test. Put a little coniine upon a watch- glass, or glass slide, and add i or 2 drops of hydrochloric acid. Evaporate to dryness and coniine hydrochloride (C 8 Hi 7 N.HCl) will remain. Immediately after evaporation examine this, resi- due with a microscope magnifying about 200 times. 1?he color- less or faintly yellow crystals are needle-like, or columnar and frequently grouped in star-shaped clusters. They show the play of color characteristic of doubly refractive substances. NICOTINE Nicotine, CioHuNz, is a colorless hygroscopic liquid which soon turns yellow and then brown upon exposure to air and in time becomes resinous. It is miscible with water in all proportions (distinction from coniine) C CH 2 CH an( * * reel y sol uble in ethyl alcohol, ether, amyl alcohol, /\ | | benzene and petroleum ether. Ether extracts nicotine 0C CHa CH 2 from aqueous solution. It has a sharp, burning taste TTP PTT TM an( ^ stron S odor of tobacco especially when warm. \^ Chemically pure nicotine is said to be almost inodorous. N CH 8 ^be so-called tobacco odor is developed after the alka- loid has been for some time in contact with air. The free alkaloid is strongly Isevo-rotatory, []D = -161.55, but its salts are dextro- rotatory. NON-VOLATILE POISONS 91 Constitution. Nicotine is a rather strong di-acid, ditertiary base and forms well-crystallized salts with one or two equivalents of acid. Like ditertiary bases it combines with two molecules of methyl iodide 1 forming a di-iodo-methylate, CioHuNz.sCHsI. Oxidized with chromic acid, nitric acid or potassium per- manganate, nicotine is converted into nicotinic acid, or /3-carboxy-pyridine. This shows that nicotine is a pyridine derivative having a side-chain in the /3- position with respect to the pyridine nitrogen. H H C CH 2 CH 2 C ' S\ I I S\ HC /3C CHa CH 2 HC C COOH I I! \/ I II HC CH N gives on oxidation HC CH \/ I . \/ N CH 3 N Nicotine Nicotinic acid This formula for nicotine proposed by Pinner was confirmed several years later by Ame" Pictet's synthesis of this alkaloid. Physiological Action. Nicotine is one of the most powerful poisons and scarcely inferior to hydrocyanic acid in toxicity and rapidity of action. It ap- pears to be toxic to all classes of animals. It is absorbed from the tongue, the eye and the rectum even in a few seconds and from the stomach somewhat more slowly. Absorption of nicotine is also possible from the outer skin. Elimina- tion takes place through the lungs and kidneys. In concentrated form nicotine is a local irritant, though, owing to the rapidity of its toxic action, it does not behave like a true corrosive nor does it cause inflammation of the mucous lining of the stomach even after a lethal dose. Nicotine, after causing stimulation for a brief period, then paralyzes the central nervous system and spinal cord, finally affecting various organs such as the heart, eyes and intestinal tract. Its poisonous influence probably extends to all parts of the brain, medulla oblongata and spinal cord. Huchard states that nicotine causes a general convulsion of the circulatory system which is apparent in chronic nicotine poisoning. In chronic tobacco poisoning the general condition of health is disturbed and quite frequently the eyes are affected. In acute nicotine poisoning death ensues from paralysis of the respiratory center. An action upon the heart is also always in evidence even in non-fatal cases. 1 In methyl iodide as well as in other alkyl haloids we have an excellent means of recognizing the tertiary nature of a nitrogen base. Like trimethylam- ine, tertiary cyclic amines, as pyridine and quinoline, also give similar iodo- methylates which are ammonium iodides with quinquivalent nitrogen: H H CH 3 \ni rS 3 \v CHAN + CH 3 .I = N.I; Trimethylamine C HC CH C HC CH 1 II - HC m CH h CH 3 .I = | || HC CH N Y Pyridine H/\ 92 DETECTION OF POISONS Detection of Nicotine Ether or low-boiling petroleum ether will extract nicotine from an aqueous alkaline solution. Spontaneous evaporation of the solvent leaves the alkaloid as an oily liquid having the odor of tobacco and a strong alkaline reaction. General alka- loidal reagents will precipitate nicotine from quite dilute solu- tions, in which respect this alkaloid is very different from coniine. Phospho-molybdic acid and potassium bismuthous iodide precipitate nicotine even in a dilution of ,1:40,000; potassium mercuric iodide in 1:15,000; gold chloride in i: 10,000; and platinum chloride in i : 5000. 1. Crystallization Test. Evaporate nicotine on a watch- glass with a few drops of concentrated hydrochloric acid. This will yield a yellow, varnish-like residue which microscopic ex- amination will show to be entirely amorphous (distinction between nicotine and coniine). If kept for a long time in a desiccator over sulphuric acid, it will become indistinctly crystalline. 2. Roussin's Test. Dissolve a trace of nicotine in ether, using a dry test-tube. Add to this solution about the same volume of ether containing iodine. Stopper and set the test- tube aside. The mixture will become turbid and deposit a brownish red resin which will gradually become crystalline. After some time, ruby-red needles with a dark blue reflex will crystallize from, the ether. These are "Roussin's crystals." If nicotine is old or resinous, it will not as a rule give these crystals. 3. Melzer's Test. 1 If a drop of nicotine is heated to boiling with 2-3 cc. of epichlorohydrin, 2 the mixture becomes dis- tinctly red. This test applied to coniine causes no color. 1 Zeitschrift des allgemeinen Oesterreichen Apotheker-Vereins 54, 65. CH 2 Cl - CH \ 2 Epichlorohydrin, | \Q, prepared by the action of i mol. of CH2 caustic alkali on a-dichlorohydrin, CH 2 C1-CH(OH)-CH 2 C1, or a,^-dichloro- hydrin, CH 2 (OH)-CHC1-CH 2 C1, is a colorless liquid insoluble in water and freely soluble in alcohol and ether. It has an odor like chloroform and a burning, sweetish taste. NON-VOLATILE POISONS 93 4. Schindelmeiser's Test. J If nicotine that is not resinous is treated first with a drop of formaldehyde solution free from formic acid and then with a drop of concentrated sulphuric acid, the mixture takes on an intense rose-red color. If nicotine and formaldehyde are in contact for several hours, the solid residue obtained gives even a finer color reaction with a drop of nitric acid. Only a little formaldehyde should be used, otherwise the solution becomes green after a while and decomposition takes place. Under the same conditions, trimethylamine, piperidine, pyridine, picoline, quinoline and aniline gave no color. Nor did extracts from putrefying horse- flesh and the entrails of animals, poisoned by arsenic or mercury, give the test, at least not when these extracts were prepared according to the Stas-Otto method. 5. Physiological Test. When very small quantities of nico- tine are present, the physiological test should accompany the chemical tests. A very characteristic picture is given by frogs after administration of small doses of nicotine. First there is stimulation, then paralysis of the brain and respiratory muscles and apparent curare-action (tetanic convulsions). The toxic action of pure nicotine should be studied first. The experiment with a frog's heart, which shows temporary cessation of diastole, is also very characteristic. ANILINE Aniline, C 6 H 5 .NH 2 , upon evaporation of the ether extract from the alkaline solution, will usually appear as reddish or brownish globules. Dissolve some of this residue in water and apply the aniline tests already described on page 45. A further test for aniline consists in mixing some of the residue with a few drops of concentrated sulphuric acid, and adding a few drops of potassium dichromate solution. If aniline is present, an evanescent blue color will appear. VERATRINE Pure officinal veratrine is an intimate mixture of two isomeric alkaloids having the composition Cs2H 49 NO9. These are cevadine, also called crystallized vera- 1 Pharmazeutische Zentral-Halle. 40, 703 (1899). 94 DETECTION OF POISONS trine, which is nearly insoluble in water; and amorphous veratridine which is soluble in water. Even small quantities of the crystalline alkaloid will render veratridine insoluble in water. On the other hand, veratridine will prevent cevadine from crystallizing. Consequently the crystalline base cannot be iso- lated by recrystallizing officinal veratrine from ethyl alcohol or from any other solvent; nor can the water-soluble alkaloid be obtained by simple extraction with water. Separation of Cevadine and Veratridine. E. Schmidt uses the following method to isolate the crystalline and the water-soluble veratrine from officinal veratrine. Place the officinal preparation in a beaker and dissolve in strong ethyl alcohol. Heat this solution to 60-70 and add enough warm water to pro- duce a permanent turbidity. Cautiously add just enough ethyl alcohol to clear the solution and allow evaporation to take place slowly at 60-70. A white, crystalline precipitate will presently appear. Filter with suction, wash the pre- cipitate with a little dilute ethyl alcohol and recrystallize from hot ethyl alcohol. This is crystalline veratrine. Clear the filtrate from the crystalline precipitate by adding a little ethyl alcohol and evaporate at 60-70. This will give a, second crop of crystals. By repeating this process several times one may obtain in a crystalline condition about one-third of the veratrine taken. Finally evaporate the filtrate from the crystalline deposit at the given temperature until there is no longer any odor of ethyl .alcohol. A consider- able quantity of a resinous mass which is a mixture of both alkaloids will separate. The aqueous filtrate from this deposit will contain veratridine which may be obtained by rapidly evaporating the solution in vacua over sulphuric acid. Properties of Officinal Veratrine. Veratrine appears as a white, amorphous powder which is crystalline under the microscope. It has a sharp, burning taste and the minutest quantity introduced into the nostrils excites protracted sneez- ing. It is almost insoluble in boiling water and the aqueous extract always has a f aintly alkaline reaction; fairly soluble in ether ( i : 10), benzene, petroleum ether and amyl alcohol; and freely soluble in ethyl alcohol (i : 4) and chloroform (1:2). All these solutions have a strong alkaline reaction. Officinal veratrine melts at 1 So-i 55 to a yellowish liquid which solidifies to a transparent, resinous mass. If the veratrine solution is faintly acid, ether will extract a very little of the alka- loid. Under the same conditions, chloroform and amyl alcohol will extract more. The alkaloid is usually deposited from ether as a white, amorphous pow- der. Phospho-molybdic acid, iodo-potassium iodide, tannic acid and potassium mercuric iodide give distinct precipitates with an aqueous veratrine solution containing hydrochloric acid and diluted i : 5000. Chlorides of gold and plati- num and picric acid fail to show the alkaloid in this dilution. Constitution. Heated with saturated barium hydroxide, or alcoholic potas- sium hydroxide solution, crystallized veratrine (cevadine) is hydrolyzed into angelic acid and cevine: C,2H4 9 N0 9 + H 2 = CsHsOz 1 + C 27 H 43 NO 8 Oevadme Angelic Cevine acid 1 Angelic acid (I) and tiglic acid (II) are stereo-isomers : I. CHr-C-H II. H-C-CHa CH 3 -C-COOH CH 3 C COOH NON-VOLATILE POISONS 95 M. Freund 1 has shown that cevadine takes up only one acetyl or benzoyl group, whereas cevine takes up two. The following formulae show these relationships: /O.C 6 H 7 /OH C 27 H 41 N0 6 < - C 2 7H 41 N0 6 < X OH \OH Cevadine Cevine I i /O.CO.CH 3 C 27 H 4 ,N0 6 < C 2 7H 41 N0 6 < X O.CO.CH 3 X O.CO.CH 3 Acetyl-cevadine Diacetyl-cevine By means of hydrogen peroxide M. Freund has converted cevine into cevine oxide, C 2 7HNO 9 , which crystallizes well and contains one more atom of oxygen. V This compound must belong to the class of the amino-oxides, R = N = O, for sulphurous acid easily converts it into cevine. Detection of Veratrine 1. Concentrated Sulphuric Acid Test. Pour a few drops of concentrated sulphuric acid upon a trace of veratrine. The alkaloid will have an intense yellow color and, if stirred, will give a solution of the same color. Gradually, this color will change to orange, then to blood-red and finally to cherry-red. Gentle heating will hasten this color change and veratrine, dis- solved in concentrated sulphuric acid, will give a fine cherry-red solution almost immediately. Frohde's and Erdmann's reagents give color changes similar to those caused by sulphuric acid. 2. Concentrated Hydrochloric Acid Test. If a trace of vera- trine is dissolved in i or 2 cc. of cold concentrated hydrochloric acid, the solution will be colorless. When this solution is heated 10 to 15 minutes in a boiling water-bath, a cherry-red color will appear. This color will last for a day and even 0.2 mg. of veratrine will produce it. 3. Concentrated Nitric Test. Concentrated nitric acid dissolves veratrine with a yellow color. 4. Weppen's Test. Thoroughly mix in a mortar i part of veratrine with about 5 parts of finely powdered cane-sugar. Add a few drops of concentrated sulphuric acid to some of 1 Berichte der Deutschen chemischen Gesellschaft 37, 1946 (1904). i 96 DETECTION OP POISONS the mixture upon a watch-glass. At first a yellow color will appear and later, beginning at the margin this will change to grass-green and finally to blue. Breathing upon the mixture will cause the color to change more quickly. Too great an excess of cane-sugar must be avoided. E. Laves 1 substitutes an aqueous furfural solution for cane- sugar in this test. Mix in a test-tube 3 or 4 drops of i per cent, aqueous furfural solution with i cc. of concentrated sulphuric acid. Add 3 to 5 drops of this solution to the substance to be tested so that it just touches the edge of the liquid. If veratrine is present, a dark streak will gradually run from the substance into the liquid. At the starting-point it will appear blue or blue-violet and farther away green. If substance and liquid are stirred with a glass rod, the liquid will become dark green. After some time, or more quickly when warmed, the color will become blue and finally violet. 5. Grandeau's Test. Direct addition of 1-2 drops of bro- mine water to the yellow solution of veratrine in concentrated sulphuric acid produces an immediate purple color almost iden- tical with that appearing when the solution of the alkaloid in concentrated sulphuric acid stands a long time or is gently warmed. 6. Vitali's Test. Dissolve veratrine in a few drops of fuming nitric acid and evaporate the solution to dryness upon the water-bath in a porcelain dish. A yellowish residue will remain. If this is cooled and then moistened with an alcoholic potas- sium hydroxide solution, the color will change to orange-red or red-violet and stirring will produce a solution having the same color. Atropine, hyoscyamine, scopolamine, as well as strychnine, respond to this test in a very similar manner. STRYCHNINE Strychnine, C2iH22N.2O2, occurs with brucine chiefly in nux vomica and Ignatius beans, constituting the larger part of the mixed alkaloids. The former contains 2.93-3.14 per cent, of these two alkaloids and the latter 3.11-3.22 per 1 Pharmaceutische Zeitung, 37, 338. NON-VOLATILE POISONS 97 4 cent. The free base strychnine forms colorless, shining prisms belonging to the rhombic system which melt at 268. The alkaloid dissolves in 6600 parts of cold and 2500 parts of hot water, giving alkaline solutions having a very bitter taste. It is nearly insoluble in absolute ethyl alcohol and in absolute ether. It dissolves in 160 parts of cold and 12 parts of boiling ethyl alcohol (go per cent, by volume>; it is also soluble in commercial ether and in benzene; but most readily in chloroform (6 parts at 15). Strychnine diluted with water 1:600,000 can be recognized by its bitter taste. Strychnine is a monacid base combining with one equivalent of acid and form- ing salts which are usually crystalline. These salts have a very bitter taste and are very poisonous. The best known strychnine salt, and one used medicinally, is the nitrate, C2iH22N2O2.HNO3. The combination of one molecule of strych- nine with one molecule of an alkyl haloid, for example, methyl iodide, to form strychnine iodo-methylate, C2jH22N 2 O 2 .CH3.I, shows that ' the alkaloid is a tertiary base. Sodium methylate (CH 3 .ONa) in alcoholic solution converts strychnine into strychnic acid which is probably an imino-carboxylic acid. Strychnic acid loses a molecule of water, when its solutions are boiled in presence of mineral acids, and is changed to strychnine. Because of this behavior Tafel regards strychnine as an inner anhydride of sfrychnic acid, one containing a group of the character of an acid imide : N Strychnine Strichnic acid On the basis of Tafel's strychnine formula, strychnine iodo-methylate would be expressed as follows: CH 3 (C2oH 22 0)-CO H Physiological Action. Strychnine increases reflex irritability of the brain and spinal cord. Even the slightest stimulus, especially if acoustic, optical, or tactile, may cause powerful reflexes after large doses of this alkaloid. Convulsions may follow each stimulus, if the dose is sufficient. Very large doses of strychnine cause curare-like paralysis of the peripheral ends of motor nerves in frogs and other warm-blooded animals. It may also affect the muscles of the heart. Strychnine diminishes the motile power of leucocytes and then arrests their motion. The poison also affects plant protoplasm, at least that of Mimosa pudica, in that the plant's motor organs lose their elasticity and flexibility. Aside from the saliva, bile and milk, the urine is the main channel through which strychnine is eliminated from the organism. Human urine may contain even the unaltered alkaloid. Elimination begins during the first hour, is slight after 2 days but is not complete until much later. More unaltered strychnine is eliminated after large than after small doses. In the former case 70-75 per cent. of the alkaloid may remain undecomposed. The liver, kidneys, brain and spinal cord may store up unchanged strychnine. (See R. Robert, "Intoxikationen.") 7 98 DETECTION OF POISONS Detection of Strychnine Sodium and potassium hydroxide, ammonia and alkaline carbonates precipitate the free base strychnine from aqueous solutions of its salts as a white crystalline solid: C 2 iH 22 N 2 2 .HN03 + NaOH = C 2 iH 22 N 2 O 2 + H 2 O + NaNO 3 . Ether will extract strychnine from an alkaline solution and deposit the alkaloid on evaporation in fine crystalline needles. Chloroform takes up the alkaloid more freely, since strychnine is considerably more soluble in this solvent than in ether. Even very dilute solutions of strychnine salts give precipitates with most of the alkaloidal reagents. Tannic acid, potassium mer- curic iodide and phospho-tungstic acid produce white precipi- tates; gold chloride and phospho-molybdic acid yellow precipi- tates; and iodo-potassium iodide brown precipitates. To obtain tests with these reagents, the residue from ether should first be dissolved in very dilute hydrochloric acid. Concentrated sulphuric acid, Erdmann's and Frohde's reagents dissolve perfectly pure, brucine-free strychnine without color. Strychnine is soluble in concentrated nitric acid with a yellow- ish color. Potassium dichromate, added to solutions of strychnine salts, precipitates strychnine dichromate, (C 2 iH 22 N 2 O 2 ) 2 .- H 2 Cr 2 O 7 , in the form of fine yellow crystalline needles which upon recrystallization from hot water appear as shining orange- yellow needles. Potassium ferricyanide, added' to solutions of strychnine salts, precipitates golden-yellow, crystalline strychnine ferri- cyanide (C 21 H 22 N 2 O 2 ) 2 .H 3 Fe(CN) 6 + 6H 2 O. Special Reactions I. Sulphuric Acid-Dichrcmate Test. Dissolve a very little strychnine in 2 or 3 drops of concentrated sulphuric acid upon a watch-glass. The solution should be colorless. Add a fragment of potassium dichromate and hold it firmly in one place upon the glass. Intense blue or blue-violet streaks will come from the potassium dichromate, if the watch-glass is tilted up and down. If the entire mixture is stirred, the sul- NON-VOLATILE POISONS 99 phuric acid will have a beautiful evanescent blue or blue- violet color. This test may also be made by scattering upon the surface of the solution of strychnine in concentrated sulphuric acid a few particles of coarsely powdered potassium dichromate and mix- ing well with a glass rod. In this way the blue to blue- violet color reaction is given very beautifully. The blue color is not permanent. It soon changes to red and finally to dirty green. 1 Strychnine chromate and ferricyanide give this test especially well. To prepare the former salt, pour a very dilute potassium dichromate solution over strychnine upon a watch-glass. When the two substances have been in contact for some time, pour the remaining liquid from the strychnine chromate. Wash the precipitate once with a little water. Put some strychnine chromate upon the end of a glass rod and draw it through a few drops of concentrated sulphuric acid upon .a watch-glass. This will produce violet and blue streaks in the acid. Mandelin's reagent, 2 that is to say, vanadic-sulphuric acid, gives this strychnine test very well. The blue or violet color given by this reagent with strychnine is more permanent than that produced by potassium dichromate. The color finally changes to orange-red. Other oxidizing agents may be substituted for potassium dichromate, as potassium permanganate, lead peroxide, manganese dioxide, 'potassium ferri- cyanide (see above), cerium oxide and vanadic acid (Mandelin's reagent). But neither potassium nitrate nor nitric acid can be used, as these reagents even pre- vent this test. Consequently strychnine nitrate does not give the test. 2. Physiological Test. Dissolve the ether residue in a little very dilute hydrochloric acid. Evaporate the filtered solution to dryness upon the water-bath. Dissolve the residue in pure water (about i cc.) and inject this solution into the lymph-sac on the back of a lively frog. Keep the experimental frog in a large, loosely covered beaker. Toxic symptoms will appear in 5 to 30 minutes, depending upon the quantity of strychnine. According to Tafel (Annalen der Chemie und Pharmazie, 268, 233 (1892), this color reaction is characteristic of many anilides and is due to the presence of the group -CO-N =. 2 See page 321 for the preparation of this reagent. 100 DETECTION OF POISONS Strychnine does not increase reflex irritability for all kinds of stimuli but only for tactile, optical and especially for acoustic stimuli. When the dose of strychnine is sufficiently large, each kind of stimulus mentioned will produce convulsions like those caused by tetanus. For example, if the beaker contain- ing the "strychnine frog" is gently tapped, this slight acoustic stimulus is sufficient to produce convulsions. Detection of Strychnine in Presence of Brucine More than traces of brucine prevent detection of strychnine with concentrated sulphuric acid and potassium dichromate. Under certain conditions Mandelin's reagent will show strych- nine more or less distinctly in presence of brucine. Dissolve the ether residue in concentrated sulphuric acid, if brucine is present, and add a trace of concentrated nitric acid. A red color indicates brucine. When the color has changed to yellow, add a fragment of potassium dichromate and stir. The mixture will become blue or reddish violet, if strychnine is present. Solid potassium permanganate, stirred with concentrated sulphuric acid alone, will give a dark green solution which is yellowish green in a thin layer and assumes with time a red to violet color on the margin. The same procedure used to estimate these two alkaloids quantitatively will permit detection of strychnine even in pres- ence of considerable brucine. Dissolve the residue containing brucine in about 2 cc. of dilute sulphuric acid, add 2 drops of concentrated nitric acid and let the mixture stand 4 hours. Render alkaline with excess of sodium hydroxide solution and extract thoroughly with ether. The residue from ether will be brucine-free or nearly so. Strychnine thus treated will give very satisfactory tests with concentrated sulphuric acid and potassium dichromate and with Mandelin's reagent. BRUCINE Brucine, Cj3H26N2O4, crystallizes in transparent, monoclinic prisms or shining leaflets. Crystals from water contain either 4 or 2 molecules and from ethyl alcohol 2 molecules of water of hydration. ' It melts in its water of hydration only a few degrees above 100, whereas the anhydrous base melts at 178. Brucine is more readily soluble than strychnine both in water and in ethyl alcohol and there- fore remains dissolved in the mother-liquors from the preparation of strychnine. NON-VOLATILE POISONS 101 It is also more soluble than strychnine in ether. Brucine solutions have a very bitter taste and a strong alkaline reaction. Benzene, but especially chloroform and amyl alcohol, are excellent solvents for brucine. Brucine differs from strych- nine in being deposited usually amorphous by evaporation of its ether solution. Brucine is a monacid, tertiary base and as such forms addition-products with one molecule of an alkyl iodide. For example, with methyl iodide it gives brucine iodo-methylate, C23H 2 6NO4.N.CH 3 L With one equivalant of acid brucine gives in part crystalline salts. Brucine nitrate, C 23 H 2 N 2 O4.HNO3. 2H 2 O, crystallizes in rectangular prisms. Brucine may be shown by Zeisel's method 1 to contain two methoxyl groups (-OCH 3 ). Heated in sealed tube to 80 with sodium and ethyl alcohol, until solution is complete, brucine is converted into brucic acid, CasH^s^Os.HzO, which contains an imino-group ( = NH) in its molecule since it forms a nitrosamine. Taf el and Moufang 2 express the relationship between brucine and brucic acid as follows: N N C 2 oH 2 o(OCH 3 )20 CO + H 2 CMC 2 oH 2 o(OCH 3 ) 2 O COOH \l \ N NH Brucine Brucic acid Heated with water, brucic acid is converted into brucine. Consequently brucic acid is related to brucine as strychnic acid is to strychnine. Detection of Brucine Ether, benzene or chloroform will extract brucine from an alkaline solution. Evaporation of the ether extract usually leaves the alkaloid in an amorphous condition. The sensitive- ness of the alkaloidal reagents toward brucine is as follows : lodo-potassium iodide (i 150,000), Potassium bismuthous iodide (i : 5000), Potassium mercuric iodide (i 130,000), Phospho-molybdic acid (i : 2000), Gold chloride (i : 20,000), Tannic acid (i : 2000), Platinic chloride (i : 1000). 1 Many alkaloids contain one or more, sometimes three or more, methoxyl groups ( OCH 3 ) united with a benzene nucleus. The determination of the number of such groups in the molecule is of the greatest importance as a step in establishing the constitution of an alkaloid, because in this way some of the car bon, oxygen and hydrogen atoms are at once disposed of. The method employed for this purpose depends on the fact that all substances containing methoxyl groups are decomposed by hydriodic acid, yielding methyl iodide and a hydroxyl compound. By estimating the methyl iodide obtained from a given quantity of a compound of known molecular weight, it is easy therefore to determine the number of methoxyl groups in the molecule. This method was first applied by Zeisel and is of general application. (Perkin and Kipping, "Organic Chem- istry," page 498.) 2 Annalen der Chemie und Pharmazie 304, 28 (1899). 102 DETECTION OF POISONS 1. Nitric -Acid-Stannous Chloride Test. Concentrated nitric acid dissolves brucine and its salts with a blood-red color. This color, however, is slightly stable and soon changes to yellowish red and finally, especially with heat, to yellow. Add a few drops of freshly prepared, dilute stannous chloride solution to this yellowish red or yellow solution. An intense violet color will appear. Heat usually changes this violet color again to yellowish red, but addition of a few more drops of stannous chloride solution will cause the violet color to reappear. The smaller the quantity of nitric acid, the more likelihood that this test will give a good result. Colorless ammonium sulphide solution may be substituted for stannous chloride. 2. R. Mauch's Modification of Nitric Acid-Stannous Chloride Test. An excellent result can be obtained with this test in the following manner. Dissolve brucine in 60 per cent, aqueous chloral hydrate solution and put about 0.5 cc. of this solution into a test-tube. Add very little dilute nitric acid and thor- oughly mix the two solutions. Add this mixture to 3 times its volume of concentrated sulphuric acid so that the former is on the surface. A yellowish red to deep red zone, depending upon the quantity of brucine, will appear immediately. When the upper layer becomes yellow introduce by a pipette a little stannous chloride solution 1 as a top layer. A brilliant, intensely violet zone will appear between the two upper layers. The intensity of this color will gradually increase, especially if the test-tube is gently tilted to and fro. ATROPINE H 2 C CH CH 2 CH 2 OH N.CH, CH O.CO CH H 2 C-CH CH, CHs Atropine, Ci 7 H 23 NO s , crystallizes in shining pointed needles which melt at 115 and dissolve in 600 parts of water, -50 parts of ether and 3.5 parts of chloro- form. It is also soluble in ethyl alcohol, amyl alcohol and benzene. The aqueous solution of the alkaloid is alkaline and has a lasting, unpleasant, bitter taste. Unlike the optically active hyoscyamine, atropine is inactive. 1 Prepare stannous chloride solution by dissolving i part of stannous chloride m 9 parts of hydrochloric acid having a specific gravity of 1.12 (about 24 per cent. HC1). NON-VOLATILE POISONS 103 Constitution. Heated with hydrochloric acid at 120-130. atropine is decom- posed into tropic acid and tropine: HOH H H H 2 C C CH 2 | CH 2 OH H 2 C C CH 2 | H 3 C N HC O| C CH = H 3 C N HC OH + H 2 C C CH 2 I O C 6 H 5 H 2 C- H Atropine CH 2 OH HO C CH II I O C 6 H 5 Tropic acid Heated with barium hydroxide solution, atropine yields atropic acid which is unsaturated and differs from tropic acid by one molecule of water: CH 2 .OH CH 2 CH.C 6 H 5 - H 2 O = C.C 6 H 5 I I COOH COOH Tropic acid Atropic acid Since the structure both of tropine and tropic acid has been determined by synthesis as well as by decomposition, that of atropine is also known. Nitro- gen in atropine is in the tertiary condition. Hyoscyamine is the stereo-isomer of atropine. The former, heated at 110 out of contact with air, or allowed merely to stand in alcoholic solution with addition of a few drops of an alkaline hydroxide solution, is changed to inactive atropine. . Atropine most likely is the racemic form, whereas hyoscyamine is the laevo-rotatory modification of this isomeric base. The degree of rotation of hyoscyamine is [a]o = 20.97. Toward alkaloidal reagents and when heated with concentrated sulphuric acid, hy- oscyamine behaves like atropine. It also resembles the latter in giving Vitali's reaction (see below). Putrefaction. Ipsen 1 has found atropine very resistant in presence of putre- fying material. Even after 2 years he could detect the alkaloid which had been exposed to the influences of decomposition. He experimented with 0.05 gram of atropine sulphate in respectively 300 cc. of blood, urine and beer and with pure atropine in 300 cc. of blood. Detection of Atropine Ether, benzene or chloroform will extract atropine from a solution alkaline with sodium hydroxide or carbonate solution. In a special search for atropine use sodium carbonate solution and extract with chloroform which is a better solvent than ether. 1 Vierteljahrsschrift fur gerichtliche Medizin und offentliches Sanitatswesen, 3i, 38. 104 DETECTION OF POISONS Evaporate the solvent and test the residue, which is usually amorphous, as follows : 1. Vitali's Test. Dissolve the alkaloid in a few drops of fuming nitric acid, and evaporate the solution in a porcelain dish to dryness upon the water-bath. Moisten the yellowish residue when cold with a few drops of a solution of potassium hydroxide in absolute ethyl alcohol. An evanescent violet color will appear, if atropine is present. Hyoscyamine and scopolamine also give Vitali's test. Strychnine and vera- trine behave similarly. This test therefore is characteristic of the atropine alkaloids only in the absence of the two latter alkaloids. 2. Para-Dimethylamino-Benzaldehyde Test. 1 The required reagent is prepared by dissolving 2 grams of para-dimethyl- CHO (i) amino-benzaldehyde, CeH^ , in 6 grams of con- X N(CH 3 ) 2 (4) centrated sulphuric acid and carefully adding 0.4 gram of water. The dark yellow solution obtained keeps well for two weeks. Place a trace of atropine on a watch-glass, add i drop of the reagent and warm gently. A very intense red-violet color is produced. This is an exceedingly delicate test. Hyoscyamine and scopolamine also give this test. The color in the cold with morphine and codeine is a clear red; with quinine red; with physostigmine and veratrine green; and with narcotine and papaverine orange. 3. Odor Test. Heat a little atropine in a dry test-tube until a white vapor appears. An agreeable odor will arise at the same time. Then add about i cc. of concentrated sulphuric acid, and heat until the acid begins to darken. Dilute at once with about 2 cc. of water. During the foaming there will be an in- tense, sweetish odor like that of honey. By this test, which was formerly the only method of identifying* atropine, o.oi gram of the alkaloid can be detected. 4. Physiological Test. Atropine acts in a very characteristic manner upon the pupil of the eye, and this behavior can be employed as a test. One drop of an atropine solution diluted i : 130,000 will produce a noticeable enlargement of the pupil. 1 Chemical Abstracts n, 1518 (1917). NON-VOLATILE POISONS 105 Dissolve a small portion of the ether residue in 4 or 5 drops of very dilute sulphuric acid, and introduce a drop of this solution into a dog's or a cat's eye. The enlargement of the pupil often persists for several hours. The utmost care should be taken in performing this test, if applied to the human eye. The following alkaloidal reagents are especially sensitive toward atropine: iodo-potassium iodide, phospho-molybdic acid (i : 10,000), gold chloride, phospho-tungstic acid, potas- sium mercuric iodide, potassium bismuthous iodide. Picric acid, added to solutions of atropine salts that are not too dilute, will precipitate atropine picrate as yellow leaflets. Platinic chloride gives monoclinic prisms. HOMATROPINE Homatropine, CieH^iNOs, is the tropyl ester of phenyl-glycolic or mandelic acid. The hydrochloride of this base is obtained by heating a mixture of tropine, jj _ jj _ jj jj mandelic acid and hydrochloric acid, the latter i| | | acting as a dehydrating agent. N.CH 3 CH.OOC CH The hydrobromide of homatropine (Ci 6 H 2 i- I I I NOs.HBr) is used in medicine as a substitute for atropine. Its action on the pupil is nearly as strong as that of the natural alkaloid and its effect disappears in 12-24 hours, whereas that of atropine often lasts 8 days. Moreover it is less toxic than atropine. Homatropine is a strong tertiary base which forms neutral salts with acids. This alkaloid does not give Vitali's test. It melts at 92-96; hyoscya- mine at 108; and atropine at 115.5. Officinal homatropine hydrobromide may be distinguished from the hydro- bromides of atropine and hyoscyamine by warming the substance in a test-tube with a little chloroform. This solvent dissolves the latter two salts in every pro- portion but homatropine hydrobromide is insoluble. An alternative procedure consists in dissolving the. given salt in a little water, precipitating the base with sodium carbonate solution, and extracting with ether. Dehydrate the ether extract with potassium carbonate and evaporate slowly in a moderately warm place. This method will give crystals of the alkaloid. Dry these crystals in aacuo over concentrated sulphuric acid and determine their melting-point. COCAINE Cocaine, Ci7H 2 iNO 4 , crystallizes from ethyl alcohol in large, colorless, mono- clinic prisms which melt at 98. It has a bitterish taste and, placed upon the tongue, causes temporary, local anaesthesia. ii.ii The alkaloid is only slightly soluble in water N.CH 3 CH OOC C 6 H 5 (i : 700), but easily soluble in ethyl alcohol, I I ether, chloroform, benzene and acetic ether. " H2 Its solutions are strongly alkaline and laevo- rotatory. Dilute acids easily dissolve cocaine and in most cases form readily 106 DETECTION OP POISONS crystallizable salts. The fixed alkalies, ammonia and alkaline carbonates precipi- tate the free base from solutions of its salts. Constitution. Cocaine is a monacid, tertiary base, since it adds a molecule of CH 3 I. On distillation with barium hydroxide, this alkaloid loses methyl amine (CHs.NH 2 ), thus proving the attachment of a methyl group to nitrogen. Co- caine must therefore contain the group = N CHs. This base is also the methyl ester of an acid and at the same time the benzoyl derivative of an alcoho,! for it is decomposed into benzoyl-ecgonine and methyl alcohol when heated with water. If mineral acids, barium hydroxide or alkalies are used instead of water, the primary product, benzoyl-ecgonine, is further decomposed into ecgonine, benzoic acid and methyl alcohol. Taking the structural formula proposed by Willstatter, we may express this reaction as follows : Ecgonine Bengnc Methyl H 2 C CH 2 C 6 H 5 CH 3 I CH I CO O HC N CH - | H I H I O I H H 2 C C CH + + O CO H O H Ecgonine (I) heated with phosphorus oxychloride loses a molecule of water and passes into anhydro-ecgonine (II). The latter heated to 280 with fuming hydrochloric acid loses carbon dioxide and is converted into tropidine (III). Tropidine heated with a caustic alkaline solution adds a molecule of water and passes into tropine (IV), the basic cleavage-product of atropine. Evaporation of tropine with tropic acid in dilute hydrochloric acid solution yields atropine (V). Thus it is possible to start with the alkaloid cocaine and synthesize the alkaloid atropine. The series of changes involved is as follows:' H.C-CH CH.COOH H 2 C-CH CH.;COOj H N.CH, CHjOHJ - H 2 N.CH 3 CH - CO 2 :-CH CHJH :! ' H 2 C-CH CH Ecgonine (I) Anhydro-ecgonine (II) H 2 C H,C H S C-CH CH 2 H 2 C-CH CH 2 CH 2 .OH CH +H 2 o N.CH, CH.O|H + Hbioc CH: "Y ' NON-VOLATILE POISONS 107 H 2 C CH CH 2 CH 2 .OH III I N.CH 3 CH.O.CO.CH + H 2 O. H 2 C CH Cn 2 Atropine (V) CH 2 C 6 H B Behavior in the Animal Organism. Experiments upon dogs and rabbits show that the former animal eliminates through the kidneys not more than 5 per cent, of the cocaine as such and the latter none at all. As the urine of these animals also contains no ecgonine, the supposition is that the alkaloid is profoundly changed in the animal organism. The same is true of the human organism. Proells 1 was able to detect cocaine in cadaveric material at most after 14 days. In the living organism the alkaloid is said to be changed rapidly into ecgonine. Detection of Cocaine Ether, chloroform or benzene will extract cocaine from an alkaline aqueous solution. Most of the alkaloidal reagents will precipitate cocaine even from very dilute solutions of its salts. The reagents especially sensitive are: iodo-potassium iodide, phospho-molybdic and phospho-tungstic acids, potassium mer- curic iodide, potassium bismuthous iodide, gold and platinum chlorides, and picric acid. Pure concentrated sulphuric and nitric acids, as well as Erd- mann's, Froehde's and Mandelin's reagents, dissolve cocaine without color. i. Precipitation Test. If i or 2 drops of potassium hydroxide solution are added to an aqueous solution of a cocaine salt not too dilute, it will become milky. First, resinous globules and later fine, crystalline needles of the free base, cocaine (melting- point 98), separate from solution: Ci 7 H 21 N0 4 .HCl 2 + KOH = dyHziNO, + H 2 O + KC1. In applying this test to the ether residue, dissolve a consider- able quantity in a few drops of dilute hydrochloric acid and add potassium hydroxide solution drop by drop until alkaline and 1 Apotheker-Zeitung 16, 779, 788. 2 Cocaine hydrochloride crystallizes from a concentrated aqueous solution in fine prisms containing 2 molecules of water which are easily given off. This salt crystallized from ethyl alcohol is anhydrous and has the formula CnH^NOi.HCl. The anhydrous compound is the officinal salt. 108 DETECTION OF POISONS cool well by setting in ice. Special care must be taken to have the alkaloid pure enough when dry for a melting-point determination. This test is not characteristic of cocaine (except the melting- point which, however, requires considerable pure material), because most of the alkaloids are precipitated by potassium hydroxide solution in much the same way. 2. Potassium Pemanganate Test. Add saturated potassium permanganate solution drop by drop to a concentrated aqueous solution of a cocaine salt. This reagent will give a violet, crystalline precipitate of cocaine permanganate. In applying this test to the ether residue, dissolve a considerable quantity in 2 drops of dilute hydrochloric acid and evaporate the solution upon the water-bath. Dissolve the residue in as little water as possible and add potassium permanganate solution. 3. Chromic Acid Test. Add a few drops of a 5 per cent, chromic acid solution, or potassium dichromate solution of corresponding concentration (7.5 per cent.) to a solution of a cocaine salt. Each drop will produce a precipitate which will immediately disappear if the solution is shaken. Then add to the clear solution about i cc. of concentrated hydrochloric acid which will produce an orange-yellow precipitate more or less crystalline. 4. Detection of Benzoyl Group. This test requires at least 0.2 gram of cocaine. First, digest the cocaine a few minutes in a test-tube with 2 cc. of concentrated sulphuric acid upon a boiling water-bath. Cool and dilute with a little water, all the while keeping the mixture cold. A white crystalline pre- cipitate of benzoic acid will appear. Collect and dry this precipitate upon a filter. Benzoic acid may be recognized by subliming the precipitate, or, if the quantity is sufficient, by determining the melting-point (120). Benzoic acid may also be extracted with ether. Mix the residue, obtained by evaporating the solvent, with i cc. of absolute ethyl alcohol and the same quantity of concentrated sulphuric acid. The characteristic odor of ethyl benzoate, C 6 H 6 .CO.OC 2 H5, will be recognized. NON-VOLATILE POISONS 109 5. Reichard's 1 Test. Addition of a concentrated aqueous solution of sodium nitroprusside, Na 2 Fe(CN) 5 NO.2H 2 O, drop by drop to a cocaine salt solution, containing at least 4 mg. of cocaine per cc., causes an immediate turbidity which appears under the microscope as well-formed reddish crystals. These crystals will dissolve, if the liquid is warmed, and appear again if the solution is well-cooled. Morphine does not give this test. 6. Deniges 2 Test. If an equal volume of 5 per cent, sodium perchlorate (NaC10 4 ) solution is added to 0.5 per cent, solution of a cocaine salt, a precipitate consisting of very fine, long needles is produced. This precipitation may be observed under the microscope, when the quantity of material is very small. 7. Pisani's 3 Test. Heat cocaine or its hydrochloride witji a few drops of concentrated sulphuric acid containing 2 per cent, of formamide (H.CO.NH 2 ). A wine-red color, increasing in intensity as the temperature rises, is produced. This color soon disappears and a brownish gray precipitate remains. This test will detect o.ooi gram of cocaine. Atropine, quinine, cinchonine, brucine, strychnine, morphine, apomorphine, codeine and narcotine do not give this test. Papaverine gives a wine-red colora- tion which changes to yellow, reddish brown and orange. 8. Physiological Test. Dissolve the material (the residue from the ether extract) in a few drops of dilute hydrochloric acid and evaporate the solution to dryness upon the water- bath. Dissolve the residue in a little pure water and apply this solution to the tongue. Cocaine produces a temporary anesthesia. R. Robert ("Intoxikationen") has found small frogs suffi- ciently sensitive for use in the physiological test for cocaine. The effects to be observed are dilatation and fixedness of the pupil, enlargement of the palpebral fissure and also stimulation 1 C. Reichard, Chemiker-Zeitung 28, 299 (1904). Pharmazeutische Zeitung 1904, Nr. 29. Pharmazeutische Zentralhalle 45, 645 (1904). 2 Chemical Abstracts 9, 234 (1915). 3 Chemical Abstracts 9, 511 (1915). HO DETECTION OF POISONS of the nervous system. Administer the same quantity of co- caine hydrochloride to animals for comparison. PHYSOSTIGMINE Physostigmine, CuHziNsC^, also called eserine, occurs in the Calabar bean, the seed of Physostigma venenosum. This alkaloid is deposited from benzene solution upon spontaneous evaporation of the solvent in large, apparently rhombic crystals melting at 105. Though but slightly soluble in water, it dissolves freely in ethyl alcohol, ether, benzene or chloroform. Physostigmine solutions are strongly alkaline, almost tasteless and laevo-rotatory. It is a strong monacid tertiary base, forming salts with acids that easily undergo decomposition and crystallize with difficulty. Light and heat cause acid and alkaline solutions of this alkaloid to turn red. Owing to this tendency of physostigmine to undergo decomposition, care must be taken during its isolation to keep it from light and air and also to avoid rise of temperature. Exclusion as far as possible of free min- eral acids and caustic alkalies is also desirable. Detection of Physostigmine Concentrated sulphuric and nitric acids dissolve physostigmine with a yellow color which soon changes to olive-green. The alkaloid evaporated upon the water-bath with fuming nitric acid leaves a residue having a green margin. Water, ethyl alcohol and sulphuric acid dissolve this residue with a green color. 1. Ammonia Test. If a small quantity of a physostigmine salt is evaporated to dryness upon the water-bath with ammonium hydroxide solution, a blue or blue-green residue will remain. This will dissolve in ethyl alcohol with a blue color. Excess of dilute mineral acid, or acetic acid added to this solution will change the color to red. The solution is also strongly fluorescent. Examined spectroscopically, the blue alkaline solution shows one absorption-band in the red; and the red acid solution one absorption-band in the yellow. A drop of concentrated sulphuric acid, added to the blue residue from evapora- tion with ammonia, will give a green solution. The green color diluted with ethyl alcohol will change to red. If the alcohol is evaporated, the green color will reappear. 2. Rubreserine Test. If an aqueous solution of a physostigmine salt is shaken for some time with an excess of potassium or sodium hydroxide solution, a red coloring-matter, rubreserine (CisHnNjC^), is formed. This compound separates as red needles which become greenish blue on further oxidation owing to forma- tion of eserine blue. Barium hydroxide solution may be substituted for the caustic alkali. This reagent first produces a white precipitate which soon becomes red on being shaken. Sometimes this change occurs even in the cold but invariably takes place with heat. 3. Physiological Test. The marked action of physostigmine in causing con- traction of the pupil is very characteristic. It is advisable to use the cat's eye for this test. Even o.i mg. of this alkaloid will produce noticeable contraction. NON-VOLATILE POISONS 111 CODEINE Codeine, Ci 7 Hi 8 (CH 3 )N03, the methyl ether of morphine, crystallizes from water, or from ether containing water, in colorless, transparent octahedrons jj which are often very large. These crystals are quite H H 2 | easily soluble in water. One part of the free base C C N is soluble at 15 in 80 parts of water and at 100 in ^\/\/\ 15 parts. Codeine differs from most of the other I || I I 2 alkaloids, morphine, for example, in its relatively high CH 3 O.C C C CH 2 solubility in water. Ethyl alcohol, ether, amyl alcohol, \/\^\/ chloroform and benzene also dissolve codeine freely. C C CH j t j S; however, practically insoluble in petroleum O-HC CH ether. Aqueous codeine solutions are strongly alka- \/ line and bitter. Pure codeine does not reduce iodic C acid, nor does it immediately produce a blue color or _/ X-VTT a blue precipitate in a mixture of potassium ferri- cyanide and ferric chloride solutions. A pure codeine solution is also not colored blue by ferric chloride solution alone. (Dif- ference between morphine and codeine.) Phospho- molybdic acid, iodo- potassium iodide, potassium bismuthous iodide and potassium mercuric iodide give precipitates even with very dilute codeine solutions. On the other hand, tannic and picric acids, gold and platinum chlorides are less sensitive. Detection of Codeine 1. Sulphuric Acid Test. Concentrated sulphuric acid dis- solves codeine without color. After long contact or upon appli- cation of gentle heat, the solution will have a reddish to bluish violet color. The solution of codeine in concentrated sulphuric acid, heated to about 150 and then cooled, is colored deep red by a drop of concentrated nitric acid. 2. Nitric Acid Test. Cold nitric acid (25 per cent.) will con- vert codeine into nitro-codeine (Ci 8 H 2 o(NO2)NO 3 ). At the same time the acid will dissolve the alkaloid with a yellow color which soon changes to red. Concentrated nitric acid dissolves codeine with a reddish brown color. 3. Oxidation Test. Mix a little codeine upon a watch-glass with four times the quantity of finely powdered potassium arsen- ate (KH 2 AsO 4 ). Add a few drops of concentrated sulphuric acid and then warm gently over a small flame. The acid will have a deep blue or blue- violet color, if the codeine is not quite pure. Excess of potassium arsenate does not affect the test. 112 DETECTION OF POISONS If water or sodium hydroxide solution is added, the blue color will change to orange-yellow. A trace of ferric chloride solution may be substituted for potassium arsenate. Sulphuric acid containing i drop of ferric chloride solution to 10 cc. of acid is prescribed by the German Pharmacopceia for detecting the alkaloid in codeine phosphate. 4. Froehde's Test. This reagent dissolves codeine with a yellowish color which soon changes to green and finally to blue. Gentle warming of the solution over a very small flame will hasten this change of color. R. Mauch warms 2 or 3 drops of a chloral hydrate solution of codeine with i drop of Froehde's reagent. An intense blue color finally appears. 5. Formalin-Sulphuric Acid Test. 1 Concentrated sulphuric acid containing formalin dissolves codeine with a reddish violet color which changes to blue-violet. This color is per- sistent. The spectrum shows an absorption of orange and yellow. 6. Furfural Test. 2 Dissolve codeine in a few drops of con- centrated sulphuric acid and warm very gently with a drop of cane-sugar solution which must not be in excess. This will produce a purple-red color. This test may also be made by mixing a drop of sugar solution with codeine, dissolved in about 5 drops of 50-60 per cent, aque- ous chloral hydrate solution, and then adding 1-2 cc. of con- centrated sulphuric acid as an under layer. A carmine-red ring will appear at the zone of contact. The color is quite permanent and increases in intensity upon standing. If the sulphuric acid and 'chloral hydrate solution are thoroughly mixed, the entire liquid will be red. After a time the shade of color will be more of a red-brown. 7. Pellagri's Test. Both codeine and morphine give this test. Dissolve codeine in concentrated hydrochloric acid and 1 See preparation of reagents, page 321. 2 This test depends upon furfural formed by the action of concentrated sul- phuric acid upon cane-sugar. Very dilute aqueous furfural solution (i: 1000) may be substituted for cane-sugar. Excess of furfural unlike cane-sugar does not interfere with the test. Tr. NON-VOLATILE POISONS 113 add at the same time 3-4 drops of concentrated sulphuric acid. Expel hydrochloric acid upon the water-bath and heat the resi- due about 15 minutes. Dissolve the dirty red or violet residue in 2-3 cc. of water, add a few drops of hydrochloric acid and neutralize with acid sodium carbonate. Then add alcoholic solution of iodine drop by drop (2 to 4 drops) and shake thor- oughly for several minutes. An emerald-green solution indi- cates codeine. Extract the green solution with ether. The color of the ether will be red, whereas that of the aqueous solu- tion will remain green. This is a test for apomorphine (see page 128) formed from codeine by the mineral acid. Ci7H 18 (CH 3 )N0 3 + HC1 = CnHnNOs + CH 3 .C1 + H 2 O. Codeine Apomorphine 8. Mecke's Test The reagent, consisting of selenious acid and concentrated sulphuric acid, 1 dissolves codeine with a blue color quickly changing to emerald-green and finally becoming a permanent olive-green. NARCOTINE Xarcotine, CszH^NO?, crystallizes in shining prisms or in tufts of needles which are nearly insoluble in cold water but readily soluble in boiling ethyl alcohol or chloroform. Separation of alkaloid from the cold alcoholic solution is almost complete. At 15 narcotine dissolves in 170 parts of ether; 31 parts of acetic ether; and 22 parts of benzene. Solutions of narcotine are not alkaline nor bitter. In these respects narcotine is very OCH 3 different from the other opium alkaloids. Salts I of narcotine do not crystallize, their stability is ^\ slight and their solutions react acid. Salts with HC C.OCH 3 volatile acids are decomposed, when their solutions are evaporated, with separation of narcotine. So- dium acetate precipitates free narcotine from its C solution in hydrochloric acid. Constitution. Narcotine is a monacid, tertiary HC O base and as such combines with i mol. of CH 3 .I, CH O C CH forming narcotine methyl iodide (C 22 H 23 NO 7 .- 3 ^\ /\ CH 3 I). This compound is formed at ordinary / O C C N.CHs temperatures but the reaction is hastened by Cx heat. Narcotine heated with hydriodic acid loses ~C ^ CH 2 3 methyl groups wh ich form CH 3 I. The al- ^ kaloid must therefore contain 3 methoxyls, H H 2 3(CH 3 O-), in the molecule. Heated with water to 140, with dilute sulphuric acid, or even with See preparation of reagents, page 322. 114 DETECTION OF POISONS barium hydroxide solution narcotine is hydrolyzed into nitrogen-free opianic acid and into the basic and consequently nitrogenous hydrocotarnine: C 22 H 23 NO 7 + H 2 O = CioH 10 p 6 + Ci 2 Hi 6 N0 3 Narcotine Opianic Hydrocotarnine acid By oxidative cleavage, that is, by treatment of narcotine with such oxidizing agents as nitric acid, manganese dioxide and sulphuric acid, lead dioxide and ferric chloride, cotarnine and opianic acid are the products : C 22 H 23 N07 + (H 2 O + O) = CioHiqOs + Ci 2 H 16 NO 4 Narcotine Opianic Cotarnine acid Evidently these cleavage-products show that this alkaloid is made up of two complexes, one nitrogen-free and the other containing nitrogen. The chemical constitution of these cleavage-products has been determined and is expressed by the following formula?: H CH 3 C | H 2 S\ C C CH 3 C CH /O C C N CH 3 C-C=0 H 2 C< | || | H \0 C C CH 2 CH 3 C C C=O H 2 C \/ C HO C=O H H 2 Opianic Acid Hydrocotarnine CH 3 O O I /" C C H -C C NH.CH 3 H 2 C<( | || | X C C CH 2 YY H H 2 Cotarnine On the basis of these results, Roser and Freund have proposed the structural formula for narcotine given above. They consider the constitution of this alka- loid as definitely settled. If the formula of narcotine is compared with that of hydrastine (see page 116), a great similarity in structure will be seen. In fact narcotine is a methoxylized hydrastine. Detection of Narcotine Narcotine is so feebly basic that chloroform will extract the alkaloid completely from an aqueous tartaric acid solution. Consequently its separation from the rest of the opium alkaloids as well as from other alkaloids is easy. Naturally ether or chloroform will also extract narcotine from an aqueous alkaline NON-VOLATILE POISONS 115 solution. The alkaloid as it comes from its ether solution is usually a slightly colored, varnish-like residue which hardens after a time to a mass of radiating crystals. Narcotine is precipitated from its hydrochloric or sulphuric acid solution by iodo-potassium iodide, phospho-molybdic acid, potassium mercuric iodide, potassium bismuthous iodide even in consider- able dilution (i : 5000). 1. Sulphuric Acid Test. Dissolved with stirring in concen- trated sulphuric acid, narcotine produces a greenish yellow color which gradually changes to reddish yellow and finally after several days to raspberry-red. 2. Dilute Sulphuric Acid Test. A solution of narcotine in dilute sulphuric acid (i 15), evaporated on the water-bath in a porcelain dish or over a very small flame, has a reddish yellow color, changing with stronger heat to crimson-red. As the acid begins to^ evaporate, blue- violet streaks radiate from the margin and finally the entire liquid has a dirty red-violet color (Dragen- dorff 's reaction) . The same color changes appear, if the yellow- ish solution of narcotine in concentrated sulphuric acid is heated very carefully. 3. Froehde's Test. This reagent dissolves narcotine with a greenish color. If concentrated Froehde's reagent is used, the green color changes immediately to cherry-red, especially upon application of gentle heat. This color is quite persistent. 4. Couerbe's Test. Dissolve narcotine in cold concentrated sulphuric acid and mix a trace of nitric acid with this solution after 1-2 hours. A red color will appear and gradually become more and more pronounced. Erdmann's reagent gives the same color change. 5. Wangerin's Test. 1 Place a mixture of o.oi gram of narcotine with 20 drops of pure concentrated sulphuric acid and 1-2 drops of i per cent, cane-sugar solution upon a watch- glass and heat upon the water-bath with stirring about i minute. At first the solution has a greenish yellow color which passes through yellow, brownish yellow, brown and brown-violet into an intense blue-violet. 1 Pharmazeutische Zeitung, 48, 607 (1903). 116 DETECTION OF POISONS The intensity of this color increases somewhat upon standing and the blue-violet color persists several hours. Applied to apomorphine, atropine, brucine, quinine, codeine, caffeine, hydras- tine, morphine, physostigmine, pilocarpine and strychnine, this test gives solutions that are colorless or nearly so. Only the morphine solution after a while has a pale pink color. Coniine and narcotine have a light yellow color; narceine chestnut-brown; and picrotoxin salmon color to pale pink. Colchicin, digitalin and veratrine behave toward this reagent as toward pure concentrated sulphuric acid without the addition of the small quantity of sugar. In this test 1-2 drops of i per cent, aqueous furfural solution may be substi- tuted for the sugar solution. From yellow, brown, olive and other colors there finally emerges a deep, clear, dark blue. The brilliancy of this color increases somewhat on standing. After several hours there is a gradual change to a pure green color. For the detection of traces of narcotine (o.ooi gram) use a i per cent, sugar solution. 6. Selenious Acid-Sulphuric Acid Test. This reagent dis- solves narcotine with a greenish steel-blue color which after a time becomes cherry-red. Heat immediately discharges the cherry-red color. HYDRASTINE Hydrastine, C 21 H 2 iNO 6 , occurs together with berberine, C 2 oH 17 NO 4 , and cana- dine, CjjoHziNCh, in hydrastis root, the root of Hydrastis canadensis, to the amount of 1.5 per cent, and more. The fluid ex- O.CH 3 tract prepared from this root and used in medicine ^ contains 2-2.5 per cent, of hydrastine. ^\ Preparation. Extract hydrastis root with hot HC C.OCH 3 water containing acetic acid. Filter the solution, I II evaporate to a thin extract and add 3 vols. of dilute \/ sulphuric acid (1:5). Nearly all the berberine C separates out in fine yellow crystals as acid sul- phate, C 2 oHi7NO4.H 2 SO 4 . Precipitate hydrastine H C -- from the mother-liquor of berberine sulphate by JJ means of ammonium hydroxide solution and purify the alkaloid by crystallization from acetic ether or p N.CHj ethyl alcohol. Hydrastine crystallizes from ethyl \O__ p p ptr alcohol in rhombic prisms melting at 132. It is \/\ / nearly insoluble in water but freely soluble in hot C C etli yl alcohol, benzene or chloroform. This alkaloid H H 2 has a bitter taste and its solutions are alkaline. Hydrastine solutions are optically active. In chloro- form this alkaloid is laevo-rotatory, whereas in dilute hydrochloric acid it is dextrorotatory. C NON-VOLATILE POISONS 117 Constitution. The constitution of hydrastine is entirely analogous to that of narcotine (see page 113). On oxidation with dilute nitric acid hydrastine gives opianic acid and hydrastinine : C 2 iH 21 N0 6 + (H 2 + O) = C 10 H 10 5 + C u Hi 3 NO 3 Hydrastine Opianic acid Hydrastinine Hydrastine is a monacid base which is shown to be a tertiary base by its behavior toward alkyl iodides, for example, with CH 3 I it forms hydrastine methyl iodide, C2iH2iNO6.CHsI, which crystallizes in needles. Hydrastine contains two methoxyl groups, because when heated with hydriodic acid according to Zeisel's method two such groups are removed. Since the chemical nature of opianic acid has long been known, the only problem is the explanation of the nature of hydrastinine, the other cleavage- product. The constitution of hydrastinine, as well as that of many other alka- loids, has been determined by A. W. Hofmann's method of exhaustive methyla- tion. 1 Hydrastinine (I) is a secondary base which forms, when heated with an excess of CHsI, hydrastinine hydriodide and trimethyl-hydrastyl-ammonium iodide (II). Heated with alkalies, this ammonium iodide is decomposed into trimethylamine, hydriodic acid and nitrogen-free hydrastal (III). The latter on oxidation gives hydrastic acid (IV) which was recognized as the methylene ether of nor-meta-hemipinic acid (V) : /CH:0 + 2 CH 3 I = (I) (CH 2 2 )C6H 2 < X CH 2 .CH 2 .NH.CH 3 /CH:O (CH 2 2 )C 6 H 2 < V X CH 2 .CH 2 .N(CH 3 ) 3 I Hydrastinine Trimethyl-hydrastyl- annnonium iodide /CH:0 (II) (CH 2 2 )C 6 H 2 < X CH 2 .CH 2 .N(CH 3 ) 3 I + KOH = /CH:O (CH 2 2 )C 6 H/ + KI + H 2 + N(CH S ) 3 CH:CH 2 Hydrastal 1 When the nitrogen of an organic base becomes quinquevalent, it is more sub- ject to change. Hofmann (Liebig's Annalen, 78, 263 (1851) showed, for example, that tetra-ethyl-ammonium hydroxide breaks up on heating into triethylamine, ethylene and water: CH 2 C 2 H 5 \ P 2 J} 5 >N-OH =|| + C 2 HAN + H 2 0. C 2 H 5 2 Nitrogen in alkaloids on treatment with an alkyl haloid (e.g., CH 3 I) combines with it in many instances, forming compounds having a structure analogous to that of tetra-ethyl-ammonium hydroxide. This process is called "exhaustive methylation." Upon decomposition these derivatives yield products which often throw light upon the structure of the alkaloid. 118 DETECTION OF POISONS CH-0 /COOH (III) (CH,0 2 )CH<( ; CH Oxidized = ( CH ') C ' H < COOH Hydrastic acid Hydrastic acid and nor-meta-hemipinic acid are identical. The latter has the structure (V): H C O C C.COOH < | || \0 C C.COOH (V) H 2 C< | || C. C H Nor-meta-hemipinic acid From these and other relations it has been determined that cotarnine is a methoxy-hy drastinine : H CH 3 .0 H H / II C C=0 C C=0 /O C C NH.CH, /QC C NH.CH 3 H 'o4 I! k H ' C i! YY YY H H 2 H H 2 Hydrastinine Cotarnine The alkaloid narcotine is a methoxy-hydrastine (see page 144). Detection of Hydrastine 1. Concentrated Sulphuric Acid dissolves hydras tine without color but upon being gently warmed the solution becomes violet. 2. Froehde's Reagent dissolves hydrastine with a green color which gradually changes to brown. 3. Mandelin's Reagent dissolves hydrastine with a rose- red color which immediately changes to orange-red and gradu- ally fades. 4. Fluorescence Test. Dissolve hydrastine in dilute sul- phuric acid, shake vigorously and add drop by drop very dilute potassium permanganate solution. Hydrastinine is formed and the solution shows a beautiful blue fluorescence. The ether extract of the alkaline solution on evaporation leaves hydrastinine in a crystalline condition. NON-VOLATILE POISONS 119 QUININE Quinine, CaoH24N 2 O 2 , is precipitated amorphous and anhydrous from solutions of its salts by caustic alkalies, alkaline carbonates or ammonia. On standing, H however, it gradually becomes crystalline, forming a C hydrate with 3 molecules of water of hydration. x There are also other hydrates of quinine. Anhy- _, CH.CH:CH 2 drous quinine melts at 173; the trihydrate at 57. i 2 An ether solution on evaporation usually deposits HO.C CH 2 CH 2 tnis alkaloid as a resinous, or varnish-like, amorphous \ | / residue. Quinine is soluble in about 2000 parts of cold and 700 parts of boiling water; and freely solu- -jj ble in ethyl alcohol, ether or chloroform. Solutions of H quinine in sulphuric, acetic or tartaric acid exhibit a C beautiful blue fluorescence. In the case of the sul- ==, phate this fluorescence is distinctly visible in a dilu- MU U L.UL.J13 H/l J.1 -C, HC C CH Hydrochloric, hydrobromic and hydriodic acid do not give fluorescent solutions of quinine. These acids N C even discharge the fluorescence, if added to a fluor- escent quinine solution. Constitution. Quinine is a diacid, ditertiary base, the salts of which with i and 2 equivalents of acid are usually crystalline. The salts with i equivalent of acid are the more stable. Quinine hydrochloride, C 20 H 24 N 2 O 2 .HCJ.2H 2 O, used in medicine, crystallizes in long delicate tufts of needles. The diter- tiary character of quinine is shown by the fact that it unites with 2 mole- cules of methyl iodide, for example, to form quinine dimethyliodide, C 2 oH 2 4N2O2.2CH3l. Quinine must contain an hydroxyl group, since it can form a mono-benzoyl and a mon-acetyl-quinine. Moreover one methoxyl group has been found in the quinine molecule. The difference empirically between cinchonine, Ci9H 2 2N 2 O, and quinine, C2oH24N2O 2 , is CH 2 O. Every investigation of these substances has shown that quinine is a methoxy-cincho- nine. For example, on oxidation with chromic acid, cinchonine gives cinchonic acid which was recognized as quinoline -y-carboxylic acid; whereas quinine under the same conditions gives quinic acid, or p-methoxy-cinchonic acid: COOH(-y) COOH( 7 ) HI' H C C C C HC 7 C X CH (p)CH 3 O.C / C \H ni c in ^ H H Cinchonic acid Quinic acid Both alkaloids on oxidation also give the nitrogenous compounds mero- quinene, cincholoiponic acid and loiponic acid. Consequently there is no doubt that cinchonine and quinine contain two nitrogenous nuclei, one of which 120 DETECTION OF POISONS is a quinoline complex. The second nucleus is connected with the latter in the 7-position, as the formation of cinchonic and quinic acids shows. Meroquinene, cincholoiponic acid and loiponic acid, derived by oxidation with chromic acid from the so-called "second half" of the cinchonine and quinine molecules, form a continuous series of oxidation products, since meroquinene can be oxidized to cincholoiponic acid and the latter to loiponic acid. The following formulae best explain the chemical behavior of these three compounds: CH 2 .COOH CH 2 .COOH COOH A H CH C \ H ' H 2 C CH CH : : CH 2 H 2 C CH.COOH H 2 C CH.COOH H 2 C CH 2 H 2 C CH 2 H 2 C CH 2 \/ \/ \/ N N N H H H Meroquinene Cincholoiponic acid Loiponic acid The structural formula already given for quinine was propose^ by W. Koenigs 1 and is based on the results of his own experiments as well as on those of \V. V. Miller and of Skraup. Cinchonine has hydrogen in place of the methoxyl group in the quinoline nucleus; otherwise the two alkaloids are identical in structure. Detection of Quinine Ether, benzene or chloroform will extract quinine from an aqueous alkaline solution. Ether on evaporation deposits the alkaloid as a resinous, amorphous varnish in which its presence may be recognized by the following tests: 1. Fluorescence Test. Dissolve the residue from the ether extraction of the alkaline solution in a little dilute sulphuric acid. If quinine is present, this solution will exhibit blue fluorescence. 2. Thalleioquin Test Dissolve quinine in a few drops of very dilute acetic acid and add 5-10 drops of saturated chlorine water. The colorless solution has a faint, blue fluorescence. Excess of ammonium hydroxide solution will produce an emer- ald-green color. A solution containing considerable quinine will give a green precipitate. This precipitate (thalleiioquin) is always an amorphous substance, the composition of which has not been determined. It is soluble in ethyl alcohol and chloroform but not in ether. 1 Meroquinene and the Structure of the Cinchona Alkaloids; Annalen der Chemie und Pharmazie 347, 147 (1906^. NON-VOLATILE POISONS 121 E. Polacci recommends the following procedure for the thal- le'ioquin test. Gradually heat quinine (about o.oi gram) to boiling with a little lead dioxide (PbO 2 ) , 2-3 cc. of water and 2 drops of dilute sulphuric acid. Let the solution settle and either decant or filter. Finally, carefully add 5-6 drops of ammonium hydroxide solution as a top layer. A beautiful green ring will appear at the zone of contact. Interferences with the Thalleioquin Test. Antipyrine interferes with this test. Mixtures of i per cent, solutions of antipyrine and quinine give finally a beautiful red instead of a green color. This interference does not cease until these two substances are in the proportion of 0.25 parts of antipyrine to 5 parts of quinine. Caffeine also interferes with the thalleioquin test, when the proportion is 2 parts of quinine to 3 parts of caffeine. Other compounds like urea prevent the appear- ance of this color, whereas morphine, pilocarpine, cocaine, atropine, codeine, strychnine, carbolic acid and chloral hydrate have no effect upon the thalleioquin test. H. Fiihner 1 has shown that the thalleioquin reaction is connected with the p-oxyquinoline complex. Chlorine passed into a solution of pure p-oxy-quino- line cooled with ice produces a white crystalline precipitate. This substance crystallizes from petroleum ether in colorless prisms or tabular crystals melting at 58. Structurally it is 5,5-dichloro-6-keto-quinoline. Solutions of this di- chloro-keto-quinoline and of its hydrochloride are colored a pure green or blue by ammonium hydroxide. Fiihner thinks 5,6-quinoline quinone is probably formed and gives the green color with ammonia. H H C C H Cl, C C H C C J^\. y/\ HC C C.OH(p) HC C CH HC C CO 1 II 1 - HC C CH HC C CO HC C CH N C H p-Oxy-quinoline N C H 5,5-Dichloro-keto- quinoline N C H 5,6-Quinoline quinone 3. Herapathite Test. Mix 30 drops of acetic acid, 20 drops of absolute ethyl alcohol and i drop of dilute sulphuric acid. Add 20 drops of this mixture to o.oi gram of quinine and heat to boiling. Finally add i drop of an alcoholic solution of iodine (i : 10) or 2 drops of o.i n-iodine solution. When the solution has stood for some time, green leaflets with a metallic luster will 1 Berichte der Deutschen chemischen Gesellschaft 38, 2713 (1905). 122 DETECTION OF POISONS form. This is an iodine compound of quinine called "Hera- pathite," having the constant composition This substance can be recrystallized from boiling ethyl alcohol. Herapathite crystals are pale olive-green by transmitted light but by reflected light they have a beautiful, cantharidin- green, metallic luster. Caustic alkalies, ammonia, sulphurous acid and hydrogen sulphide decompose herapathite. A. Christensen recommends keeping on hand the following reagent for the herapathite test: Parts Iodine i Hydriodic acid (50%; i Sulphuric acid 0.8 Ethyl alcohol (70%) 50 Add a few drops of this reagent to the alcoholic solution to be tested for quinine. 4. Hirschsohn's Test. 1 If i drop each of 2 per cent, hydro- gen dioxide and 10 per cent, copper sulphate solution are added to a neutral solution of quinine hydrochloride or sulphate at boiling temperature, a more or less intense raspberry-red color will appear. This color soon passes through blue-violet into blue and after a time into green. A quinine solution (i : 10,000) will still give a distinct red- violet color. Excess of acid as well as of ethyl alcohol interferes with this test. The behavior of a solution of aloes toward this test is similar to that of quinine. Of the alkaloidal reagents potassium bismuthous iodide is especially recommended as a precipitant of quinine. With quinine sulphate solutions this reagent produces precipitates having an intense yellowish red color. Shaken with sodium hydroxide solution this precipitate is decomposed and unaltered quinine can be obtained by extraction with ether and evapora- tion of the ether solution. H. Thorns 2 has made use of this reaction in the quantitative separation of quinine from mixtures. CAFFEINE Since caffeine (see page 84) is a weak base, ether will extract only a little of the alkaloid from the tartaric acid solution. The 1 Pharmazeutische Zentral-Halle 43, 367 (1902). 2 Berichte der Deutschen pharmazeutischen Gesellschaft 16, 130 (1906). NON-VOLATILE POISONS 123 greater part will be in the ether extract of the alkaline solution. Ether usually deposits caffeine in white, shining needles ar- ranged in clusters. Caffeine dissolves in ether with some difficulty and the alkaline solution should be extracted several times. For the tests characteristic of this alkaloid see page 85. ANTIPYRINE Most of the antipyrine (see page 82) is obtained by extract- ing the alkaline solution with ether. It is usually purer from the acid than from the alkaline solution and frequently appears in crystalline leaflets. Antipyrine differs from most alkaloids in having only a faintly bitter taste and in being freely soluble in water. To identify antipyrine, dissolve the ether residue in a little water and divide the solution into two equal parts. Test one portion with ferric chloride solution and the other with fuming nitric acid. Detection of Antipyrine in Urine. The color of urine after administration of antipyrine is intensely yellow to blood-red. Part of the antipyrine in the organ- ism appears in the urine as oxy-antipyrine-glycuronic acid and another part is unchanged and can usually be detected directly in urine by ferric chloride solu- tion. A safer procedure is to add excess of ammonia to a considerable quantity of urine and extract with chloroform. Evaporate the solvent, dissolve the residue in a little water and test the filtered solution for antipyrine with ferric chloride solution and with fuming nitric acid. Antipyrine is easily absorbed. The urine may show a reddish color, even an hour after the drug has been taken, and give a test with ferric chloride solution. The red color disappears in about 24 hours but the elimination of antipyrine is not complete in that time. Its detection is still possible after 36 hours. A con- venient procedure is to add to the urine as an upper layer very dilute ferric chlo- ride solution. A red ring will appear if the urine contains antipyrine. Jonescu 1 states that antipyrine in the human organism passes unchanged into the urine. Only a small portion and large doses of the drug must have been taken is eliminated in conjugation with sulphuric acid. Conjugation with glycuronic acid 2 (see above) according to Jonescu does not occur in the human organism. 1 Berichte der Deutschen pharmazeutischen Gesellschaft 16, 133 (1906). H\ 2 Glycuronic acid, C 6 Hi O: = ^C(CH.OH) 4 COOH, may be regarded as a ? derivative of glucose. Possibly it occurs in normal urine in small quantity as a conjugated acid. After administration of various alcohols, aldehydes, ketones, phenols (chloral hydrate, camphor, phenol, thymol, menthol, borneol), there takes place in the animal organism often after oxidation or reduction a con- jugation of these substances with glycuronic acid. 124 DETECTION OF POISONS PYRAMIDONE Pyramidone, or 4-dimethyl-amino-antipyrine, Ci 3 Hi 7 N 3 O, has been exten- sively used in medicine of late as an antip>retic and anodyne. It is a white, crystalline powder, nearly tasteless and readily soluble in water. It melts at 108. Its aqueous solution has jsj a neutral reaction. Ether removes only traces of pyramidone from acid solution, but extracts it easily CH 3 N 2 sCO an d completely from alkaline solution. Ether usually H _Qs = 4(i N(CH 3 ) 2 de P sits this substance in fine needles. Pyramidone is also freely soluble in ethyl alcohol, ether, chloroform or benzene. It is a strong reducing agent and in this respect differs from antipyrine. For example, pyramidone will reduce gold chloride even in the cold, whereas antipyrine and tolypyrine require heat. Preparation. Antipyrine dissolved in concentrated acetic acid is converted by treatment with potassium nitrite into nitroso-antipyrine which appears as green crystals. This compound dissolved in ethyl alcohol may be reduced by zinc and acetic acid to amino-antipyrine. The latter, dissolved in methyl alcohol and treated with methyl iodide and potassium hydroxide, is converted into dimethyl- amino-antipyrine, or pyramidone. C 6 H 5 C 6 H 6 C 6 H 6 I I I N N N CH 3 .N CO CH 3 .N CO ' CH 3 .N CO + 2 CH 3 I - | | +4H-+ | - CH 3 .C=C|H HOj.NO CH 3 .C=C.NO CH 3 .C=C.NH 2 2 KOH Antipyrine Nitroso-antipyrine Amino-antipyrine C 6 H 5 N ' * / CH 3 .N CO CH 3 .C=C.N(CH 3 ) 2 Pyramidone Behavior in the Organism. Human urine, if neutral or faintly acid, usually has a bright purplish red color after administration of pyramidone. After stand- ing for some time it will deposit a sediment consisting of red needles soluble in ether or chloroform but especially in acetic ether. Jaffe 1 recognized this com- pound as rubazonic acid, a pyrazolone derivative. Isolation of rubazonic acid from urine may be brought about as follows. Acidify fresh urine with hydro- chloric acid and let it stand in an open dish. The acid will appear as small red. particles. Ferric chloride solution produces a blue- violet color in the acid liquid filtered from rubazonic acid. This filtrate contains most of the product formed from pyramidone in animal metabolism, namely, crystalline antipyryl-urea melting at about 245. ^erichte der Deutschen chemischen Gesellschaft 34, 2737 (1901); and 35, 2891 (1902). NON-VOLATILE POISONS 125 C 6 H 8 A CH 3 .N CO CH 3 .C=C.NH.CO.NH 2 Antipyryl-urea Detection of Pyramidone 1. Ferric Chloride Test. Ferric chloride solution added to pyramidone produces a blue-violet color which soon changes to reddish violet and then disappears. 2. Fuming Nitric Acid Test. A few drops of fuming nitric acid, added to a solution containing pyramidone, give a blue to blue-violet color. 3. Bromine Water Test. This reagent imparts a grayish color to pyramidone solutions. With concentrated solutions it produces an inky color. 4. Iodine Test. Tincture of iodine colors an aqueous pyra- midone solution blue. 5. Guglialmelli's 1 Test. Either of the following two reagents may be used, but (6) usually gives the better result: (a) Arseno-tungstic Solution. Dissolve 25 grams of sodium tungstate (Na 2 WO4.2H 2 O) in 200 cc. of cold distilled water, adding 20 grams of pure arsenic trioxide (As2Os) and boiling the solution for 1.5 hours under a reflux-con- denser. Filter the resulting light, bluish green solution when cold and bring the volume to 250 cc. (b) Arseno-tungsto-molybdic Solution. Boil in the same manner 10 grams of sodium tungstate, 2 grams of sodium molybdate (Na2MoO4-ioH2O) and 10 grams of pure arsenic trioxide for 1-2 hours with 75 cc. of distilled water. Bring the cold solution to a volume of 100 cc. Added to aqueous pyramidone solutions, these reagents produce white spots. Those from reagent (a) are soluble in alkali with an intense blue color; whereas those from reagent (b} give an intense indigo color. Pyramidone produces these colors in a dilution of i : 750,000. These reagents produce white spots with antipyrine solutions but they are soluble in alkali without color. 1 Chemical Abstracts 12, 664 (1918). 126 DETECTION OF POISONS 6. Palet's 1 Test. A few drops of a freshly prepared solution of potassium ferricyanide and ferric chloride, added to an aqueous pyramidone solution, produce the characteristic blue color and precipitate of Prussian blue. The reaction is very sensitive. Morphine gives the same test (see page 135). With antipyrine the reagent gives a blood-red color and precipitate and is negative with phenacetine, acetanilide and caffeine. If pyramidone and anti- pyrine are together, a little hydrochloric acid should first be added. C. Extraction of the Ammoniacal Solution with Ether and Chloroform (a) Ether Extract. Apomorphine and traces of morphine. 2 (/3) Chloroform Extract. Morphine and narceine. (It may also contain antipyrine and caffeine. 3 ) The aqueous alkaline solution (see page 86) , separated from ether, must be tested further for the substances under a and 0. Apomorphine may be recognized by the green color of the aqueous acid solution. Excess of sodium hydroxide solution causes oxidation, especially if the solution is exposed for any length of time to air, and gradually changes the color to deep purple-red. Moreover, the ether extracts, both of the acid and alkaline solutions, are red or violet-red when apomorphine is present. Solutions, examined by the Stas-Otto method, not having these characteristics, need not be tested for apomorphine. In that case proceed at once with the morphine and narceine tests. To extract apomorphine, morphine and narceine with the proper solvent, the aqueous solution separated from ether, which is alkaline from sodium hydroxide solution (see page 86), must be rendered alkaline with ammonium hydroxide solution. First acidify the solution with dilute hydrochloric acid (test with blue litmus paper) and then add ammonium hydroxide solution until alkaline. 1 Chemical Abstracts, 13, 216 (1919). 2 Ether dissolves traces of freshly precipitated, amorphous morphine. Antipyrine and caffeine, though freely soluble in chloroform, dissolve with lifficulty in ether. The latter solvent frequently fails to extract these substances completely from aqueous solution. They will then appear in the chloroform NON-VOLATILE POISONS 127 (a) If there is any indication of apomorphine, first extract the ammoniacal solution repeatedly with ether and then several times with hot chloroform for the morphine and narceine tests. (/3) If there is no indication of apomorphine, extract the ammoniacal solution several times direct with hot chloroform (see below). APOMORPHINE Constitution. Apomorphine, CiTHnNOz, is a monacid, tertiary base with two phenol hydroxyl groups. According to R. Pschorr 1 it has the structural formula here given. Properties. Apomorphine is an amorphous base W (9) readily soluble in ethyl alcohol, ether, benzene or C C* N 3 chloroform and colored green in contact with air. ,/\/\/\ Aqueous and alcoholic apomorphine solutions, origi- HC C CH CH 2 nally colorless, soon turn green in the air from oxida- iwn r r r rw ^ on ' Solutions of apomorphine thus changed by ' >\ /v xx s 2 oxidation are emerald-green. Ether and benzene solu- C C C(8) tions are purplish violet; those in chloroform blue- 1 || | violet. Being phenolic in character, apomorphine (4) HO HC CH resembles morphine in its solubility in sodium hy- 0^ droxide solution. Alkaline solutions of the alkaloid jj absorb oxygen from the air and become brown or even black in color. Apomorphine differs from mor- phine in being more soluble in water and in ethyl alcohol, but especially in being soluble in ether, benzene and cold chloroform, in which morphine is almost insoluble. Formation and Preparation. Sulphuric, hydrochloric, phos- phoric and oxalic acids, the alkalies and zinc chloride have mainly a dehydrating action upon morphine and convert it into apomorphine: C, 7 Hi9N0 3 = H 2 + CirHuNO* Morphine Apomorphine Codeine, the methyl ether of morphine, also gives apomor- phine when heated at 140 with concentrated hydrochloric acid. l8 2 3 HCl = H 2 O + CH 3 C1 + CirHnNOi Codeine Apomorphine Apomorphine is prepared by heating morphine (i part) with concentrated hydrochloric acid (20 parts) for 3 hours in an autoclave at 130-150. 1 Berichte der Deutschen chemischen Gesellschaft 39, 3124 (1906); and 40, 1984 (1907). 128 DETECTION OF POISONS (a) Detection of Apomorphine in the Ether Extract Ether will not extract apomorphine from a solution contain- ing tartaric acid but will dissolve its colored oxidation products. This solvent behaves similarly toward solutions of this alkaloid in sodium or potassium hydroxide solutions. Ether or chloro- form will extract apomorphine only from a solution alkaline with ammonium hydroxide. Ether solutions of apomorphine usually deposit a greenish residue. A characteristic of this alkaloid is its strong reducing action. For example, it will re- duce iodic acid with liberation of iodine and produce a purple color with gold chloride. Apomorphine gives the following tests: 1. Sulphuric and Nitric Acids. Concentrated sulphuric acid dissolves apomorphine without color. Addition of a drop of concentrated nitric acid to such a solution produces an eva- nescent violet color that soon changes to blood-red and finally to yellowish red. With concentrated nitric acid alone this alka- loid gives a violet-red color that soon becomes red-brown and finally brownish red. 2. Pellagri's Test. Dissolve apomorphine in dilute hydro- chloric or sulphuric acid and first add acid sodium carbonate in excess. Then add drop by drop 1-3 drops of an alcoholic iodine solution and shake for several minutes. The solution will have a blue-green or emerald-green color. Extract with a little ether and the solvent will become violet, whereas the aqueous solution will remain green. 3. Froehde's Test. This reagent dissolves pure apomorphine with a green color. If the alkaloid has been acted upon by air to any extent, the color is violet. 4- Wangerin's 1 Test. L Prepare a fresh solution of apomor- phine hydrochloride (about i per cent.). Add 4 drops of potas- sium dichromate solution (0.3 per cent.) to i cc. of this solution and shake for about i minute. The solution will have an in- tense dark green color. Then add 10 cc. of acetic ether and shake again. This solvent will become violet. Finally add 1 Pharmazeutische Zeitung 47, 599 and 739-740 (1902). NON-VOLATILE POISONS 129 from a pipette about 5 drops of stannous chloride solution 1 (i per cent.) and shake well. The color of the acetic ether layer will change to green and, upon further addition of a few drops of potassium dichromate solution, the acetic ether will again be- come violet. If 10 cc. of chloroform are substituted for acetic ether in this test, the oxidation product of apomorphine will im- part the same violet color to the chloroform. But if stannous chloride solution is added carefully, the color will change to pure indigo-blue and persist upon further agitation with potassium dichromate solution. '5. E. Schmidt's Tests. 2 (a) A drop of very dilute ferric chloride solution (i : 100) will color 10 cc. of an aqueous apo- morphine hydrochloride solution blue even in a dilution of i : 10,000. (&) Shake 10 cc. of the same apomorphine hydrochloride solution with i cc. of chloroform. Then render alkaline with sodium hydroxide solution and at once shake with air. The aqueous solution becomes evanescent violet in color and the chloroform blue. 6. Palet's Test. 3 Add 1-2 drops of apomorphine solution to 1-2 cc. of Guglialmelli's reagents. 4 After shaking the mixture for 2-3 minutes, add 5-10 cc. of a cold, saturated solution of pure sodium carbonate. An indigo-blue color, varying in intensity with concentration and time, appears. Divide the liquid after 5 minutes into 3 portions. These treated respectively with amyl alcohol, benzene and acetic ether give rise to an intense blue, dark violet and violet color. Further addition of 1-2 drops of 10 per cent, stannous chloride solution to the acetic ether extract changes the violet color to emerald- green as in Wangerin's test. Apomorphine diluted 1:500,000 gives a distinct blue color, and a slightly positive result when 1 Prepare this reagent as follows : Crystallized stannous chloride (SnCl 2 .2H 2 O; i gram Hydrochloric acid (25 per cent.) 50 cc. Water 50 cc. 2 Apotheker-Zeitung 23, 657 (1908). 3 Chemical Abstracts 12, 601 (1918). 4 See page 125 for the preparation of these reagents. 130 DETECTION OF POISONS diluted i : 1,000,000. In all cases the intensity of the color increases on standing. Morphine gives the same blue color which is not extracted by solvents. Nar- cotine and narceine do not give this reaction. (/3) Examination of the Chloroform Extract Preliminary Morphine Test. As a preliminary test for mor- phine, acidify a small portion of the aqueous alkaline solution separated from ether (see page 85) with dilute sulphuric acid, add iodic acid solution and extract' with a little chloroform. If the latter has a violet color from dissolved iodine, morphine may be present. But a final conclusion regarding the presence of morphine must not be drawn from a positive test, since there are many other organic substances besides this alkaloid that will reduce iodic acid. 1 This is a delicate preliminary test for morphine and that is its only value. If it is negative, morphine is probably absent. To detect morphine and narceine positively, render the aque- ous solution alkaline with ammonium hydroxide and extract at once as already directed (see page 126) with considerable hot chloroform 2 in a capacious flask. Separate the two liquids as usual in a separatory funnel. Several extractions of the aque- ous solution with fresh portions of hot chloroform are necessary because of the slight solubility of morphine even in boiling chloroform. Should the chloroform and the aqueous solution form a refractory emulsion that will not separate, add a few drops of ethyl alcohol, set the flask on a warm but not boiling water-bath and carefully turn the flask from time to time. This procedure usually causes the immediate separation of the two liquids. Place the combined chloroform extracts in a dry flask, add a few crystals of dry sodium chloride or anhydrous sodium sulphate to remove adherent water, pour the chloroform when clear through a dry filter and evaporate in not too large a glass 1 In testing animal matter that contained no morphine, the author has repeat- edly obtained extracts that strongly reduced iodic acid. 2 C. Kippenberger (Zeitschrift fur analytische Chemie 39, 201, 290) uses chloroform, containing 10 per cent, of ethyl alcohol by volume, to extract morphine. NON-VOLATILE POISONS 131 dish placed upon a warm water-bath. The chloroform may also be filtered directly into the dish as fast as it evaporates. If the residue is bitter and can be scraped together with a platinum spatula or a pocket-knife, test for morphine and narceine. 1 In testing for morphine use Froehde's, Husemann's and Pellagri's tests as well as those given by formalin-sulphuric acid and iodic acid. The presence of morphine is not established unless all these morphine tests give positive results. If the quantity of the residue from chloroform permits, test for morphine with ferric chloride solution. This test is very characteristic of morphine but requires more than traces for a satisfactory result. Purification of Impure Morphine When the chloroform residue is too impure, especially if red or biown, it must be purified. Dissolve in hot amyl alcohol and shake the solution thoroughly with several portions of hot water containing a few drops of dilute sulphuric acid. The acid dissolves the morphine, whereas the amyl alcohol retains most of the coloring matter. Add ammonium hydroxide solution in 'excess to the acid solution and extract several times with hot chloroform. The morphine obtained by evaporation of the chloroform should be nearly pure. MORPHINE Morphine, CirHigNOs, crystallizes from dilute ethyl alcohol in shining prisms which are colorless and transparent and but slightly soluble in water (i: 5000 at 15; and 1:500 at 100). These solution. CHs are very bitter and have an alkaline reactions H HZ | Crystalline morphine is insoluble in ether and x,^ xx xx benzene. The amorphous alkaloid is soluble in JJQ CH cfj 2 amyl alcohol, hot chloroform and acetic ether. ill | | Solutions of the hydroxides of ammonia, potassium C C CHz or sodium and sodium carbonate solution precipi- ^^C ^p rvrr tate free morphine from solutions of morphine | | | salts. ~~/9 , CH!! Constitution. Morphine is a mon- H C acid, tertiary base whose nitrogen is in jj OH union with three atoms of carbon. The three oxygen atoms have different func- tions. One is a phenolic hydroxyl and gives to morphine th 1 Antipyrine and caffeine may also be in this residue (see above). 132 DETECTION OF POISONS character of a monatomic phenol. Conseqeuntly when sodium hydroxide solution is added drop by drop to a morphine salt solution, there is first a precipitate of crystalline morphine (a) which is freely soluble in excess of alkali (0) but is again precipitated on addition of ammonium chloride solution (7) : (a) Ci 7 H l8 N0 2 (OH).HCl + NaOH = CnHisNCMOH) + H 2 O + NaCl, 09) Ci 7 Hi 8 N0 2 (OH) + NaOH = CwHwNOrfONa) + H 2 O, (y) CnHisNCMONa) + (H 4 N)C1 = CnH l8 NO 2 (OH) + NH 5 + NaCl. Hydrogen of this phenolic hydroxyl may be replaced also by alkyl groups and acid radicals. In codeine this hydrogen is replaced by methyl. A second oxygen atom of morphine is alcoholic and the third is indifferent. The latter like the oxygen of an ether is combined with two carbon atoms and forms a so- called bridge-oxygen atom. Of the 17 carbon atoms of morphine 14 belong to the phenan- threne nucleus, 1 since the nitrogen-free cleavage-products of morphine and codeine, namely, morphol and morphenol, have been identified as phenanthrene derivatives. R. Pschorr has synthesized morphol which is 3,4-dioxyphenanthrene. Mor- phenol contains two hydrogen atoms less and may be converted into morphol by i eduction with nascent hydrogen. These two phenanthrene derivatives have the following structural formulae : H H C C /\ /\ HC CH HC CH i L;; t li H/V H/VX II I II I o HC C HC C / \/\ \S\/ C C.OH( 4 ) C C (i)HC C.OH( 3 ) HC C.OH \/ V (2)C C H H Morphol Morphenol / \ /CH = CH\ Phenanthrene, Ci 4 H 10) HC^ >C C/ >CH, occurs C - CH^ in coal-tar together with anthracene. It forms colorless crystals which melt at 99 and boil at 340. It is readily soluble in ether or benzene and with difficulty in ethyl alcohol. Phenanthrene solutions exhibit bluish fluorescence. NON-VOLATILE POISONS 133 By distillation over zinc dust morphenol may be reduced to phenanthrene. The structural formula of morphine written above was pro- posed by R. Pschorr 1 and seems to explain most satisfactorily the reactions of this alkaloid. Morphine is easily oxidized. This may be brought about in alkaline solution by atmospheric oxygen. Potassium per- manganate or ferricyanide and ammoniacal copper solution may also be used. As a result the non-toxic oxy-dimorphine, also called pseudomorphine, which is soluble in caustic alkali, is formed: 2 C 17 Hi9N0 3 + O = (Ci7H 18 NO ? )2 + H 2 O. Morphine Oxydimorphine Detection of Morphine 1. Nitric Acid Test. Concentrated nitric acid dissolves mor- phine with a blood-red color which gradually changes to yellow. Stannous chloride or ammonium sulphide solution will not re- store the violet color of a solution that has become yellow. (Distinction from brucine.) 2. Husemann's Test. Dissolve morphine upon a watch- glass in a few cc. of concentrated sulphuric acid. The solution is colorless. Heat for 30 minutes upon the water-bath, or over a small flame for a very short time until white fumes arise. A reddish or brownish color appears. Cool and add 1-2 drops of concentrated nitric acid. A fugitive, reddish violet color ap- pears and soon changes to blood-red or yellowish red. This color gradually disappears. A preferable procedure is to dissolve morphine in cold con- centrated sulphuric acid and add a trace of concentrated nitric acid after the solution has stood in a desiccator 24 hours. A small crystal of potassium nitrate or chlorate may be substi- tuted for nitric acid. Frequently impure morphine is obtained from the chloroform extract of a solution prepared from animal material., Such a residue gives a more or less 1 Berichte der Deutschen chemischen Gesellschaft 40, 1984 (1907). 134 DETECTION OF POISONS highly colored solution with sulphuric acid. Heat usually intensifies the color. But even under these conditions it is possible to detect the red color caused by nitric acid or potassium nitrate. 3. Pellagri's Test. Proceed as described for codeine. (See page 112.) Avoid excess of alcoholic iodine solution, otherwise the latter may mask the green color. 4. Froehde's Test. This reagent dissolves morphine with a violet color which passes through blue to dirty green and finally to faint red. These colors vanish on addition of water. 5. Formaldehyde-Sulphuric Acid Test. The solution used for this test is called Marquis' reagent. 1 With a trace of mor- phine it produces a purple-red color which changes to violet and finally becomes pure blue. This blue solution, kept in a test-tube and only slightly exposed to air, retains its color for some time. Codeine and apo morphine give the same violet color. Narcotine also gives violet solutions but they become olive-green and finally yellow. Oxy-dimorphine gives a green color. 6. lodic Acid Test. Shake a solution of morphine in dilute sulphuric acid with a few drops of iodic acid and chloroform. Morphine will liberate iodine which will dissolve in chloroform with a violet color. Obviously this delicate test is conclusive for morphine only in the absence of other reducing substances. 7. Ferric Chloride Test. Add 1-2 drops of neutral ferric chloride solution to a neutral solution of a morphine salt. A blue color appears. In testing the chloroform residue, dissolve in a little very dilute hydrochloric acid. Evaporate this solu- tion to dryness upon the water-bath, dissolve the residue in pure water and add a drop of ferric chloride solution. 8. Lloyd's Test. Lloyd has found that a mixture of morphine, hydrastine and concentrated sulphuric acid alone without potassium dichromate will produce the same violet color given by the latter with a solution of strychnine in concentrated sul- 1 Mix 2-3 drops of 40 per cent, formaldehyde solution with 5 cc. of concen- trated sulphuric acid and use a few drops of this mixture for the morphine test. NON-VOLATILE POISONS 135 phuric acid. Lloyd's reaction is of value in the detection of mor- phine or hydrastine only when more than traces of both alka- loids are present. A. Wangerin 1 considers these reactions characteristic only when 0.0050.01 gram of morphine and 0.002-0.01 gram of hydrastine are present. Make an intimate mixture of about these quantities of both alkaloids upon a watch-glass. Add 5 drops of pure concen- trated sulphuric acid and stir the mixture for 10 minutes over a white background. In the center the color-tone is a clear red- violet and more or less of a blue-violet in the thinner marginal region. Apomorphine hydrochloride, treated in the same way with hydrastine and concentrated sulphuric acid, gives almost the same reaction as morphine. 9. Prussian Blue Test. Add a few drops of a dilute mixture of ferric chloride and potassium ferricyanide solutions to a morphine salt solution. A deep blue color appears. Consid- erable morphine produces a precipitate of Prussian blue. Potassium ferricyanide oxidizes morphine to oxy-dimorphine : + 2KOH + K 6 Fe 2 (CN)i 2 = aH 2 O + (CnHuNO^ + 2K 4 Fe(CN) Morphine Potassium Gxy-dimorphine Potassium ferricyanide ferrocyanide Potassium ferrocyanide then forms Prussian blue with ferric chloride. 10. Silver Test. Warm a morphine salt solution with silver nitrate and excess of ammonium hydroxide solution. Mor- phine produces a gray precipitate of metallic silver. 11. Bismuth Test.^Dissolve morphine in concentrated sulphuric acid and sprinkle a little bismuth subnitrate on the surface of the solution. A dark brown color appears. 12. G. Fleury's Test. 2 Dissolve morphine in a little very dilute sulphuric acid (about 0.05 normal), add some lead di- oxide (PbO 2 ) and shake for 6-8 minutes. A pale rose color appears. Addition to the nitrate of ammonium hydroxide solution in excess produces a brown color which persists for 1 Pharmazeutische Zeitung 46, 57 (1903;. 2 Annales de Chimie analytique appliquee 6, 417 (1907). 136 DETECTION OF POISONS several hours. When the quantity of substance is very small, stir on a porcelain color-plate for 6-8 minutes with a drop of dilute sulphuric acid and a minute particle of lead dioxide. When the insoluble matter has settled, tilt the porcelain plate so that the clear solution runs up the side. A drop of am- monium hydroxide solution now gives a brown color. 13. Dan Radulescu's Test. 1 Add a small particle of sodium nitrite to a very dilute morphine salt solution, then a dilute acid and render alkaline with concentrated potassium hydroxide solution before all the gas has escaped. The solution when con- centrated has a pale rose to a deep ruby-red color. Acids dis- charge but alkalies restore this color. This reaction is said to be characteristic of morphine bases and especially adapted for the detection of morphine in mixtures. 14. Diazonium Test. 2 - In presence of alkalies, morphine and its salts form dyestuffs with diazonium compounds. The reagent is a solution of diazotized sulphanilic acid. Diazonium Reagent. Dissolve 0.2 gram of sulphanilic acid / /NH 2 (i)\ I C 6 H 4 <^ n-HCl and 10 cc. o.i n-NaNOj. /NH 2 /N = N C6H ^SO H + HCI + NaN 2 = CeH4 \ ' Dissolve 0.243 gram of morphine sulphate in 100 cc. of water. Render 5 cc. of this solution alkaline with sodium carbonate or bicarbonate and add an equal volume of diazonium reagent. A red color, changing to orange on addition of acid, immediately appears. The color diluted i : 10,000 is faint red; i : 100,000 distinctly yellow; and i : 2ooo 3 intensely dark red in a thin layer. No opium alkaloid except morphine gives this reaction; not even the synthetic derivatives of this alkaloid (dionine, peronine and heroine). Other alkaloids 1 Chemisches Zentralblatt 1906, i, 1378. 2 Lautenschlaeger: Chemical Abstracts 13, 22^2 (1919). 3 If the above proportions are used and the test is made as directed, the dilu- tion will be i : 2000. lin 80 cc. of water, cooling with ice. Then add 10 cc. o.i NON-VOLATILE POISONS 137 giving colors are: emetine and physostigmine (red); sparteine and piperidine (yellow); coniine and nicotine (bright yellow). But the morphine color above is stable in acid solution. General Alkaloidal Reagents. The reagents of this class especially sensitive toward solutions of morphine salts are: lodo-potassium iodide, Potassium bismuthous iodide, Phospho-tungstic acid, Phospho-molybdic acid, Potassium mercuric iodide, Gold chloride. Plantinic chloride after some time causes a granular orange- yellow precipitate. Tannic acid causes no precipitate, or at most only a very slight cloudiness which becomes somewhat more pronounced with time. Behavior of Morphine in the Animal Organism. The mucous lining of the stomach, rectum or respiratory passages as well as open wounds absorb mor- phine. The alkaloid injected hypodermically acts more rapidly and more po- tently than when absorbed from the stomach. Marquis 1 found that morphine disappears very quickly from the blood but is firmly retained by certain organs like the brain. Some absorbed morphine is conjugated with glycuronic acid and some is oxidized but the rest of the alkaloid is eliminated unchanged. Faust has found that morphine is transformed or destroyed only in men and animals habituated to the poison but is eliminated unchanged nearly quantitatively in the faeces in the case of organisms not immunized. Morphine appears in the urine only in very small quantity after medicinal doses. In men and dogs a not insignificant quantity of the morphine taken is eliminated by the glands of the gastro-intestinal tract, even when the alkaloid has been subcutaneously injected. Marquis found that more than 30 per cent, of intravenously injected mor- phine is deposited in the liver in the course of 15 minutes. The alkaloid is present at first in this organ in the free state and then is soon combined or trans- formed. The conjugation of morphine in the brain also begins very soon. Free morphine is also rapidly changed in the blood, spleen, kidneys and in the mucous lining of the intestines. Marquis states that always in acute and even more so in chronic morphine poisoning a large quantity of the poison leaves the blood and is stored in the salivary glands, mucous lining of the stomach and large intestine, kidneys, spleen, liver and is withdrawn by these organs from the brain and spinal cord. Morphine is quite resistant to putrefaction. The author 2 detected this alka- loid positively in animal material containing morphine (stomach and intestines together with contents) which had stood for 15 months in a glass vessel and had completely putrefied in presence of insufficient air. Doepmann 3 obtained the 1 Arbeiten des Dorpater Instituts, ed Robert, 14 (1896). 2 Berichte der Deutschen Pharmazeutischen Gesellschaft n, 494 (1901). 3 Chemical Abstracts 9, 1663 (1915). 138 DETECTION OF POISONS same result by mixing definite quantities of morphine hydrochloride with lean horse meat and allowing putrefaction to take place from one to eleven months. NARCEINE OCH 3 Narceine, C 23 H 27 N6 8 .3H 2 O, crystallizes from water or ethyl alcohol in prisms which melt at /,--. 165 when air-dried. The alkaloid has a faintly HC C.OCHs bitter taste. Though only slightly soluble in cold water, it is freely soluble in hot. When a hot H( ^ C.COOH satura ted aqueous solution of narceine is cooled, Q it solidifies to a crystalline mass. Narceine is in- soluble in ether, benzene or petroleum ether and CO is soluble only with difficulty in cold ethyl alcohol , CH 3 O. | amyl alcohol or chloroform. In detecting nar- A>\ / 2 jj ceine it is important to know that it is not ex- /O.C C N<^ tracted by ether, benzene or petroleum ether from H 2 C<(' | || I ^CH 3 a solution rendered alkaline by potassium or -C , C . CH 2 sodium hydroxide solution. It is, however, Q extracted by hot chloroform or amyl alcohol H H 2 from an aqueous solution rendered alkaline by ammonium hydroxide solution. Constitution. Narceine is a weak tertiary base in which two methyl groups are attached to nitrogen. By means of Zeisel's method it may be shown that the molecule also contains three methoxyl groups. Narceine, being soluble in caustic alkalies and forming esters with alcohols, must contain a carboxyl group. The alkaloid must also contain a carbonyl group (CO), since it forms a hydrazone with phenyl-hydrazine. The nar- ceine formula above may therefore be resolved into : C 23 H 2T NO 8 = CieHuON (CH 3 ) 2 (OCH 3 ) 3 (CO) (COOH). The narceine molecule contains neither an alcoholic nor a phenolic hydroxyl group, since it forms no acetyl derivative with acetic anhydride. There is a close relationship between narceine and narcotine. By heating narcotine iodo-methylate with sodium hydroxide solution Roser converted this compound into a base called pseudo-narceine. Freund has recently shown that Roser's pseudo-narceine is identical with the opium alka- loid narceine and explains the conversion of narcotine into nar- ceine by saying that the iodo-methylate loses i molecule of hydriodic acid and takes up i molecule of water: NON-VOLATILE POISONS 139 OCH 3 OCH 3 HC Hi C.OCH 3 Ico CH 3 JHi C O / ai 1 /O.C C N CH 3 H 2 C< | || |\CH, X O.C C CH 2 \/ \S C C H H 2 Narcotine iodo-methyolate + H 2 -HI CH 3 H 2 C HC C.OCHs I II HC C.COOH V/ c CO CH 2 CH 3 Y Cns H 3 CH 2 H H 2 Narceine All the reactions and transformations of narceine can easily be explained on the basis of this structural formula. Detection of Narceine 1. Sulphuric Acid Test. Concentrated sulphuric acid dis- solves narceine with a grayish brown color, which gradually changes to blood-red. This reaction takes place at once with heat. 2. Dilute Sulphuric Acid Test. Narceine, warmed in a porce- lain dish upon the water-bath with dilute sulphuric acid until a certain concentration is reached, gives rise to a fine violet color which changes after long heating to cherry-red. 3. Froehde's Test. At first a solution of narceine in this reagent has a brownish green color which gradually changes to green and finally to red. Gentle heat hastens this reaction. 4. Iodine Test. Aqueous iodine solution (iodine water) or iodine vapor colors solid narceine blue. Morphine interferes with or entirely prevents this reaction. 5. Erdmann's Test. This reagent, as well as concentrated nitric acid, dissolves narceine with a yellow color which heat changes to dark orange. 6. Chlorine -Ammonia Test. Pour a few drops of chlorine water upon narceine and add, while stirring, a few drops of 140 DETECTION OF POISONS ammonium hydroxide solution. A deep red color immediately appears. 7. Resorcinol-Sulphuric Acid Test. 1 Mix thoroughly upon a watch-glass resorcinol (o.oi to 0.02 gram) with 10 drops of pure concentrated sulphuric acid. Add a trace of narceine (about 0.002 to 0.005 gram) and, while stirring, warm the in- tensely yellow solution upon a boiling water-bath. A carmine- red to cherry-red color appears. As the solution cools, this color begins at the margin to change gradually to more of a blood-red and finally after several hours to orange-yellow. 8. Tannin-Sulphuric Acid Test. Mix narceine (0.002 to o.oi gram) with tannin (o.oi to 0.02 gram) and 10 drops of pure concentrated sulphuric acid. Heat with constant stirring upon the water-bath and the color of the solution, which is yellowish brown at first, soon becomes pure green. If heat is applied for some time, the green color changes to blue-green and finally through a more or less blue tone to a dirty green. Tannin-sulphuric acid gives a similar color test with narcotine and hydras- tine which closely resemble narceine in constitution. Of the general alkaloidal reagents potassium zinc iodide 2 precipitates narceine even in a dilution of i : 1000. It is a white, filiform precipitate which after a time becomes blue. This blue color appears immediately, if a trace of iodine solu- tion is added to the reagent. Of the other general reagents iodo-potassium iodide, potassium mercuric iodide, potassium bismuthous iodide and phospho-molybdic acid are char- acterized by considerable delicacy toward narceine. SYNOPSIS OF GROUP II Stas-Otto Method A. Ether Extract of Acid Solution may Contain : Picrotoxin. Very bitter. Reduces Fehling's solution with heat. Melzer's test: red streaks radiating from picrotoxin with alcoholic benzaldehyde + cone. H 2 SO 4 . 1 A. Wangerin, Pharmaceutische Zeitung, 47, 916 (1902). 2 See page 319 for the preparation of this reagent. NON-VOLATILE POISONS 141 Cone. H 2 S0 4 : soluble with yellow or orange-red color; drop of K^C^OT + Aq has brown margin. Langley's test: picro toxin + 3 parts KNO 3 , moistened with cone. H2SO4, red with excess of saturated NaOH + Aq. Colchicin. Very bitter. Yellowish and amorphous. Dilute mineral acids render aqueous solutions intensely yellow. Cone. HNO 3 : soluble with dirty violet color changing to brownish red and finally to yellow; excess of KOH + Aq renders orange-yellow or orange-red. Zeisel's test: boil yellow colchicin solution in cone. HC1 in test- tube 2-3 minutes with 2 drops of FeCl 3 + Aq. Green or olive- green when cold, especially if diluted with equal volume of water. Picric Acid. Very bitter. Yellow. Material and extracts more or less intensely yellow. Isopurpuric acid test: aqueous picric acid, gently warmed with a few drops of saturated KCN + Aq, gives red color. Picramic acid test: aqueous picric acid, warmed with few drops of (H 4 N) 2 S + Aq, becomes red. Dyeing test: aqueous picric acid dyes wool and silk intense yellow but not cotton. Acetanilide. Faint, burning taste. Indophenol test: heat with a few cc. of cone. HC1 and evap- orate to about 20 drops. Cool, add aqueous phenol solution and then calcium hypochlorite solution drop by drop. Mix- ture, shaken with excess of ammonia, becomes dirty red to blue-violet and blue. Phenylisocyanide test: boil with KOH + Aq and then add a little chloroform. Odor of phenylisocyanide. Isolation of aniline: boil several minutes with alcoholic KOH, dilute with water and extract with ether. Evaporation of solvent leaves oily drops of aniline. Dissolve in water and test with calcium hypochlorite. Phenacetine. Tasteless. Gives indophenol but not phenyl- isocyanide test. Cone. HNO 3 : yellow color when cold. Dil. HNO 3 dissolves with yellow or orange-yellow color, if heated. Yellow nitro- phenacetine crystallizes as saturated solution cools. 142 DETECTION OP POISONS Salicylic Acid. Sweet, acidulous, harsh taste. FeCl 3 + Aq: aqueous solutions colored blue-violet; if dilute, more of a red- violet. Millon's test: red color upon warming. Br 2 _|_ Aq: yellowish white, crystalline precipitate. Veronal. Bitter. Crystalline. Dissolve ether residue in very little NaOH + Aq or (H 4 N)- OH + Aq, filter and acidify filtrate with dil. HC1. Veronal crystallizes. Wash with a little cold water, dry and determine melting-point (187-188). The crystals mixed with pure veronal should have same melting-point. Antipyrine. Mild, bitter taste. Examine aqueous solution of ether residue for antipyrine. FeCl 3 + Aq: red color. HNO 3 : green color with 1-2 drops of fuming acid. Heat and a few more drops of fuming acid change green color to red. Most of the antipyrine in ether extract of alkaline solution (see B). Caffeine. Faintly bitter. Cl2 + Aq: evaporated upon water-bath with saturated Cl2 + Aq, gives red-brown residue which turns purplish red moistened with very little (H 4 N)OH -f Aq. Most of the caffeine in ether extract of alkaline solution (seeB). Cantharidin. 1 Rhombic leaflets from ether solution. Physiological test: triturate residue with few drops of almond oil and test mixture as vesicant by applying to upper part of the arm. B. Ether Extract of Alkaline Solution may Contain : Coniine. Yellow oil drops with penetrating odor. Cold saturated aqueous solution becomes milky when warmed. 1 Cantharidin is taken up in Chapter IV of this book upon page 203. Ether extracts this compound from acid solution but it dissolves with difficulty in this solvent (o.n : 100 at 18). NON-VOLATILE POISONS 143 Spontaneous evaporation with a drop of HC1 gives coniine hydrochloride as doubly refractive crystals which are needle or prism-shaped and sometimes in star-like clusters. Physiological test: paralysis of peripheral nerves. Nicotine. Liquid. Remains dissolved in residual water upon evaporation of ether and has fajnt tobacco odor. Melzer's test: red color, heated with 2-3 cc. of epichlorohy- drin. Schindelmeiser's test: nicotine, after standing several hours with a drop of formaldehyde solution, gives an intense red color with a drop of cone. HNOs. Roussin's test: ether solution of iodine after some time pro- duces ruby-red, crystalline needles. Aniline. Yellow, reddish or brownish oil drops from evapora- tion of ether extract. (See page 60, "Synopsis of Group I," for further details.) Veratrine. Cone. H 2 SO 4 : soluble with yellow color, gradually changing to orange, then to red and finally to cherry-red. Gen- tle heat hastens these changes. Solution at first shows greenish yellow fluorescence. Froehde: same color changes as with cone. H 2 SO 4 . Cone. HChvery stable red color when heated in test-tube upon water-bath. Weppen's test: mixed with 6 times the quantity of cane- sugar + a few drops of cone. H 2 SO 4 , gradually becomes green and finally blue. Cone. H 2 SO 4 containing furfural may be used instead. Vitali's test: same as for atropine (see below). Strychnine. Fine, crystalline needles having a very bitter taste upon evaporation of ether extract. Oxidation test: colorless solution in cone. H 2 SO 4 becomes evanescent blue or blue-violet with a little solid K 2 Cr 2 O7. Same color given by Mandelin's reagent but more permanent. Brucine. Cone. HNO 3 : dissolves with blood-red color soon changing to reddish yellow and yellow. Dilution of yellow solution in a test-tube with a little water and addition drop by drop of dilute SnCl 2 + Aq changes yellow to violet. 144 DETECTION OF POISONS Careful addition of solution in dil. HNO 3 to cone. H 2 SO 4 as upper layer produces red or yellowish red zone. Atropine. Vitali's test: evaporated upon water-bath in por- celain dish with a little fuming HNO 3 , gives yellowish residue which becomes violet when moistened with alcoholic KOH. Hyoscyamine and scopolamine also give this test. Strychnine and veratrine behave similarly. Para-Dime thylamino-Benzaldehyde test: intense red- violet color. Hyoscyamine and scopolamine also give this test. Physiological test: enlargement of pupil of eye caused by a single drop of solution i : 130,000. Cocaine. Free base, precipitated by KOH -f Aq from not too dilute cocaine salt solution, forms oil drops soon becoming solid and crystalline. Benzoyl group: heat 5 minutes in a test-tube upon boiling water-bath with i cc. cone. H 2 SO 4 . Odor of methyl benzoate upon addition of 2 cc. of water. Upon cooling, benzoic acid separates. This acid, washed and dried, recognized by melting- point (120) and by tendency to sublime. Physiological test: anesthesia of the tongue. Codeine. Cone. H 2 SO 4 : soluble without color. Reddish or more bluish upon long standing, or at once upon gentle warming. Oxidation: deep blue or blue- violet, when warmed with cone. H 2 SO 4 and KH 2 As0 4 , or with a little FeCl 3 + Aq. Froehde: yellowish color soon changing to green and to blue upon gentle warming. Sugar test: purple-red color upon gently warming with cone. H 2 SO 4 and a little cane-sugar. Due to fuifural formed. Formalin test: dissolves in cone. H 2 SO 4 containing formalde- hyde with reddish violet color soon changing to permanent blue- violet. Pellagri's test: given by codeine (see apomorphine, page 146). Hydrastine. Froehde: dissolves with fairly permanent blue- color later changing to brown. Mandelin: dissolves with reddish color gradually changing to orange-red. Fluorescence: intense blue fluorescence (characteristic) upon NON-VOLATILE POISONS 145 shaking dil. H2SO4 solution with very dilute KMnO 4 + Aq added carefully drop by drop. Quinine.- Amorphous varnish having very bitter taste from ether. Fluorescence: blue fluorescence in dil. H 2 SO 4 . Thalleioquin test: emerald-green color, upon adding i cc. saturated C1 2 + Aq to dilute acetic acid solution and then at once excess of (H 4 N)OH + Aq drop by drop. Herapathite test: heat to boiling with 10 drops of mixture (30 drops acetic acid + 20 drops absolute alcohol + i drop dil. H 2 SO 4 ) and add i drop alcoholic iodine solution (1:10). Shining, olive-green leaflets, appearing cantharides-green by reflected light. Antipyrine. Freely soluble in water. Neutral. Mildly bitter. Dissolve ether residue in little water and test for antipyrine as directed in A (see page 142). Pyramidone. Fine needles from ether. Freely soluble in water. Neutral. FeCls + Aq: aqueous solution blue- violet or more red- violet. HNOs: fuming acid renders aqueous solution blue to blue- violet. Caffeine. Concentric clusters of shining needles from ether. Mild, bitter taste. Fairly soluble in water. Neutral. Apply tests described under A (see page 142). Physostigmine. (H 4 N)OH + Aq: evaporated with (H 4 N)- OH + Aq, gives blue residue soluble in alcohol with same color. Physiological test: causes contraction of pupil of eye. Narcotine. Not bitter. Neutral. Froehde: soluble with green color. A concentrated reagent (0.05 gram (H 4 N) 2 MoO 4 to i cc. cone. H 2 SO 4 ) gives greenish color at first which gradually changes to cherry-red and to a blue from margin toward center. Erdmann: soluble with fine red color. Papaverine. Tasteless, colorless, neutral prisms. Cone. H 2 S0 4 : pure alkaloid soluble without color. Heat produces dark violet color. 146 DETECTION OF POISONS Froehde: soluble with green color, soon changing when warmed to blue, violet and finally cherry-red. HNO 3 -H 2 SO 4 test: cone. H 2 SO 4 containing HNO 3 , or cone. HNO 3 itself, gives a dark red solution. Thebaine. Tasteless, colorless, a'lkaline prisms. Cone. H 2 SO 4 : soluble with deep red color. Froehde and Erdmann behave similarly. C. Ether Extract 1 of Ammonia Solution may Contain: Apomorphine. Residue amorphous and usually green. H^SCVHNOs test: solution in cone. H 2 SO 4 colored evanes- cent violet, then reddish yellow or orange by drop of cone. HNO 3 . Froehde: soluble with green or violet color. Pellagri's test: dissolve in dil. HC1, add excess of NaHCO 3 , shake well and add i drops alcoholic iodine solution. Blue or emerald-green color soluble in ether with violet color. Wangerin's test: 1-2 drops K 2 Cr 2 O 7 + Aq (0.3 per cent.), added to apomorphine hydrochloride solution, gradually pro- duces dark green color. Chloroform added becomes violet. Addition of dil. SnCl 2 + Aq produces pure indigo-blue color. D. Chloroform Extract of Ammonia Solution may Contain : Morphine. Very bitter. Usually amorphous. Rarely crys- talline. Froehde: soluble with violet color gradually changing to dirty green and finally to pale red. Formaldehyde-H 2 SO 4 : soluble with purple-red color later be- coming blue-violet and almost pure blue. Husemann's test: dissolve in cone. H 2 SO 4 , heat over very small flame until abundant white fumes appear, cool and add i drop cone. HNO 3 . Very evanescent, red-violet color which soon changes to blood-red or reddish yellow. 1 Unless the tartaric acid and alkaline solutions, as well as their ether extracts, behave as described on page 126, that is to say, have a green or red color, omit this extraction. NON-VOLATILE POISONS 147 Pellagri's test: see apomorphine. FeCl 3 +Aq: dissolve in few drops very dilute HC1, evaporate to dryness upon water-bath, dissolve in little water and add drop FeCl 3 +Aq. Blue color. Bismuth test: dissolve in cone. H 2 S0 4 and sprinkle bismuth subnitrate on surface of solution. Dark brown color. Antipyrine and Caffeine. Being soluble in ether with some difficulty, but readily soluble in chloroform, these substances may appear in the residue from D, if they have not been pre- viously completely extracted with ether. Narceine. I 2 test: blue color with I 2 +Aq. Resorcinol-H 2 SO 4 test: dissolves in resorcinol-H 2 SO 4 , giving intense yellow solution which becomes carmine-red or cherry- red, if warmed upon the water-bath and stirred. Tannin-H 2 SO 4 test: dissolves in tannin-H 2 SO 4 , giving yellow- ish brown solution which becomes pure green, if warmed upon the water-bath. CHAPTER III METALLIC POISONS Destruction of Organic Matter The analyst cannot rely upon tests for poisonous metals, if animal or vegetable matter is present. Consequently complete destruction of interfering organic substances is absolutely essential to success. Description of a few of the more important methods used for this purpose will suffice. i. Fresenius-v. Babo Method 1 The residue left after removal of volatile poisons by steam distillation may be used in this part of the analysis, as it must contain poisonous metals if any are present. A portion of the original material, 2 previously finely chopped and well mixed in a large flask with enough water to produce a fluid mass, may also be used. According to the quantity of material, add 10, 20 or 30 cc. of pure concentrated hydrochloric acid. 3 Finally add 1-2 grams of potassium chlorate, shake well and set the flask upon a boiling water-bath. Nascent chlorine should come into contact with the material as intimately as possible. When the mixture is hot enough, add 0.3-0.5 gram of potas- sium chlorate at 5 minute intervals and shake the flask fre- quently. Continue in this manner, until most of the organic matter is dissolved and the solution is pale yellow. Further 1 Annalen der Chemie und Pharmazie 49, 306 (1844). 1 Cadaveric material should be divided as finely as possible, then brought to a thin mixture by stirring with 12.5 per cent., arsenic-free hydrochloric acid and heated with frequent shaking with 1-2 grams of potassium chlorate as directed above. If the material is heated on the water-bath in a porcelain dish, it should be stirred constantly. 3 In laboratory experiments 5-10 cc. cone, hydrochloric acid is usually suffi- cient. A large excess of hydrochloric acid should be avoided. 148 METALLIC POISONS 149 addition of potassium chlorate and longer heating should produce no real change. Fat especially resists the action of chlorine. When organic matter is completely destroyed, dilute with hot water, adding a few drops of dilute sulphuric acid to pre- cipitate possible barium, shake and pour the liquid through a wetted filter. If the excess of free hydro- chloric acid is not too large, satuiate the filtrate direct with hydrogen sulphide as directed on page 152. Other- wise, evaporate the solution in" a porcelain dish upon the water-bath nearly to dryness to remove most of the free hydrochloric acid. This step frequently gives rise to a dark brown color which a few crystals of potassium chlorate will discharge. In testing for lead, cadmium and copper, it is advisable to evaporate, because hydrogen sulphide precipitates the first two metals incompletely, or not at all, from solutions contain- ing too much hydrochloric p IG . I2 . acid. An alternative procedure consists in removing part of the free hydrochloric acid from the filtrate, obtained after treat- ment with hydrochloric acid and potassium chlorate, by first evaporating to smaller volume and then adding ammonium hydroxide solution until alkaline. Add dilute nitric acid until the solution is faintly acid and saturate with hydrogen sulphide (seepage 152). 150 DETECTION OF POISONS The residue upon the filter may contain silver chloride, barium sulphate and lead sulphate in addition to fat. Examine as directed under "Metallic Poisons IV" (see page 170). H. Thorns 1 destroys organic matter in the apparatus shown in Fig. 12. Oxidation is carried on in an ordinary fractioning flask (A) with the tubulus (B) bent upward. A separating funnel (C), held in the neck of the flask by a stopper, contains an aqueous solution of potassium chlorate (i : 20) saturated at room temperature. The organic matter is in the flask as a thin mixture with 12.5 per cent, hydrochloric acid. Add about i gram of solid potassium chlorate and warm the flask on a boiling water-bath. When the mass in the flask is warm, let the potassium chlorate solution run in drop by drop and shake -constantly. Care must be taken not to add too much of this solution at once; otherwise the procedure is identical with that previously described. Notes. Potassium chlorate and hydrochloric acid evolve chlorine (a and @), part of which acts upon the organic material and part in contact with water forms oxygen and oxygen-acids of chlorine (HOC1) (C = C >C = N(CH 3 ) 2 C1 (quinoidal form) (CH 3 ) 2 N.C,H/ \CH = CH/ or V (CH 3 ) 2 N.C 6 H 4X /C6H 4 .N(CH 3 ) 2 C1 (CH 3 ) 2 N.C 6 H 4 ' 2 Methyl orange = Dimethyl-amino-azobenzene-4-sulphonic acid: (CH 3 ) 2 N. C 6 H 4 .N = N. C 6 H 4 . S0 2 OH. The sodium salt of this sulphonic acid also appears in commerce under the name "methyl orange." 3 See page 321 for the preparation of this reagent. 182 POISONS NOT IN THE THREE MAIN GROUPS 183 5. Sulphocyanate Test. Add a little potassium sulphocya- nate solution to ferric acetate solution and dilute with water until yellow. Then add the solution to be tested. Free mineral acid produces a blood-red color. Traces of free mineral acid, especially if considerably diluted, do not give a red color until several minutes have elapsed. One or more of these general tests, which furnish evidence of a free mineral acid, must always accompany the special tests to be described later. Not only free mineral acids give the special tests but in certain cases their salts. Chlorides, sulphates and nitrates are normal constituents of nearly all vegetable and animal materials. As a rule an examination of cadaveric material for mineral acids is necessary only when the autopsy points conclusively to such poisoning. That is to say, when there are characteristic corrosions and discolorations about the face, mouth, cesophagus and stomach. If general tests show the presence of free mineral acid, make special tests for the particu- lar acid. Hydrochloric Acid 1. Chlorine Test. Warm a little of the aqueous extract, if not too dilute, with finely powdered manganese dioxide. Free hydrochloric acid yields chlorine, recognized by its color and odor, or by passing the gas into potassium iodide solution and liberating iodine. Hydrochloric acid exclusively does not give this test. A chloride (NaCl) and free sulphuric acid give chlor- ine under the same conditions. 2. Distillation. If possible, separate hydrochloric acid from other substances by distillation. The concentration of the acid is especially important, since dilute hydrochloric acid upon distillation at first yields only water. Hydrochloric acid 1 itself does not begin to distil until the concentration is about 10 per cent. Since a dilute hydrochloric acid is usually ex- amined, distil the material mixed with water, or preferably a 1 In the distillation of 100 cc. of i per cent, hydrochloric acid, the first 90 cc. of distillate will contain only traces of hydrochloric acid, whereas the last portion will contain most of the acid. 184 DETECTION OF POISONS filtered aqueous extract, nearly to dryness. In such a dis- tillation apply heat by means of an oil-bath. To detect hydro- chloric acid in the distillate, acidify with dilute nitric acid and add silver nitrate solution. Frequently a quantitative estima- tion of hydrochloric acid is required. In the absence of other acids, titrate the distillate with o.i n-potassium hydroxide solution, using phenolphthalein as indicator. Otherwise, esti- mate the acid gravimetrically, precipitating with silver nitrate and weighing silver chloride, or volumetrically by Volhard's method. In the latter case, precipitate hydrochloric acid with o.i n-silver nitrate solution in excess and subsequently estimate that excess by titration with o.i n-ammonium sulphocyanate solution, using ferric alum as indicator. Since the human stomach normally contains 0.1-0.6 per cent, of free hydro- chloric acid, an examination of stomach contents for this acid must always include a quantitative estimation. Nitric Acid The human body normally contains only a very small amount of nitrates. When present, they are due usually to vegetable foods which contain small quantities of nitrates. Human urine almost always shows traces of the salts of nitric and nitrous acids. The chemical examination of cadaveric material need not include tests for nitric acid, unless the autopsy affords evidence of poisoning by this acid, as distinct signs of corrosion about the lips, mouth, oesophagus and stomach and sometimes perforation. These parts are more or less yellow or yellowish brown. A yellow froth is said to exude from the mouth and nose of the cadaver. Also the stomach contents are yellow in concentrated nitric acid poisoning. If the concentration of the acid is less than 20 per cent., these specific changes may not appear in the gastro-intestinal tract. Nitric acid taken inter- nally, dilute or concentrated, appears at once in the urine. Detection of Nitric Acid i. Distillation. If possible, extract the material direct with water, filter and test the filtrate for nitric acid in the usual way. When the quantity of nitric acid is large, separate it from other substances by distilling the filtered aqueous extract. Apply heat by means of an oil-bath. Nitric acid 1 does not distil, until 1 If ioo cc. of i per cent, nitric acid are mixed with bread crumbs and distilled, most of the acid will be in the final 10 cc. of distillate. POISONS NOT IN THE THREE MAIN GROUPS 185 it reaches a definite concentration. At the same time a large part of the acid combines with organic substances, if any are present, forming nitro-derivatives, xanthoproteic acid, etc. Nitric acid may also cause oxidation. Consequently the dis- tillate does not contain all the acid originally present. The residue from such a distillation is usually distinctly yellow. Toward the end of distillation brown vapors of nitrogen peroxide often appear. Such a distillate, added to starch paste and po- tassium iodide, produces an immediate blue color in presence of dilute sulphuric acid. To detect nitric acid in the distillate, employ the following tests: 2. G. Fleury's Procedure. 1 Extract the finely divided material with absolute alcohol, filter and add slaked lime in excess to the filtrate. To decompose any nitric acid ester present, let the mixture stand 12 hours, filter and evaporate the filtrate to dryness. Dissolve the residue in 95 per cent, alcohol, expel alcohol from the filtered solution and finally test an aqueous solution of the residue for nitric acid. Fleury has obtained by means of this method about 20 per cent, of the nitric acid from animal material. This procedure converts the acid into its calcium salt which is soluble in alcohol. But sodium nitrate is also quite soluble in 95 per cent, alcohol (i : 50). Therefore, if the final residue gives a faint test for nitric acid, the proof of free acid in the original material is not conclusive. The following method obviates this difficulty. 3. Baumert's Procedure. 2 Neutralize the material itself, or its aqueous extract, with milk of lime, dry and extract with alcohol. Or, after neutralization with milk of lime or calcium carbonate, evaporate to a syrup, stir and mix with alcohol. Distil the filtered alcoholic extract obtained in either way, dissolve the residue in water, filter and evaporate the solution. Dissolve the residue again in alcohol and allow this solution to stand for several hours in a closed flask with about the 1 Annales de Chimie analytique appliqu^e 6, 12. 2 Lehrbuch der gerichtlichen Chemie, second edition (1907). 186 DETECTION OF POISONS same volume of ether. Filter this alcohol-ether solution, evapo- rate the solvent and dissolve the residue in a little water. Apply the following nitric acid tests to this solution: (a) Diphenylamine and Sulphuric Acid Test Blue color. Add a few drops of diphenylamine sulphate solution 1 to the aqueous extract, or distillate, and carefully pour this mixture upon pure concentrated sulphuric acid free from nitric acid. If nitric acid is present, a blue zone appears where the two liquids meet. (b) Brucine and Sulphuric Acid Test. Red color. Mix the liquid to be tested with the same volume of brucine sulphate solution 2 and carefully pour this mixture upon pure concentrated sulphuric acid. If nitric acid is present, a red zone appears where the two liquids meet. (c) Ferrous Sulphate and Sulphuric Acid Test. Saturate the liquid to be tested with pure ferrous sulphate and carefully pour this solution upon pure concentrated sulphuric acid. If nitric acid is present, a black zone appears where the two liquids meet. (d) Copper Test. Place a small piece of clean copper (wire or sheet) in nitric acid and heat. Red-brown vapors of nitrogen peroxide (NO 2 ) appear. Sulphuric Acid Nearly all animal and vegetable substances normally contain sulphates. Con- sequently an examination for free sulphuric acid must exclude its salts. There is no need of examining cadaveric material for the free acid, unless marked corro- sion and discoloration of lips, mouth, oesophagus and stomach indicate its pres- ence. There are eschars upon the lips and the mucous lining of the mouth is grayish white. The white coating on the back of the tongue may have been dissolved exposing the firm, brownish muscular tissue beneath. The tongue often looks as if it had been boiled. The mucous lining of the oesophagus is 1 Prepare this solution by dissolving i gram of diphenylamine, in 5 grams of dilute sulphuric acid and 100 cc. of water. 2 Prepare this solution by dissolving i gram of brucine in 5 grams of dilute sulphuric acid and 100 cc. of water. The sulphuric acid used must give none of the tests for nitric acid. If it does not meet this requirement, heat in a platinum dish to expel interfering nitrous substances. Or distil the acid from a small retort, rejecting the first part of the distillate. POISONS NOT IN THE THREE MAIN GROUPS 187 much wrinkled and coated gray. Externally the stomach is usually brown or slate-gray and its contents black. Frequently in sulphuric acid poisoning there is perforation of the stomach wall and brownish black masses find their way into the abdominal cavity. There may be black spots in the stomach, due according to R. "Kobert (Intoxikationen) not to charring, as previously supposed, but to brown-black haematin. Acids decompose the blood-pigment oxyhaemoglobin mainly into haematin and protein (globulin). Methaemoglobin and hsematopor- phyrin may also be formed. Acids produce the latter from haematin and in the change there is loss of iron. All three of these decomposition products of the red blood-pigment, namely, methaemoglobin, haematin and haematoporphyrin may be formed successively and then appear in the urine. The blood in the stomach wall,s is often acid and then contains chiefly methaemoglobin and haema- tin. The mucosa of the intestines even far down may be grayish white and strongly acid. Detection of Sulphuric Acid 1. Extract the finely divided material, if strongly acid, with cold absolute alcohol and after some time filter. The solution contains sulphuric acid but not sulphates. Evaporate the alcoholic filtrate upon the water-bath, or, if the volume is large, distil the alcohol. Dissolve the residue in a little water (10 cc.) and heat the solution to boiling to saponify 1 ethyl sulphuric acid. Filter and test the filtrate with barium chloride or lead acetate solution. To prove that the precipitate is a sulphate, mix with sodium carbonate and fuse upon charcoal. The sodium sulphide formed blackens metallic silver in presence of water, or gives hydrogen sulphide with acids. 2. Extract the finely divided material with water and apply the following tests to the filtrate: (a) Sugar Test. Evaporate some of the filtered extract in a porcelain dish with a small particle of sugar. Free sulphuric acid produces a black, carbonaceous residue. (b) Sulphur Dioxide Test. Concentrate the filtered extract upon the water-bath and heat in a test-tube with a few pieces of copper. Free sulphuric acid generates sulphur dioxide, recog- nized by its stifling odor. Distil the sulphur dioxide (preferably in an atmosphere of carbon dioxide) into a little water and test the distillate as follows: 1 HO.SO 2 .OC 2 H 8 + H 2 O = C 2 H 3 .OH + H 2 SO 4 . 188 DETECTION OF POISONS a. Warm some of the liquid with a little stannous chloride solution. A yellow precipitate of stannic sulphide 1 appears. |8. Add iodo-potassium iodide solution drop by drop. The color of the iodine disappears and at the same time sulphuric acid is formed: H 2 S0 3 + H 2 + I, = H 2 S0 4 + zHI. Barium chloride then precipitates barium sulphate insoluble in dilute hydrochloric acid. To estimate sulphuric acid quantitatively, either precipitate and weigh barium sulphate in the usual way, or titrate with o.i n-potassium hydroxide solution, using phenolphthalein as indicator. 1000 cc. of o.i n-potassium hydroxide solution = o.i gram- equivalent of sulphuric acid = 4.0 grams of H 2 SO4. Detection of Sulphurous Acid Sulphur dioxide acts most injuriously when inhaled. It is very irritating to the respiratory organs and also changes the blood-pigment. After death the respiratory organs are found to be profoundly altered as when acted upon by strong mineral acids. After severe poisoning by vapors containing sulphur dioxide, the blood is dirty brownish red 2 and usually gives the haematin spectrum. Human beings experience discomfort, if there are 0.015-0.02 volumes of sulphur dioxide per 1000 volumes of air. Many persons become quite ill in a few minutes, when there are 0.03 volumes of sulphur dioxide in 1000 volumes of air. The gas produces a sharp, stinging sensation in the nostrils, sneezing and coughing. In experiments upon mice, rabbits and guinea-pigs, Lehmann observed marked toxic symptoms from air containing 0.04 volume per cent, of sulphur dioxide; death ensued in 6 hours from 0.06 per cent.; and in 20 minutes from 0.08 per cent. Articles of food and drink, preserved by means of sulphurous acid or its salts, may injure the health, causing especially gastro-intestinal catarrh and other chronic derangements. For this reason it is prohibited to preserve articles of food and drink by means of sulphurous acid, sulphites and hyposulphites. If the quantity of sulphur dioxide in air is not too small, its presence may be recognized by its characteristic stifling odor. A strip of paper, moistened with 1 Sulphurous acid and sodium sulphite, added to stannous chloride solution not too strongly acid, precipitate stannous sulphite, SnSO 3 , white and readily soluble in hydrochloric acid. Warmed in presence of hydrochloric acid, sulphur dioxide acts upon a stannous salt as an oxidizing agent. A precipitate of SnOioS 2 is formed, or H 2 S is evolved and SnCl 4 formed, depending upon the amount of hydrochloric acid present. (Prescott and Johnson, Qualitative Chemical Analy- sis. Fifth edition, page 86.) 2 Neutral sulphites cause the blood to become brick-red. POISONS NOT IN THE THREE MAIN GROUPS 189 a solution of pure potassium iodate (KIO 3 ) and starch, turns blue in air contain- ing sulphur dioxide owing to the formation of a compound of iodine and starch. This reaction serves as a preliminary test for the detection of sulphurous acid and hyposulphites in chopped meat, sausage meat and other meat products. Shake the meat in an Erlenmeyer flask with phosphoric acid, suspend in the neck of the flask from the stopper (see Fig. i, page 3) a paper strip prepared as described and heat the flask upon the water-bath. The paper should not turn blue. Explanation. Sulphur dioxide reduces potassium iodate (a) . Sulphuric acid thus formed liberates hydriodic and iodic acids from their salts (/3 and y). The iodine set free by the interaction of these two acids (5) finally turns the starch blue. (a) KI0 3 + 3 H 2 S0 3 = KI + 3 H 2 S0 4 , (0) 2KI + H 2 S0 4 = 2HI + K 2 S0 4 , (7) 2KI0 3 + H 2 SO 4 = 2HI0 3 + K 2 SO 4 , (6) HIO 3 + 5HI = 312 + 3H 2 O. The official directions 1 for the detection and quantitative estimation of sulphur dioxide in meat are as follows. Mix 30 grams of finely chopped meat with 200 cc. of boiled water in a 500 cc. distilling flask. 2 Add sodium carbonate solution until the reaction is faintly alkaline. Let the mixture stand for an hour and then completely expel air from the apparatus by passing carbon dioxide through the tube extending to the bottom of the flask. Then introduce into the Peligot tube (see below) 50 cc. of iodine solution (5 grams of pure iodine and 7.5 grams of potassium iodide in a liter of water). Raise the stopper of the distilling flask and, without stopping the flow of carbon dioxide, add 10 cc. of 10 per cent, phosphoric acid solution. Then carefully heat the contents of the flask and distil half the liquid, maintaining all the while a current of carbon dioxide. Transfer the contents of the Peligot tube, which should be brown, to a beaker, rinsing it out with water to prevent loss of solution. Add a little hydrochloric acid, heat and by means of barium chloride solution completely precipitate the sulphuric acid formed from the oxida- tion of sulphurous acid by iodine. H 2 S0 3 + H 2 O + I, = H 2 SO 4 + 2HI. If this test is positive, then the meat examined contains either free sulphurous acid, sulphites or hyposulphites. In the quantitative estimation the barium sul- phate should be weighed in the usual manner. OXALIC ACID Oxalic acid and it s salts, for example, salt of sorrel, are quite toxic substances. Administration of oxalic acid has terminated fatally in the case of adults in a few 1 Measures for putting into effect the law of the German Empire of June 3, 1900, relating to the inspection of beef-cattle and meats. 2 The apparatus prescribed for official examinations is a distilling flask, having a capacity of 400-500 cc. and provided with a two-hole stopper for two glass tubes entering the flask. One tube extends to the bottom of the flask and the other only into the neck. The latter is connected with a Liebig condenser to which a Peligot tube is fastened at the other end by a tight stopper. 190 DETECTION OF POISONS minutes. Oxalic acid is very abundant in the vegetable kingdom in the form of its acid potassium salt, KHC2O4, and calcium salt. Sorrel, wood-sorrel and rhubarb are especially rich in salts of oxalic acid. Hence this acid may find access to the body through food and drugs of vegetable origin. Moreover, oxalic acid is a normal constituent in small quantity of human urine, 2-6 milli- grams being excreted in the course of a day. Consequently in examining animal material it is often necessary to supplement a positive qualitative test by a quantitative estimation of oxalic acid. Toxic Action. An important difference between mineral acids and oxalic acid is the toxicity of salts of the latter. Not only do free oxalic acid and its acid potassium salt, salt of sorrel, show poisonous properties but even very dilute solutions of neutral sodium oxalate, Na2C2C>4, act in the same way. Therefore in oxalic acid poisoning it is necessary to distinguish between local corrosion, occurring at the point of application and also in part upon elimination, and re- mote action due to absorption. Local action at the point of application is cor- rosive like that of all acids. Local action at the place of elimination depends upon the formation and insolubility of calcium oxalate. On account of the ease wjth which the organism takes up oxalic acid and its alkali salts, the action or the absorbed poison is rapid. The effects caused by its presence may be attributed 'to the fact that this acid removes in part from organs, as the heart, and from body fluids (blood) the calcium they' require for their life processes, converting it in part into insoluble calcium oxalate. Oxalates diminish the coagulating power as well as the alkalinity of blood. On the other hand they increase the quantity of sugar in the blood. In oxalic acid poisoning there is a depression of the entire metabolism. This is also the case as regards taking up oxygen and giving off carbon dioxide. The body temperature falls as the processes of metabo- lism are retarded. Owing to withdrawal of calcium from the heart, that organ is weakened and finally paralyzed. Local action upon the kidneys is due to clogging of the injured urinary tubules by deposits of calcium oxalate. The flow of urine may wholly cease in consequence of total impairment of the urinary tubules and death may ensue from anuria and uraemia. Fatal poisonings from large doses of oxalic acid are usually of short duration. R. Robert (Intoxika- tionen) describes a case where death occurred within 10 minutes. Bischoff 1 has made statements with regard to the distribution of oxalic acid in the different organs of persons poisoned by this substance. In a case, which ter- minated fatally in less than 15 minutes, the quantity of oxalic acid in each organ was determined separately and found to be: Weight Organ Oxalic Acid 2240 grams Stomach, oesophagus, intestine and contents 2.28 grams. 7 70 grams Liver 0.285 grams. 290 grams Kidneys 0.0145 grams. 180 grams Blood from the heart 0.0435 grams. 40 grams Urine 0.0076 grams. 1 Berichte der Deutschen chemischen Gesellschaft, 16, 1350 (1883). POISONS NOT IN THE THREE MAIN GROUPS 191 The quantity of oxalic acid in the liver is noticeably large. The kidneys and Urine contain only a little of the poison, owing to the short duration of life after poisoning. A striking thing about the urine excreted during oxalic acid poisoning is the abundant deposition of crystallized calcium oxalate. Detection of Oxalic Acid To detect oxalate without discriminating between the free acid, acid potassium salt or calcium oxalate, employ the follow- ing method : Add to the finely divided material 3-4 volumes of alcohol and acidify with dilute hydrochloric acid. Stir frequently and let the mixture digest 1-2 hours cold. Then filter through a plaited paper moistened with alcohol and wash the residue with alcohol. To prevent formation of ethyl oxalate during evapo- ration, add about 20 cc. of water to the total filtrate. Evapo- rate upon the water-bath until all alcohol is expelled. Pass the aqueous residue through a small filter. Extract the filtrate in a separating funnel 3-4 times with 5060 cc. portions of ether. Let the total ether extract stand for some time in a dry flask, then pass through a dry filter and distil. Dis- solve the residue in 2-3 cc. of water and pass the solution, if necessary, through a moist filter. Add am- monium hydroxide solution until alkaline and then saturated calcium sulphate solution. If there is a pre- cipitate, acidify with acetic acid and 9 FIG. 17. Calcium Oxalate let solution and precipitate stand Crystals, over night in a covered beaker. If there is still a crystalline precipitate, it can be only calcium oxalate. A microscopic examination of this precipitate is advisable. Calcium oxalate forms characteristic octahedrons having the so-called envelope-shape (Fig. 17). When thor- oughly washed, calcium oxalate may be converted by ignition into calcium oxide which may be weighed. CaO :H 2 C 2 O 4 .2H 2 O = Weight of CaO :x (56) (126) found 192 DETECTION OF POISONS Calculation. Since the quotient 56 : 126 = 0.444, multiply the weight of calcium oxide found by 0.444 to get the corresponding amount of crystallized oxalic acid. FREE ALKALIES Potassium, Sodium and Ammonium Hydroxides Free Alkalies. The same general principles used in detecting mineral acids are applicable also to the alkalies. Since potassium and sodium compounds are normal constituents of animal and vegetable organisms, and since ammonia is a decomposition product of nitrogenous organic matter, the examination must always show that the alkalies are in the free state, for they alone and their car- bonic acid salts decompose and corrode animal tissues and not their neutral salts. Poisonings due to caustic alkalies resemble those caused by corrosive acids. If taken internally, their corrosive action gives rise to pain in the mouth, throat, oesophagus, stomach and abdomen. Mineral acid corrosions are dry and brittle, whereas those from caustic alkalies are soft and greasy. The alkali albuminates formed become gelatinous, swell and may partly dissolve in presence of much water. The destructive action of the caustic alkalies extends deep and affects the parts around the corroded places. In caustic alkaline solutions gelatinous tissues, horny substances, hair and skin swell considerably and finally dissolve The stomach in alkali poisoning is softened in places, corroded and decidedly bright red in color. Detection of Alkalies Ammonia Free ammonia is usually recognized by its odor. A piece of moist red litmus paper, held over the material, becomes blue. A paper moistened with mercurous nitrate solution is blackened. Distillation. If the material is strongly alkaline, extract several times with absolute alcohol. Use a flask with a glass stopper and distil the combined extracts. Collect the distillate in a little dilute hydrochloric acid and evaporate the solution to dryness in a porcelain dish upon the water-bath. Dissolve the residue in water and test the solution for ammonia, using Nessler's reagent and chloroplatinic acid. Fixed Alkalies The residue from the above distillation may contain potas- sium and sodium hydroxides. If the residue is strongly alka- line, first add a few drops of phenolphthalein and then excess of barium chloride solution. The red color and the alkaline reac- POISONS NOT IN THE THREE MAIN GROUPS 193 tion, if due to carbonates, disappear, because two neutral salts are formed : K 2 CO 3 + BaCl 2 = BaCO 3 + 2KC1. But if alkaline hydroxides are present, the alkaline reaction and red color remain, for soluble barium hydroxide is formed: 2 KOH + BaCl 2 = Ba(OH) 2 + aKCL And the solution of this compound reddens phenolphthalein. To distinguish potassium from sodium hydroxide, neutralize the residue from distillation with dilute hydrochloric acid and test for potassium and sodium as follows: 1. Add solution of chloroplatinic acid (H 2 PtCl 6 ) which causes the precipitation of potassium in the form of the double chloride of potassium and platinum (potassium chloroplatinate, K 2 PtCl 6 ). 2. Add de Konink's reagent 1 which is a solution of sodium cobaltic nitrite, 6NaNO 2 .Co 2 (NO 2 )6. This reagent produces a yellow precipitate of potassium cobaltic nitrite, 6KNO 2 . Co 2 (NO 2 ) 6 + xH 2 O, in a solution containing a potassium salt. To hasten the reaction, add a few drops of acetic acid. 3. Test for sodium in a neutral solution by adding a few drops of freshly prepared acid potassium pyro-antimonate solution, K 2 H 2 Sb 2 O 7 . At first the solution is turbid but, if stirred, de- posits a white crystalline precipitate of sodium pyro-antimon- ate, Na 2 H 2 Sb 2 O 7 . Vitali's procedure in testing for caustic alkalies consists in shaking the alcoholic extract of the material, prepared as far as possible with exclusion of air (see above), with freshly precipi- tated and well-washed mercurous chloride. Free alkali black- ens this compound. The solubility of mercurous oxide (Hg 2 O), the black compound formed, in dilute nitric acid distinguishes it from mercuric sulphide. Quantitative Estimation of Hydroxides and Carbonates of Alkalies. To determine both the free caustic alkali and that 1 Prepare sodium cobaltic nitrite by dissolving 10 grams of pure sodium nitrite and 4 grams of cobaltous nitrate separately in sufficient water. Mix the solutions, add 2 cc. of acetic acid and dilute to 100 cc. with water. 194 DETECTION OF POISONS converted into carbonate, first determine total alkalinity by titrating a portion of the distillation residue with normal or o.i n-hydrochloric acid, using methyl orange as indicator. Then precipitate carbonate in a second portion of the distilla- tion residue with barium chloride solution and determine free caustic alkali in the nitrate. If the examination shows only alkaline carbonate, this dpes not exclude the possibility of caus- tic alkali having been originally present. POTASSIUM CHLORATE Toxic Action. Large doses (4-10 grams) of potassium chlorate, KCIOs, are decidedly toxic. During the first stage of intoxication, alteration in the shape of the red corpuscles and conversion of oxyhaemoglobin in the intact corpuscles into brown methaemoglobin take place. Then the red blood- corpuscles, at least in a case of severe poisoning, change their form, becoming shriveled and undergoing decomposition. Toxicologists (see R. Kobert, Intoxikationen) ascribe change of blood pigment and red blood- corpuscles to specific salt action possessed in high degree by potassium chlorate. This explanation also accounts for salt diuresis, 1 appearing at the beginning of potassium chlorate poisoning, whereby the blood is much thickened. But most notable is the high alkalinity of the urine, resulting in decreased alkalinity of the blood plasma. In severe chlorate poisoning so much oxyhsemoglobin is changed to methaemoglobin that the amount of oxygen in the blood may drop to i per cent. As a result human beings or animals thus poisoned may become asphyxiated from lack of oxygen. Potassium chlorate through the action of potassium weakens the heart. In chlorate poisoning the blood has a characteristic chocolate-brown color (see above) . Potassium chlorate taken by the mouth is quite rapidly eliminated by the kidneys. After administration of o.i gram of potassium chlorate, chloric acid appears in the urine in an hour. Most of the potassium chlorate passes into the urine unchanged, only a little of the salt being reduced to potassium chloride. During chlorate poisoning, the urine is usually very dark, even black, and may contain haemoglobin and methaemoglobin. It is frequently opaque and strongly alkaline. Upon long standing a dark brown sediment gradually deposits. The urine also contains considerable albumin. In suspected chlorate poisoning, the urine should if possible receive a thorough chemical and microscopical examination. An anuria lasting several days may precede death and render an examination of the urine quite impossible. Detection of Chloric Acid To isolate potassium chlorate from organic material, use a dialyzer which should be as flat as possible, because the thinner the layer in the inner container and the larger the volume of 1 Diuresis = increased secretion of urine. POISONS NOT IN THE THREE MAIN GROUPS 195 water in the outer vessel, the more rapid the diffusion. Place the material to be examined, as parts of organs and stomach or intestinal contents, in the inner container of a flat dialyzer and pure water in the outer vessel. Allow dialysis to take place 5-6 hours without changing the water in the outer vessel. Then evaporate the dialysate (contents of the outer vessel) to dryness in a porcelain dish upon the water-bath. Dissolve the residue in a little water and examine the filtered solution for chloric acid as follows : 1. Indigo Test. Add dilute sulphuric acid and a few drops of indigo solution, until there is a distinct blue color. Then introduce sulphurous acid drop by drop. If chloric acid is present, the blue color changes to yellow or greenish yellow. This is a delicate test for chloric acid, given even by o.oi gram of potassium chlorate. 2. Silver Nitrate Test. Add silver nitrate solution in excess. If there is a precipitate (AgCl), filter and add a few drops of sulphurous acid to the clear filtrate. A chlorate will cause the precipitation of more silver chloride. Silver chloride differs from silver sulphite in being insoluble in hot dilute nitric acid. Sulphurous acid reduces silver chlorate to chloride: AgClOs + 3 H 2 S0 3 = AgCl + 3 H 2 S0 4 . 3. Free Chlorine Test. A solution containing a chlorate, heated with concentrated hydrochloric acid, gives free chlorine. The gas passed into potassium iodide solution liberates iodine. Shake the solution with chloroform which dissolves iodine with a violet color. This test indicates chloric acid only in the ab- sence of substances like chromic acid and dichromates which also give chlorine with hydrochloric acid. If the material is a - powder, dissolve in water and filter if necessary. A direct test for chloric acid is usually possible with such a solution. Quantitative Estimation of Chloric Acid To estimate potassium chlorate quantitatively in urine, dialy- sates and other liquids, reduce with zinc dust, or employ Scholtz's method. 196 DETECTION OF POISONS 1. Zinc Dust Method. Divide the solution into two equal parts. Determine chloride gravimetrically in one portion by precipitating and weighing AgCl, or volumetrically by titrating according to Volhard's method. Determine chloride and chlorate together in the second portion. Add 5-10 grams of zinc dust and a little dilute sul- phuric or acetic acid, and heat the mixture 0.5-1 hour upon a boiling water-bath. Filter and wash the residue with boiling water. Acidify the nitrate with nitric acid and precipitate chloride with silver nitrate. More chlorine appears in the sec- ond than in the first determination. Calculate the percentage of potassium chlorate from the difference between the two chlorine determinations. One molecule of KClOs upon re- duction yields i molecule of KC1 and therefore i atom of chlorine. Zinc dust in presence of sulphuric or acetic acid reduces potassium chlorate to chloride: () KC10 3 + 3Zn = KC1 + 3 ZnO, (0) ZnO + 2 CH S .COOH = H 2 O + Zn(CH 3 .COO) 2 . 2. Method of M. Scholtz. 1 This method makes use of the reducing action of nitrous acid upon chloric acid : HC10 3 + 3HN0 2 = HC1 + 3HNO 3 . Add to the solution 10 cc. of nitric acid (sp. gr. 1.2 = 32 per cent.) and 10 cc. of 10 per cent, sodium nitrite solution. Let the mixture stand for 15 minutes at room temperature. Then add 30-50 cc. of o.i n-silver nitrate solution and 5 cc. of satu- rated iron alum solution, (I^N^SO^FesCSOOs^HsO. Ti- trate excess of silver with o.i n-ammonium sulphocyanate solution. 1000 cc. of o.i n-AgNO 3 = o.i KC1O 3 gram = 12.245 grams of KC1O 3 . The slight excess of nitrous acid has no effect upon the deli- cacy of the reaction. Liquids like dialysates of stomach con- tents and organs always contain chloride. In that case first 1 Archiv der Pharmazie 243, 353 (1905). POISONS NOT IN THE THREE MAIN GROUPS 197 determine the amount of chloride in another portion by Vol- hard's method. H. Hildebrandt 1 has adapted Scholtz's method to the ex- amination of urine. First completely precipitate chloride in a measured volume of urine with silver nitrate in presence of nitric acid. Add sodium nitrite solution to the clear, chloride- free filtrate, as well as more silver nitrate solution, until there is no longer a precipitate. Determine as usual the weight of silver chloride obtained. In the case of urine a larger quantity of nitrous acid is decomposed by the urea : CO(NH,), + 2 HN0 2 = C0 2 + 2N 2 + 2H 2 O. Consequently do not use too little sodium nitrite. Behavior of Potassium Chlorate in Putrefaction C. Bischoff states that potassium chlorate, mixed with moist, organic substances, especially blood, is very soon reduced to chloride! Bischoff describes several cases, in which poisoning by potassium chlorate had undoubtedly occurred, and yet chloric acid could not be detected chemically in parts of the cadaver. In an experiment, too grams of blood, 0.5 gram of potassium chlorate and 100 grams of water were allowed to stand for 5 days at room temperatures. Not a trace of chloric acid could be detected in the dialysate. Bischoff concludes from this experiment that potassium chlorate, mixed with moist organic substances, especially with blood, is soon reduced. Conse- quently, chloric acid may not be detected, even in cases of rapidly fatal poisoning by potassium chlorate. Detection of Chlorate in Meat The German law of June 3, 1900, relating to the inspection of beef-cattle and meat, forbids the use of chlorates in preserving meat, sausage and fat. The official directions prescribed for the chemical examination of meat and fats are as follows: Let 30 grams of finely divided meat stand i hour in the cold with 100 cc. of water and then heat to boiling. Filter when cold and add silver nitrate solution 1 Vierteljahrsschrift fur gerichtliche Medizin 32, 8 1 (1906). 198 DETECTION OF POISONS in excess to the filtrate. Add 2 cc. of 10 per cent, sodium sulphite solution and 2 cc. df concentrated nitric acid to 50 cc. of the clear filtrate from the silver pre- cipitate and then heat to boiling. If there is a precipitate, insoluble in more hot water and consisting of silver chloride, chlorate is present. SANTONIN, SULPHONAL AND TRIONAL These substances do not find a place in the Stas-Otto process on account of their behavior toward cold tartaric acid solution and ether. Use the following method for their detection. Extract the material, neutral or faintly acid with tartaric acid, under a reflux condenser with boiling absolute alcohol. Filter hot and evaporate the filtrate to dryness upon the water- bath. Dissolve the residue in hot water. If the solution is colored, digest for some time upon the water-bath with bone- black and stir frequently. Filter the hot solution. All of the above substances, if present in considerable quantity, crystallize in part as the solution cools. -Extract the filtrate and any crystals thoroughly with chloroform several times. Pass the chloroform extract through a dry filter. The residue from chloroform may contain santonin, sulphonal and trional, as well as acetanilide and phenacetine. The chloroform residue may also contain those substances extracted in the Stas-Otto process from the acid solution by ether. Chloroform completely extracts substances like anti- pyrine, caffeine, acetanilide, phenacetine and salicylic acid. As a rule they are purer from this solvent than from ether. The chloroform residue may also contain the weak base narco- tine. SANTONIN Santonin, CuH 18 Oj, crystallizes in colorless, inodorous, shining leaflets which are bitter and melt at 170. Santonin dissolves in 5000 parts of cold and 250 parts of boiling water; in 44 parts of ethyl alcohol; and in 4 parts of chloroform. All these solutions are neutral. It is slightly soluble in ether (1:150). Light turns these crystals yellow. Upon evaporation, an alcoholic solution of the yellow modification deposits white santonin. Constitution. Santonin is the internal anhydride (lactone) of santonic acid, CuH 2 oO 4 . Caustic alkalies, as well as calcium and barium hydroxides, dissolve santonin forming salts of this acid. In this case, as with all lactones, the lactone ring is broken as follows : POISONS NOT IN THE THREE MAIN GROUPS 199 CH 3 CH 3 c c 2 c c 2 H 2 C C CH O\ H. HzC c CH OH >CO + OK = | | | OC C CH CH/ OC C CH CH COOK \/ V I \/ \/ I C C CH 3 C C CH 3 H 2 | H 2 CH 3 CH 3 Santonin Potassium santonate A solution of a santonate, acidified with hydrochloric acid, first gives free santonic acid. To isolate this compound from the mixture, extract at once with ether. Otherwise, the acid loses i molecule of water upon standing and passes into its internal anhydride, santonin. Santonin is also a ketone. As such it forms a hydrazone, CisHi 8 O 2 = N- NH.CsHs, with phenylhydrazine and an oxime, CuHisOz = NOH, with hy- droxylamine. According to the structural formula above, santonin is a derivative of hexa- hydro-dimethyl-naphthalene. Fused with potassium hydroxide, santonic acid gives hydrogen, propionic acid and a naphthalene derivative, namely, dimethyl-/?-naphthol. Behavior in the Organism. Santonin seems to be incompletely absorbed in the body. M. Jaffe 1 has administered quite large quantities of santonin to dogs and rabbits. He obtained a new substance, called a-oxysantonin (CnHuO<), from the urine of the dog, amounting to 5 or 6 per cent, of the santonin administered. He extracted with chloroform considerable quantities of unaltered santonin from the faeces of the dog. Rabbits can usually tolerate being fed with santonin for weeks, and a-oxysantonin is formed only in very small quantity. In the ether extract of the rabbit's urine, Jaffe found a second santonin derivative, /3-oxy- santonin, isomeric with a-oxysantonin, with considerable unaltered santonin. In these experiments only about half the santonin administered was absorbed by the rabbit. After administration of santonin, a red pigment called santonin red appears in human urine. Even after medicinal doses santonin urine is red, or becomes at least scarlet-red to purple on addition of potassium or sodium hydroxide solution. Urine containing santonin also becomes carmine-red on addition of calcium hy- droxide solution. Detection of Santonin Ether, benzene, or better chloroform, extract santonin only from acid solutions. The organic solvent fails to remove this compound from an alkaline solution, as it is then in the form of a santonate. Santonin is not an alkaloid and forms no pre- x Zeitschrift fur physiologische Chemie 22, 537 (1896-1897). 200 DETECTION OF POISONS cipitates with the general alkaloidal reagents, but it gives several more or less characteristic color reactions. 1. Alcoholic Potassium Hydroxide Test. Pure santonin, heated with an alcoholic solution of potassium hydroxide, gives a fine carmine-red color, which gradually changes to reddish yellow and finally fades entirely. In this test yellow santonin dissolves at once with a yellowish red color. 2. Sulphuric Acid-Ferric Chloride Test. Heat santonin with concentrated sulphuric acid and add a drop of ferric chloride solution. The mixture becomes violet. Use about i cc. of sulphuric acid to o.oi gram of santonin. 3. Furfural-Sulphuric Acid Test. Mix 2-3 drops of alcoholic santonin solution with 1-2 drops of 2 per cent, alcoholic fur- fural solution and 2 cc. of pure concentrated sulphuric acid. Warm this mixture in a small porcelain dish upon the water- bath. A purple-red color appears and changes with continued heating to crimson-red, blue-violet and finally to dark blue (Thater 1 ). Only a few alkaloids and glucosides give distinct color reactions with furfural and sulphuric acid. Substances behaving similarly are veratrine, picrotoxin (violet) and piperine (green to blue-green, finally indigo-blue). The colors given by a- and /3-naphthol with furfural and sulphuric acid are also characteristic. SULPHONAL Sulphonal, C?Hi 50482, crystallizes in colorless, inodorous and tasteless prisms, melting at 125-126 and distilling with slight decomposition at 300. It is soluble in 500 parts of cold and 15 parts of boiling water; in 135 C H! > parts of ether; and in 65 parts of cold and 2 parts of CH 3 C SO 2 C 2 H 6 boilm 8 eth yl alcohol. Sulphonal is freely soluble in chloroform. Especially characteristic of this compound SO 2 .C 2 H 6 are the ease with which it crystallizes and its great stability in presence of chemical reagents. The halogens, halogen acids, alkaline hydroxides and carbonates, concentrated sulphuric and nitric acids are without action in the cold. Preparation. The condensation of ethyl mercaptan (2 molecules) with acetone (i molecule) by means of dry hydrogen chloride gas, or concentrated sulphuric acid, results in the formation of the ethyl-mercaptole of acetone. The latter com- 1 Archiv der Pharmazie 235, 410 (1897). POISONS NOT IN THE THREE MAIN GROUPS 201 pound, shaken with a saturated solution of potassium perman- ganate in presence of dilute sulphuric acid, undergoes oxidation with formation of sulphonal: 1 H 3 C X HSC 2 H 5 H 3 C\ /SC 2 H 5 + 2 H 3 C\ /SO 2 C 2 H 6 >C = O + =H 2 O+ C > C H,(X HSC 2 H 6 H 3 C/ \SC 2 H 5 + O 2 H 3 C/ \SO 2 C 2 H 5 Acetone Ethyl Ethyl-mercaptole Sulphonal mercaptan of acetone Detection of Sulphonal Ether, or better chloroform, extracts sulphonal from acid, neutral and alkaline solutions. Test the residue left upon evaporating these solutions as follows : 1. Melting-point Test Determine the melting point (125- 126) of the perfectly pure substance. Crystallization from boiling water with the use of a little bone-black easily gives a pure product. A mixture of these crystals with pure sul- phonal should also melt at 125-126. 2. Reduction Test. Sulphonal heated in a test-tube with powdered wood charcoal gives the characteristic odor of ethyl mercaptan. 3. Detection of Sulphur. (a) With Sodium. Fusion of sulphonal in a dry test-tube with a little metallic sodium pro- duces sodium sulphide. Dissolve cautiously (unaltered metallic sodium!) the cold melt in a little water, filter and test the filtrate with sodium nitroprusside solution for sulphide (see page 23). (6) With Potassium Cyanide. Fuse i part of sulphonal and about 2 parts of pure potassium cyanide in a dry test- tube. Note the penetrating odor of ethyl mercaptan (C 2 H 5 .SH). Potassium sulphocyanate is also a product of the reaction. An aqueous solution of the melt, acidified with dilute hydrochloric acid, becomes deep red with 1-2 drops of ferric chloride solution. (c] With Powdered Iron. Sulphonal heated with pure pow- dered iron free from sulphur gives a garlic-like odor. Add 1 Sulphur in the sulphone group = SO 2 is most likely sexivalent, corresponding to the atomic grouping I, and not quadrivalent, as in II: VI /& IV /O i. = sf ; n. = s< | X) X O 202 DETECTION OF POISONS hydrochloric acid to the residue. Hydrogen sulphide evolved blackens lead acetate paper. Detection of Sulphonal in Urine Sulphonal is cumulative in its action. Therefore continuous administration for a long time of large doses may result in the collection of a considerable quantity of this compound in the organism. Most of the sulphonal taken ap- pears in the urine as ethyl-sulphonic acid, C2H5-SO2OH. 1 The formation of this acid causes an increase of ammonia in the urine during sulphonal intoxication, as does administration of mineral acids. Sulphonal occurs in urine in detectable quantity only following considerable doses, especially when they have been taken without interruption. Such urine is often dark red to garnet-brown from haematoporphyrin. But this decom- position product of blood pigment appears in urine only succeeding severe poisoning by sulphonal, and even then its occurrence is rare. To isolate sulphonal from urine, evaporate 1000 cc. to one-tenth its volume, and extract several times with large quantities of ether. Pass the ether extracts, after they have settled in a dry flask for several hours, through a dry filter and distil. Evaporate the residue with 20-30 cc. of 10 per cent, sodium hydroxide solution to dryness upon the water-bath. This will remove coloring matter, extracted from urine by ether, but will not affect the sulphonal. Extract sul- phonal from the alkaline residue with ether. Evaporate the solvent, and sul- phonal will remain pure and almost colorless. Determine the melting-point of this residue, and make the other tests for sulphonal. Detection of Haematoporphyrin in Urine Coloring matters have been observed in red, brownish red to cherry-red urines, which quite probably are identical with haematoporphyrin. The spectroscopic examination of such urine is made in the following manner. Add sodium hydroxide solution, drop by drop, to about 500 cc. of urine, until the reaction is strongly alkaline, and then add a little barium chloride solution. Filter after a while, and wash the precipitate well. Extract the precipitate upon the filter with hot alcohol, containing a few drops of dilute sulphuric acid. A spectroscopic examination of this filtrate can be made directly with a Brown- ing pocket spectroscope. Acid haematoporphyrin solutions are violet; when more concentrated, they have a cherry-red color, and show the characteristic spectrum with two absorption-bands (see page 306). If the acid, alcoholic solution is saturated with a few drops of ammonium or sodium hydroxide solution, the spectrum of alkaline haematoporphyrin solution with its four ab- sorption-bands appears. Traces of hsematoporphyrin very frequently appear 1 The structural formula of ethyl-sulphonic acid is: C 2 H 6 .S^OH. V> It should not be confused with ethyl-sulphuric acid: C 2 H 6 -O-S^OH. V) POISONS NOT IN THE THREE MAIN GROUPS 203 in normal urine. It has been observed more abundantly, at times, in urine during chronic sulphonal poisoning. TRIONAL Trional crystallizes in colorless, shining leaflets melting at 76. It is soluble in 320 parts of cold, but more easily soluble in hot water. It is also soluble in ethyl alcohol, ether and chloroform. The aqueous solution is \ c / neutral and bitter. In the latter respect it differs from C 2 jj 6 / \S0 2 C 2 Hj sulphonal which is tasteless. Trional gives the sulphonal reactions. Trional is completely decomposed in the organism and the danger of cumulative action is much less than in the case of sulphonal. Moreover, haematoporphyrin has almost never been observed, even following considerable doses of trional and after uninterrupted use for weeks. Active Organic Substances 1 Rarely Occurring in lexicological Analysis CANTHARIDIN Cantharidin, CioHi 2 O4, is the active vesicating principle of Spanish fly (Lytta vesicatoria) and is present to the extent of 0.8-1 per cent. Cantharidin H forms colorless, shining, neutral, rhombic leaflets, C CHs melting at 218 and subliming at higher temperature in white needles. It is almost insoluble even in boiling H 2 C I H 2 C ! C = O water. Acids, as tartaric acid, increase its solubility yO in water, though Cantharidin is not a base. It dis- I c = O solves with difficulty in cold ethyl alcohol (0.03 : 100 at \)X \ 18) and in ether (0.011:100). Chloroform (1.52:100), C CHs acetone and acetic ether are its best solvents. It is as good as insoluble in petroleum benzine. Constitution. According to Gadamer 2 Cantharidin has the structural formula shown above. Treated with potassium or sodium hydroxide, it loses its anhy- dride character and passes into solution as the alkali salt of dibasic cantharidic acid, CioHi 4 O 5 : H H C CHs C CHs ' /|\ / C C = O H 2 C C COOK H 2 C 1 >o KiOH = O H 2 C C C = 6 j H H 2 C C COOK \ / ' \ \ / ' \ C CH 3 OK CHs H H Cantharidin Potassium cantharidat H 2 0. Potassium cantharidate, CioHi 2 O 5 K 2 .2H 2 O, recently recommended for phthisis, and sodium cantharidate, Ci Hi 2 O 6 Na 2 .2H 2 O, are well crystallized 1 The toxic substances considered in this place have been arranged in alpha- betical order. 2 Chemical Abstracts 12, 806 (1918). 204 DETECTION OF POISONS salts. Mineral acid first sets cantharidic acid free from these salts. The latter soon loses a molecule of water, passing into its internal anhydride, cantharidin. H 2 C H C /\ / CH 3 ^C COOiH H 2 C C COjOH \/ C II H C / \ CH 3 H,C X \ / C C = < > H 2 C C C = MX \ C CH 3 + H 2 Cantharidic acid Cantharidin Cantharidin, heated with hydriodic acid at 100, or treated at room tem- perature with chlorosulphonic acid, Cl-SOj-OH, changes into the isomeric cantharic acid, CioHi2O4, crystallizing in colorless needles melting at 275 and having the following structure: HC H 2 C C COOH V H, Cantharic acid This acid is not a vesicant. Heated for 3 hours at 135 in sealed tube with acetyl chloride, cantharic acid yields another isomer of cantharidin which Ga- damer 1 has shown to be acetyl-hydrato-cantharic anhydride having the following structure: H C CH 3 /\ / HC C C = O H 2 C C C = O \/ \ C CH 3 /\ H CO CH 3 Acetyl-hydrato-cantharic anhydride The latter crystallizes from alcohol in large colorless leaflets melting at 76. There is a close relationship between o-xylene and cantharidin, for the latter, heated at 400 with calcium hydroxide, gives a dihydro-o-xylene, C 8 Hi 2 , called cantharene, and also o-xylene, C 6 H 4 (CH 3 ) 2 , and xylic acid. Finally, cantharidin, heated with an excess of phosphorus pentasulphide and distilled, gives pure o-xylene. (J. Piccard.) 2 1 Chemical Abstracts 12, 806 (1918). 2 Berichte der Deutschen chemischen Gesellschaft 12, 577 (1879). POISONS NOT IN THE THREE MAIN GROUPS 205 Detection of Cantharidin Evaporate a liquid, or material containing much moisture (organs, stomach or intestinal contents, etc.), to dryness upon the water-bath. Dragendorff directs repeated extraction of the finely divided material with alcohol containing sulphuric acid. Filter the extracts, add one-sixth their volume -of water and distil the alcohol. Extract the residue 2-3 times with chloro- form and shake the chloroform extracts with water to remove adherent acid. Finally separate the chloroform from water, distil and examine the residue for cantharidin. Since this com- pound gives no characteristic chemical reactions, employ the physiological test for identification. Dissolve the chloroform residue, unless fatty substances are present, in a few drops of hot almond oil. Bind a cloth, saturated with this solution, upon the upper arm or breast by means of adhesive plaster. Cantharidin reddens the skin and sometimes raises blisters. Even 0.14 mg. of cantharidin causes blistering. Salts of can- tharidic acid also have a vesicating action. To detect cantharidin in blood, brain, liver and other material rich in protein, E. Schmidt boils with dilute potassium hydrox- ide solution (i gram of KOH in 15 cc. of water), until the mass is homogeneous, acidifies with dilute sulphuric acid and extracts thoroughly with hot alcohol. The procedure in other respects is as described above. Cantharidin is said to resist putrefaction. CYTISINE Cytisine, CiiHuN 2 O, occurs in the ripe seeds of Golden chain (Cytisus Labur- num) which contain about 1.5 per cent. Cytisine and the alkaloid originally called ulexine, isolated from the seeds of Ulex europaeus, are identical (A. Partheil). Preparation. Extract powdered ripe laburnum seeds with 60 per cent, alcohol containing acetic acid. Distil the alcohol from the extracts, pour the residue through a moist filter and precipitate extractive and tannin substances with lead acetate solution. Filter, add potassium hydroxide solution to the clear nitrate and extract cytisine with chloroform. Distil the chloroform which usually deposits cytisine as a radiating crystalline mass. If purification is neces- sary, recrystallize the residue from absolute alcohol or boiling ligroin. Sub- limation in a partial vacuum also purifies crude cytisine. 206 DETECTION OF POISONS Cytisine crystallizes in large, colorless, tasteless prisms, melting at 152-153 and subliming at a higher temperature, if carefully heated. It dissolves freely in water, alcohol, chloroform and acetic ether; less easily in commercial ether, benzene and acetone; and is insoluble in petroleum ether and absolute ether. Cytisine is a strong secondary base and very toxic. Although capable of com- bining with i or 2 molecules of hydrochloric acid, this compound behaves in other respects like a monacid base. Only the salts containing one equivalent of acid crystallize well. Nitrous acid converts this secondary base into nitroso- cytisine, CnHisON-NO, which crystallizes in needles. Nitrous fumes appear, if cytisine is warmed upon the water-bath with twice the amount of concentrated nitric acid, and the solution at once becomes reddish yellow to brown. This solution poured into water gives a precipitate of nitro-nitroso-cytisine, CnHi 2 ON- (NO Z )N-NO. This compound crystallizes from water in pale yellow scales melting at 242-244. Toxic Action. Cytisine produces convulsions, its action in this respect being very similar to that of strychnine. But unlike the latter alkaloid it also irri- tates the gastro-intestinal mucosa even causing bloody inflammation. Cytisine also differs from strychnine in stimulating the vomiting center. Consequently, after doses of cytisine or laburnum preparations, human beings and animals capable of emesis thus rid the organism of a large part of the poison. Like strychnine, cytisine stimulates the respiratory and vaso-motor centers. Finally as in strychnine intoxication death results from paralysis of these two centers. A part of the cytisine leaves the organism unchanged and appears in the urine (R. Robert). Detection of Cytisine Prepare an aqueous tartaric acid solution of stomach con- tents, vomitus or parts of organs, following the general pro- cedure for alkaloids. To remove final traces of fatty acids and fat, shake this solution well with ether. Withdraw the aqueous solution, make alkaline with sodium hydroxide solution and extract thoroughly with chloroform or isobutyl alcohol. Evap- orate the chloroform or isobutyl alcohol extracts and test the residue as follows for cytisine: 1. Van der Moer's 1 Test Ferric chloride solution colors cytisine and its salts blood-red. Dilution with water, or acidi- fication, discharges this color. Hydrogen peroxide also pro- duces the same result. The solution containing hydrogen peroxide, warmed upon the water-bath, becomes intensely blue. 2. A. Ramverda's 2 Test. A little nitrobenzene, containing 1 Berichte der Deutschen pharmazeutischen Gesellschaft 5, 267 (1895). 2 Chemisches Zentral-Blatt, 1900, II, 268. POISONS NOT IN THE THREE MAIN GROUPS 207 dinitro-thiophene, poured upon cytisine gives a fairly stable, brilliant red- violet color. A similar color given by coniine is very unstable. 3. Nitro-Nitroso-Cytisine Test. Nitro-nitroso-cytisine (see above), formed by concentrated nitric acid, serves to detect small quantities of this alkaloid. Nitro-nitroso-cytisine dis- solves with difficulty in 94 per cent, alcohol and crystallizes from this solvent in microscopic prisms. Flat, tabular crystals form from 50 per cent, alcohol which is a better solvent. The solubility of nitro-nitroso-cytisine in concentrated hydrochloric acid indicates basic properties, but they are feeble, for dilution with water precipitates this compound unchanged. THE DIGITALIS GLUCOSIDES The digitalis plant (Digitalis purpurea L.) contains in all its parts, but especially in the leaves and seeds, medicinally useful substances belonging to the glucoside group. Thus far three digitalis glucosides have been isolated as well characterized, crystalline compounds of homogeneous composition. These are digitalin in a narrower sense (= Digitalinum verum crystal- lisatum Kiliani) C 3 5H 56 Oi4; digitoxin, C 34 H54Oii; and digitonin, C 55 H94O 2 8 or C 5 4H9 2 O 2 8. A fourth glucoside called digitalein seems not to have been obtained wholly pure as yet. Digitonin, CssH^C^s or C54H92O28, 1 occurs almost exclusively in digitalis seeds, the leaves containing at most only traces. Digitonin, classified at present with the saponins (see page 220), crystallizes from alcohol in fine needles soluble in 50 parts of 50 per cent, alcohol. Even very dilute hydrochloric acid hydrolyzes digitonin into digitogenin, dextrose and galactose: 2 2 H 2 O = CsiHsoOe + 2C 6 H 12 8 + 2C 6 H 12 O(?). Digitonin Digitogenin Dextrose Galactose Digitonin crystallizes from alcohol in fine needles which soften at 235 and become yellow. Digitonin is not a cardiac poison. Pure digitonin and concentrated sulphuric acid, upon addition 1 The results obtained by A. Windhaus (Berichte der Deutschen chemischen Gesellschaft 42, 238 (1909) favor the formula C^^tOzs for digitonin. 2 H. Kiliani, Berichte der Deutschen chemischen Gesellschaft 24, 340 (1891). 208 DETECTION OF POISONS of a little bromine water, give a color which becomes intensely red. Digitoxin, C 3 4H 5 4Oii, occurs almost exclusively in digitalis leaves. This very active and highly toxic compound is almost wholly insoluble in water and ether but soluble in alcohol and chloroform. Consequently ether precipitates it from chloro- form solution. Digitoxin crystallizes from 85 per cent, alcohol in leaflets melting at 145. Alcoholic hydrochloric acid hy- drolyzes it forming digitoxigenin and digitoxose : C 3 4H 5 4p u + H 2 O = C 22 H 32 04 + 2C 6 H 12 4 . Digitoxin Digitoxigenin Digitoxose Digitoxin dissolves in concentrated sulphuric acid with a brownish or greenish brown color unchanged by bromine. Kiliani's Digitoxin Test. 1 Dissolve a trace of digitoxin in 3-4 cc. of glacial acetic acid containing iron (100 cc. of glacial acetic acid and i cc. of 5 per cent, ferric sulphate solution). Cautiously add sulphuric acid containing iron (100 cc. of sul- phuric acid and i cc. of 5 per cent, ferric sulphate solution) in about the same quantity as an under layer. A dark zone ap- pears where the two solutions meet, above which after a few minutes a blue band is visible. After some time the entire acetic acid layer becomes deep indigo-blue. Digitalin, CssHseO^, according to Kiliani occurs only in digitalis seeds. It is soluble in water (i : 1000) and very active. Boiling with very dilute hydrochloric acid hydrolyzes its alco- holic solution into digitaligenin and two sugars, namely, dex- trose and digitalose : 2 CssHseOn + H 2 O = C 22 H 30 q 3 + C 6 Hi 2 O 6 + aCrHuOs Digitalin Digitaligenin Dextrose Digitalose Test for digitalin as follows : i. Concentrated sulphuric acid colors pure digitalin orange- yellow. This solution soon becomes blood-red, changing upon addition of a little bromine water to cherry and blue-red. A drop of nitric acid or ferric chloride solution will do as well as bromine water. This test after 1-2 hours is surer and more 1 Archiv der Pharmazie 234, 273-277 (1896). 2 H. Kiliani, Berichte der Deutschen chemischen Gesellschaft 31, 2454 (1898). POISONS NOT IN THE THREE MAIN GROUPS 209 permanent, if a trace of digitalin is dissolved direct in concen- trated sulphuric acid and nothing else is added. 2. Concentrated hydrochloric acid dissolves digitalin with a golden yellow color, changing with heat to garnet or violet-red. At present nothing definite is known regarding the fate of digitalis glucosides in the human organism, or the products into which they are changed or the forms in which they are eliminated. In the case of human beings elimination of the three active substances has never been observed. Moreover, R. Robert has not been able to detect anything active in the urine of animals except in isolated cases. Thus far it has not been possible to find any of the digitalis compounds mentioned above in blood or animal organs. In a toxicological analysis especial attention would have to be given to vomitus and the contents of the gastro-intestinal tract. But there is slight chance of detecting the digitalis bodies in such material. ERGOT Officinal ergot (Secale cornutum) is the sclerotium (compact mycelium or spawn) of Claviceps purpurea collected from rye shortly before the fruiting period and dried at gentle heat. Ergot is commonly known as an abortifacient and in- toxications have occurred from its use. Consequently examinations for legal pur- . poses may require its detection in powders and other mixtures. Our knowledge of the constituents of ergot is still very defective notwithstanding several ex- haustive investigations. Ergot alkaloids, as ergotine, ergotmine, cornutine, picro-sclerotine, were described long ago. But, with the possible exception of ergotinine (Tanret, C. C. Keller), the preparations were not entirely pure. Ergot contains in addition to alkaloids other peculiar chemical substances which have received but little attention. They have not the characteristic physiological action of ergot but, like the pigment sclererythrin, are useful for purposes of identi- fication. Among these substances belong sphacelic acid and sclerotic acid, ac- cording to R. Robert a very poisonous resin having acid properties. Alkaloids. The most recent 1 researches upon ergot mention as well character- ized bases ergotinine, CasHagNsOs, and hydro-ergotinine, CssH^NsOe. Barger calls the latter ergotoxine. Ergotinine crystallizes from alcohol in long needles melting at about 229 when heated rapidly. This compound dissolves in 52 parts of boiling alcohol; in i.i parts of ether; and is readily soluble in chloroform. Crystalline salts of this base have not yet been prepared. Hydro-ergotinine (= hydrate of ergotinine), obtained as a crystalline phosphate from ergotinine mother liquors by means of alcohol and phosphoric acid, is a white powder soften- ing at 155 and melting at 162-164. Though freely soluble in alcohol, it dis- solves but slightly in ether. As a rule the salts of hydro-ergotinine (ergotoxine) crystallize well. 2 By preparing a cold methyl alcohol solution of hydro-ergo- tinine and boiling this solution for several hours under a return condenser, F. 1 F. Rraft, Archiv der Pharmazie 244, 336 (1906) and G. Barger, Journal of the Chemical Society 91, 337. 2 G. Barger and F. H. Carr, Proceedings of the Chemical Society 23, 27. 14 210 DETECTION OF POISONS Kraft has converted this substance completely into ergotinine. On the other hand, ergotinine in dilute acetic acid solution passes back almost entirely into hydro-ergotinine within 10 days. As an indication of purity, a solution of hydro- ergotinine in 2 parts of cold methyl alcohol after several days standing should not deposit crystals (ergotinine) nor become green. Solutions of both alkaloids are fluorescent. According to Keller the play of colors with sulphuric acid and ferric chloride is characteristic of ergotinine (see below). Physiological Action of the Alkaloids. Ergotinine and hydro-ergotinine ac- cording to A. Jaquet produce convulsions and gangrene. They are not, however, the cause of the specific uterine contraction characteristic of ergot. Keller's coroutine according to Kraft is identical with ergotinine, according to G. Barger and H. H. Dale 1 with ergotinine, which is impure from ergotoxine (hydro- ergotinine). The English investigators believe that the physiological effects observed with ergotinine are due to adhering ergotoxine. The latter is readily formed when the difficultly soluble ergotinine is brought into solution by means of glacial acetic acid, phosphoric acid, or a little sodium hydroxide solution. Ergo- toxine according to Barger and Dale produces the effects typical of ergot, causing powerful contraction of the uterus and later abortion. Sclererythrin. This is the pigment of the outer coat of ergot. E. Schmidt gives the following directions for its isolation. Extract freshly powdered ergot with ether to remove fat. Then moisten the powder with water containing tar- taric acid, dry and extract with 95 per cent, alcohol. Filter and distil the alcohol. Extract the residue with ether. This solvent now dissolves sclererythrin which can be precipitated by means of petroleum ether. Sclererythrin is an amorphous red powder which can be sublimed. It is in- soluble in water but soluble in absolute alcohol and glacial acetic acid. This substance behaves like an acid, dissolving in caustic alkalies, ammonia, and alkaline carbonate and bicarbonate solutions with a red or red-violet color. Owing to presence of sclererythrin, ether, if shaken with powdered ergot mois- tened with tartaric acid solution, becomes red. If such an ether solution of sclererythrin is shaken with sodium hydroxide solution, the pigment dissolves in the latter which then becomes red. Solutions of this pigment show characteristic absorption- bands in the spectrum. Moreover the pigment gives blue-violet precipitates with solutions of calcium hydroxide, barium hydroxide and lead acetate. The precipitate with stannous chloride is currant-red; with copper sul- phate a pure violet; with ferric chloride a deep green; and with chlorine or bro- mine water a lemon-yellow. Detection of Ergot in Flour, Bread and Powders This examination usually consists in undertaking to detect by chemical and physical means the red pigment sclererythrin which is characteristic of ergot. The property possessed by this substance of passing from ether into a solution of an alkaline hydroxide or bicarbonate is especially valuable for purposes of identification. 1 Bio-Chemical Journals, 240. POISONS NOT IN THE THREE MAIN GROUPS 211 1. Detection of Sclererythrin. Shake frequently and let 10 grams or more of flour stand for a day in a closed flask with 20 cc. of ether and about 15 drops of dilute sulphuric acid (i : 5). Then pass the ether through a dry paper, wash the residue with a little ether and shake the filtrate thoroughly with 10-15 drops of cold saturated sodium bicarbonate solution. If the flour contains ergot, the aqueous layer separates with a violet color. R. Palm extracts the flour at 3040 with 10-15 times its volume of 40 per cent, alcohol containing a few drops of am- monia. Express the liquid, filter and add basic lead acetate solution to the filtrate. Press the precipitate between filter paper and warm while still moist with a little cold saturated borax solution. A red-violet color appears, if the flour contains ergot. 2. Spectroscopic Examination. This test gives a positive result, if the material (powdered ergot, flour, bread) contains more than o.i per cent, of ergot. Examine spectroscopically the alkaline and acid solution of the pigment. The red solu- tion, prepared in Test i by means of ether containing sulphuric acid, shows two absorption-bands. One lies in the green be- tween ,E and F but nearer E and a second broader band in the blue midway between F and G. Then render the solution alkaline with ammonia. Three absorption-bands should appear. The first lies between D and E, the second at E somewhat to the right and the third to the left of F. 3. Choline. Ergot powder, warmed with dilute potassium hydroxide solution, gives the characteristic odor of trimeth- ylamine, (CH 3 ) 3 N, 1 due to decomposition of choline in ergot. /CH 2 .CH 2 .OH (CH 3 ) 3 N< \OH Occasionally flour that does not contain ergot may give an odor when heated with potassium hydroxide solution. 4. Detection and Quantitative Estimation of Ergotinine (C. C. Keller). Dry finely powdered ergot over lime, place 25 grams 1 The so-called corn smut (Ustilago Maidis), said to cause effects similar to those of ergot, also gives the trimethylamine odor when warmed with potassium hydroxide solution, for it contains appreciable quantities of choline. 212 DETECTION OF POISONS in a Soxhlet tube and completely extract fat with petroleum ether. Dry the powder at gentle heat, add 100 grams of ether and after 10 minutes shake well with milk of magnesia (i gram of MgO and 20 cc. of water) . Shake repeatedly during an hour and then pass 80 grams of the ether solution (=20 grams of ergot) through a covered folded filter into a separating funnel. Shake the ether in succession with 25, 15, 10 and 5 cc. of 0.5 per cent, hydrochloric acid. Pour the hydrochloric acid ex- tracts, now containing the ergot alkaloids, through a small moistened filter. l Add ammonia until alkaline and extract the solution twice with about half its volume of ether. Let the ether extract settle in a dry flask, then filter into a dry weighed flask and wash the filter with a little ether. Distil the ether and dry flask and residue at 100 to constant weight. Good Ger- man ergot contains 0.13-0.16 per cent, and Russian ergot 0.22- 0.25 per cent, of the alkaloid. To detect ergot alkaloid qualitatively, proceed as follows: (a) Dissolve a part of the residue in i cc. of concentrated sulphuric acid and add a trace of ferric chloride solution. The mixture is orange-red and becomes at once deep red but the margin appears bluish to bluish green. (b) Dissolve a part of the residue in about 4 cc. of glacial acetic acid and add a trace of ferric chloride solution. Cau- tiously add this mixture to concentrated sulphuric acid as an upper layer. If ergotinine is present, a brilliant violet color appears where the two liquids meet. OPIUM Detection of Meconic Acid and Meconine Since it is comparatively easy to procure small quantities of opium preparations, especially the tincture, poisoning from this source is possible. Consequently, it is often desirable to recognize the presence of opium itself. Detection of the alkaloids narcotine and morphine, always present in opium in 1 Clarify the filtrate from these hydrochloric acid extracts, if not clear, by agitation with a little talcum powder, previously treated with hydrochloric acid and thoroughly washed with water. Then filter again. POISONS NOT IN THE THREE MAIN GROUPS 213 considerable quantity, affords partial evidence of the presence of this substance. Moreover, opium always contains two non- basic substances, meconic acid and meconine. Detection of these two compounds in conjunction with narcotine and morphine definitely determines the presence of opium. Meconic Acid, C 7 H 4 O 7 = C 5 HO 2 (OH)(COOH) 2 , is an oxy- pyrone-dicarboxylic acid (II) and therefore a derivative of pyrone (I) : o o c c HC CH HC C OH I. || || II. HC CH HOOC C C COOH Y V Pyrone Meconic acid Meconic acid crystallizes in plates or prisms with 3 mole- cules of water. It is easily soluble in hot water and alcohol. A solution of a ferric salt turns a meconic acid solution dark r,ed. To detect meconic acid, extract a portion of the material with alcohol containing a few drops of hydrochloric acid. Filter and evaporate the filtrate upon the water-bath. Dissolve the , residue in a little water and heat the filtered solution to boil- ing with excess of calcined magnesium oxide. The solution contains magnesium meconate. Filter hot to remove undis- solved magnesium oxide, evaporate the filrtate to a small volume and acidify faintly with dilute hydrochloric acid. Add a few drops of ferric chloride solution. A blood-red color appears, if meconic acid is present. Wanning with hydro- chloric acid does not discharge this red color, in which respect it differs from the red color caused by acetic acid. This color differs from that caused by sulphocyanic acid in not being affected upon addition of gold chloride. But stannous chloride reduces ferric to ferrous oxide and discharges the color. Nitrous acid, however, at once restores it. These tests permit the identification of meconic acid in an extract from only 0.05 gram of opium. Meconine, CioHi 4 . Opium contains only 0.05-0.08 per cent, of this compound. It forms small prisms, melting at 102 214 DETECTION OF POISONS and subliming at higher temperature without decomposition. Meconine dissolves freely in alcohol, ether, benzene and chloro- form, but less easily in water. Alkalies convert meconine into easily soluble salts of meconinic acid, Ci Hi2O 5 . This monobasic acid cannot exist free but changes to meconine when liberated from its salts by a mineral acid. Meconine, formed by abstracting a molecule of water from meconinic acid, is therefore the internal anhydride (lactone) of meconinic acid: CHr- 0;H CH 2 O i ! i HC C CO iOH HC C CO HC C OCH 3 HC C OCH 3 C C OCHa OCH 3 Meconinic acid Meconine To detect meconine, extract the material with alcohol con- taining sulphuric acid. Filter and evaporate the filtrate to a syrup upon the water-bath. Dissolve the residue in water and extract meconine from this acid solution with benzene. Evapo- ration of the solvent frequently gives crystals of meconine. To detect meconine, dissolve in a little concentrated sulphuric acid. The solution is green but turns red within two days. If the green sulphuric acid solution, or that which has turned red upon standing, is carefully warmed, a fine emerald-green color ap- pears, passing through blue, violet and finally back to red. Selenious-Sulphuric Acid Reagent for Opium Alkaloids 1 Prepare the reagent used in these tests by dissolving 0.5 gram of selenious acid (H 2 Se0 3 ) in 100 grams of pure concen- trated sulphuric acid. This reagent is especially delicate for opium alkaloids, detecting even traces of morphine and codeine (0.05 milligram), as well as of papaverine (o.i milligram). Selenious-sulphuric acid gives the following color reactions with the commoner opium alkaloids: 1 Mecke, Zeitschrift fiir offentliche Chemie 5, 350 (1899) and Zeitschrift fur -analytische Chemie 39, 468 (1900). POISONS NOT IN THE THREE MAIN GROUPS 215 Cold Hot Morphine Apomorphine Codeine Narceine Narcotine Blue; then permanent blue- green to olive-green. Dark blue-violet. Blue quickly changing to emerald-green and later to permanent olive-green. Faint greenish yellow; then violet. Greenish steel- blue* later Brown. Gradually dark brown. Steel-blue; then brown. Dark violet. Papaverine Thebaine cherry-red. Greenish, dark steel-blue; then deep violet. Deep orange gradually fad- ing. Intense dark violet. Dark brown. PAPAVERINE Papaverine, C2oH2iNO4, constitutes about 0.5-1 per cent, of opium. When crude it is usually mixed with narcotine. To remove the latter, prepare the acid H H C C H 3 C.O C C CH 'i i- H 3 C.O ( Y CH 2 oxalate of papaverine which dissolves with difficulty in water. Crystallize this salt from boiling water until it dissolves in concentrated sulphuric acid without color. Convert papa- verine oxalate into the hydrochloride by treat- ment with calcium chloride and then liberate the alkaloid with ammonia. This product crystallized from alcohol is pure papaverine. Papaverine crystallizes in colorless prisms melting at 147. This alkaloid is insoluble in water; soluble with difficulty in ether (i 1260), cold alcohol and benzene; but freely soluble in alcohol, acetone and chloroform. These solutions are neutral, not bitter, and optically inactive. Papaverine is a weak base which dissolves in but does not neutralize acetic acid. Ether partially extracts it from an aqueous tartaric acid solution and completely extracts it from alkaline solution. Consequently this alkaloid appears in the Stas-Otto process in ether extract B. Chloroform extracts papaverine with almost as much ease from an acid solution as from one that is alkaline. Constitution. Papaverine is a monacid, tertiary base which combines with alkyl iodides forming crystalline addition products. As it forms no acetyl deriva- HC CH HC C O. V r\c*tj UCrla O.CH 3 216 DETECTION OF POISONS tive with acetic anhydride, free hydroxyl is not present. But there are probably four methoxyl groups, for it loses four methyl groups when treated with hydriodic acid according to Zeisel's method. Consequently all the oxygen atoms in papa- verine are present as methoxyl groups. The researches of Guido Goldschmiedt, extending from 1883 to 1898, have completely explained the constitution of papaverine. Moderate oxidation with potassium permanganate and sulphuric acid gives papaveraldine, C2oHi9NOB, without breaking the carbon chain. Fu- sion with potassium hydroxide breaks the latter into nitrogen-free veratric acid and the nitrogenous base dimethoxy-isoquinoline : l 1 Isoquinoline (II) is isomeric with quinoline (I) and like the latter is a monacid, tertiary base: I. H H C C ^\ /\ HC C CH CH II. Hi i C N H Quinoline H H C C H H Isoquinoline H 3 CO C C CH H 3 CO C C N C C H I I H 3 CO C' CH I II CO + OK HC CH Hi C OCH 3 C OCH 3 Papaveraldine H 3 CO C C N Y Y H H Dimethoxy-isoquinoline COOK i H/\H HC C OCH 3 V c OCH 3 Veratric acid Detection of Papaverine ^The following general reagents precipitate papaverine in a dilution of i : 10,000: phospho-molybdic acid, potassium bis- muthous iodide and iodo-potassium iodide. The following still give precipitates in a dilution of i : 5000: tannic acid, gold chloride and potassium mercuric iodide. POISONS NOT IN THE THREE MAIN GROUPS 217 The following special tests should be made : 1. Concentrated Sulphuric Acid. The cold colorless solution of papaverine in this acid becomes dark violet upon gentle warming. But even a cold solution of impure papaverine in this acid is violet. 2. Froehde's Test. The solution of pure papaverine in this reagent is green. Blue, violet and finally a brilliant cherry-red color appear upon warming the solution. 3. L. E. Warren's 1 Test. Crush a very small crystal of potassium permanganate with a glass rod and intimately mix about 0.0005 gram of papaverine with the powder. Stir this mixture into about 0.2 cc. of Marquis' reagent. A green color, almost instantly changing to blue, appears. The latter color deepens into an intense violet-blue which after some time be- comes bluish green, green and finally a dirty brown. Of thirty-nine alkaloids tested, the only one in any way simulating papaverine was an unnamed alkaloid separated from sanguinaria. 4. Solutions of this alkaloid in concentrated nitric acid, or Erdmann's reagent, are dark red. . Heat to boiling a solution of i part of papaverine with 10 parts of nitric acid (sp. gr. i. 06 = 10 per cent. HNO 3 ). As the solution cools, yellow crystals of the nitrate of nitro-papaverine, C2oH 2 o(NO 2 )NO4.HNO3.H 2 O, appear. Yellow prisms of nitro-papaverine, C2oH2o(NO2)NO4.H2O, may be obtained from this nitrate by means of ammonia. 5. Ammonia colors the greenish solution of papaverine in chlorine water deep red-brown which becomes later almost black-brown. 6. Selenious -Sulphuric Acid Test. See page 215 for the color changes given by pure papaverine dissolved in this reagent. PILOCARPINE Pilocarpine, C n Hi6N 2 O2, occurs with isopilocarpine and probably also with pilocarpidine in the leaves of jaborandum (Pilocarpus pennatifolius 2 ). The 1 Journal of the American Chemical Society 37, 2402 (1915). 2 According to Jowett, jaborine, which has been described as another alkaloid peculiar to jaborandum leaves, is a mixture of isopilocarpine, pilocarpidine, a little pilocarpine and pigment. 218 DETECTION OF POISONS H O free base as usually obtained is semi-liquid, viscous, non- C 2 H 6 C C volatile and alkaline. It dissolves but slightly in water; No is freely soluble in alcohol, ether and chloroform; and rip Q' insoluble in benzene. Solutions of pilocarpine and its salts | HZ are dextro-rotatory. This alkaloid is a strong base neu- CH2 tralizing acids and forming salts that are usually crys- P 3 talline. Caustic alkalies, added to concentrated solutions Q -^ of pilocarpine salts, precipitate the free base which redis- II ^CH solves "* an excess f tne precipitant. Solutions of If # sodium hydroxide, or sodium ethylate (C 2 H 5 .ONa), cause a molecular rearrangement of pilocarpine. This reaction runs more smoothly, if pilocarpine hydrochloride is heated for half an hour at 200. The product of this change is isopilocarpine, CnHi6N2O2, isomeric and very likely stereo-isomeric with pilocarpine. Both isomeric pilocarpines differ in melting-points, solubilities and particularly in specific rotation. Isopilocarpine is less dextro-rotatory than pilocarpine and crystallizes in deliquescent prisms easily soluble in water and alcohol. The salts of the two bases also show similar differences: Pilocarpine nitrate, CiiHi 6 N 2 O2.HNO3; mpt. 178; []D = + 82.90. Isopilocarpine nitrate, CnHi 6 N 2 O 2 .HNOa; mpt. 159; [a]D = + 35-68. Jowett 1 has succeeded in converting isopilocarpine into pilocarpine by means of the same reagent used in converting pilocarpine into isopilocarpine. Pure isopilocarpine, heated with pure alcoholic potassium hydroxide, gives a mixture of unaltered isopilocarpine and pilocarpine. The identity of the latter with pure pilocarpine was established by preparing the hydrochloride and nitrate (mpt. 178). This reciprocal conversion of one alkaloid into the other strongly supports the idea of the stereo-isomerism of pilocarpine and isopilocarpine. Pinner was the first to show that the two nitrogen atoms of the two isomeric bases belong to a glyoxaline ring. 2 In 1905 Jowett proposed for pilocarpine and isopilocarpine the following formulae : C 2 H 5 .CH CH CH 2 C N CH 3 C 2 H 6 .CH CH CH 2 C N CH 3 \ CH \ CH OC CH 2 HC-N X OC CH 2 HC ^ V V Pilocarpine Isopilocarpine 1 Proceedings of the Chemical Society 21, 172 (1905). 2 Glyoxaline, or imidazole (C 3 H 4 N 2 ), is obtained by the action of ammonia upon glyoxal in presence of formaldehyde. It is a strong base and crystalline : = jp "I5 INH H h C = HC NH II > HC-N Glyoxaline : = p H 2 jN|H HO! Formaldehyde POISONS NOT IN THE THREE MAIN GROUPS 219 Detection of Pilocarpine Ether, chloroform or benzene extracts pilocarpine from aque- ous solutions alkaline with sodium hydroxide or carbonate. Evaporation of these solutions leaves a thick, non-crystalline, alkaline syrup. The general reagents especially delicate for pilocarpine are: iodo-potassium iodide, phospho-molybdic acid, phospho-tungstic acid and potassium bismuthous iodide. 1 . Place a particle of potassium dichromate and 1-2 cc.- of chloroform in a test-tube. Then add pilocarpine itself, or its solution and i cc. of 3 per cent, hydrogen peroxide. Shake for several minutes. The mixture yellowish at first gradually darkens and in 5 minutes is dark brown. Depending upon the amount of pilocarpine, the chloroform is blue-violet, dark or indigo-blue. But the upper aqueous solution gradually fades. The chloroform mixture is an intense blue from o.oi gram of pilocarpine and blue-violet from o.ooi gram and less. The color lasts from an hour to a day (H. Helch 1 ). Apomorphine (o.oi gram) colors chloroform blue- violet even without hydrogen peroxide. Strychnine gives a barely perceptible bluish tint which changes com- pletely within a few minutes. There is a color with antipyrine only after acidification of the hydrogen peroxide. 2. Mandelin's reagent dissolves pilocarpine with a golden yellow color which gradually changes to bright green and finally to light brown. 3. The solution of pilocarpine in formalin-sulphuric acid becomes yellow, yellowish brown and blood-red, if warmed. Thus far fatal poisonings from this alkaloid have not occurred and nothing is known as to the possibility of its detection in the cadaver. PTOMAINES Ptomaines are basic substances containing nitrogen and may be toxic or non-toxic. They are produced during putrefaction 'of cadavers under the in- fluence of bacteria. They are to be regarded to some extent as products of bacterial metabolism and are nearly always present in cadavers, especially in those parts which are in an advanced state of putrefaction. Many ptomaines closely resemble alkaloids. Like alkaloids they give precipitates with the general reagents, and certain ptomaines resemble well-defined alkaloids even with special 1 Pharmazeutische Post 35, 289, 498 (1902) and 39, 313 (1906). 220 DETECTION OF POISONS reagents. Hence ptomaines are of very great importance in forensic chemistry, since their presence may easily lead to mistakes and false conclusions. These putrefactive products also resemble vegetable bases in their behavior with sol- vents. Ether extracts some of them from acid solution and others from alkaline solution, whereas certain ptomaines are removed from alkaline solution only by amyl alcohol or chloroform. Most of the ptomaines are strong reducing agents, for example, they will immediately convert potasium ferricyanide into ferro- cyanide. Consequently, they give the Prucsian blue test with a dilute mixture of solutions of ferric chloride and potassium ferricyanide. Many of the alkaloids like morphine resemble the ptomaines in this respect. The resemblance of a ptomaine to a definite vegetable base is frequently con- fined to some one reaction, and never extends to all the reactions characteristic of the particular alkaloid. In a legal-chemical investigation no precaution, which guards against mistaking a ptomaine for a vegetable base, should be omitted. It is an invariable rule to make every test characteristic of the sus- pected alkaloid, and not to be satisfied with possibly one positive test. A determination of the physiological action of the substance should supplement the chemical examination. A ptomaine may resemble a vegetable base chemi- cally, and yet the two substances may differ very decidedly in physiological action. Thus far, ptomaines have been found which show certain resemblances to coniine, nicotine, strychnine, codeine, veratrine, delphinine, atropine, hyoscyamine, morphine and narceine. Selmi has described a putrefactive product which resembles morphine. Ether failed to extract it, either from asid or alkaline solution, whereas amyl alcohol removed it with ease from an alkaline or am- moniacal solution. It liberated iodine from iodic acid, but failed to give the tests which are characteristic of morphine alone, namely, Husemann's, Pellagri's and the ferric chloride tests ! The object in such cases must be to get a result about which there can be no doubt. Every possible means must be used to isolate the alkaloid in a perfectly pure state. When this can be accomplished, the nature of the poison can always be established beyond question. SAPONmS The term saponins, or sap6nin substances, includes a large number of gluco- side-like bodies of widespread occurrence in the vegetable kingdom and having in common certain chemical, physical and especially physiological properties. Their aqueous solutions when shaken foam readily. In this respect they re- semble the soaps. Many saponin substances have a sharp, harsh taste. In powdered form they excite violent sneezing. They are capable of holding many finely divided substances in a state of emulsion. They dialyze incompletely and salts precipitate them from solution. Excepting the gluco-alkaloid solanine, which contains nitrogen and is alkaline, the saponins may be classified chemically as nitrogen-free glucosides. Most saponins are neutral and only a few are faintly acid. Neutral saponins and alkali salts of acid saponin substances dis- solve in water and hot aqueous alcohol but are insoluble in absolute alcohol and ether. Barium hydroxide and lead acetate (neutral and basic) precipitate saponins from concentrated aqueous solution. The former gives baryta saponins. POISONS NOT IN THE THREE MAIN GROUPS 221 Basic lead acetate precipitates all saponins but the neutral salt precipitates only acid saponins. Ammonium sulphate is capable of salting saponin sub- stances from solution as it does proteins. Solutions of saponins in concentrated sulphuric acid are yellow, gradually becoming red and sometimes violet and blue-green. The detection thus far of saponin substances in more than 50 plant families having over 200 monocotyledenous and dicotyledenous species shows the wide occurrence of these substances in the vegetable kingdom. Saponins occur in roots (Senega, Saponaria), tub'ers (Cyclamen), barks (Quillaja, Guaia- cum), fruits (Sapindus, Saponaria), seeds (yEsculus, Agrostemma. Thea), stems (Dulcamara) and leaves (Guaiacum). In fact almost any part of the plant organism may contain saponins. The plant families, producing saponin sub- stances in greater abundance, are the sapindaceae, caryoph'yllaceae, colchicaceae polygalaceae, sileneas and solanaceae. Quite considerable quantities of saponins may occur in the particular part of the plant. Saponin solutions, heated with dilute hydrochloric or sulphuric acid, are hydrolyzed into sugars and a non-toxic substance insoluble in water called sapogenin. The sapogenins have not been extensively investigated but they are not entirely identical. The following saponins have been more closely studied: Digitonin: in the seeds of Digitalis pupurea. Saponin: in the root of Saponaria offkinalis (4-5 per cent.). Githagin: in the seeds of the corn cockle, Agrostemma githago (6 .5 per cent.). Senegin: in Senega root, the root of Polygala senega. Struthiin: in levantine soap root, the root of Gypsophila struthium (14 per cent.). Quillaja-Sapotoxin: in the bark of Quillaja saponaria (8.8 per cent.). Sapindus-Sapotoxin: in the fruit of Sapindus saponaria. Sarsaparilla-Saponin: in the sarsaparilla root/the root of various kinds of smilax. Physiological Action of Saponins. Almost without exception saponin sub- stances are highly toxic, if introduced directly into the blood. Most saponins are absorbed with difficulty. Consequently healthy individuals may take dilute saponin solutions by the mouth in considerable quantities without ill effects. Toxic saponins act in common as protoplasmic irritants. In larger doses saponin substances kill protoplasm. They manifest in various ways their power of acting as protoplasmic poisons. Saponins act upon blood-corpuscles for the same reason. R. Robert and his collaborators have shown defibrinated blood, diluted 100 times with physiological salt solution (see below), to be the best and most convenient reagent for saponin substances. Saponins cause haemolysis and the blood solution becomes laky. Agglutination and formation of methsemoglobin do not occur. The freer the blood is of serum, the more pronounced the hae- molytic action of saponin substances upon blood-corpuscles. Recent investi- gations have shown that saponins act more vigorously upon blood-corpuscles isolated from serum, because blood serum contains cholesterin which has a pro- tective influence and retards haemolysis. Most likely the haemolytic action of saponins is due to removal of cell membrane lecithin, the chief 'constituent of the cell wall, from red blood-corpuscles, for lecithin-saponins are formed. Saponins 222 DETECTION OF POISONS also combine with cholesterin, as well as with lecithin, forming cholesterin- saponins. The affinities of a saponin having been satisfied by cholesterin, it no longer acts upon the lecithin of the membrane of blood-corpuscles. Thus cholesterin prevents haemolysis, which a saponin may produce, and so acts as an antidote to saponin substances. Ransam 1 has made the important discovery that addition of cholesterin checks the solvent action of a saponin upon blood- corpuscles. At first it was not known whether this antidotal action was due to a chemical reaction, or to absorption, that is to say, to a physical process. R. Kobert 2 as well as Madsen and Noguchi 3 were able to dissolve cholesterin, which is insoluble in water, in an aqueous saponin solution. They assumed that this physiologically inactive solution contained a labile saponin-cholesterin compound no longer having haemolytic power. Recently A. Windhaus 4 has definitely proved that saponin-cholesterides exist. Digitonin-cholesteride, C5sH94O28.C27H46O, crystallizes in fine needles, when a hot alcoholic solution of digitonin (i molecule) is poured into a similar solution of cholesterin (i mole- cule). This cholesteride is formed without elimination of water. Hence in this reaction between digitonin and cholesterin we are dealing most probably with the formation of a molecular compound. Saponin solutions also dissolve white blood-corpuscles but only at higher concentrations. A physiological action characteristic of many saponins is exhibited in the stupefaction and killing of fish, even in water containing only 1:200,000 of saponin substance (R. Kobert). Detection of Saponins The matter of solubility is especially important in isolating saponin substances from mixtures. All saponins are soluble in water and some in alcohol, but they are practically insoluble in ether, benzene, chloroform and petroleum ether. Employ neutral or basic lead acetate (see above) in isolating saponins. Decompose the washed precipitate with hydrogen sulphide, filter and evaporate the nitrate upon the water-bath. Pre- cipitate the saponin with absolute alcohol and ether from the concentrated solution. Solutions of most saponins in con- centrated sulphuric acid are red or yellowish red, gradually becoming violet. Saponin substances give various colors with Froehde's reagent and vanadic-sulphuric acid: brown, red- brown, blue, green and violet (see solanin). A saponin solu- tion heated with dilute hydrochloric acid, undergoes hydrolysis 1 Deutsche medizinische Wochenschrift 1901, 194. 2 R. Kobert, Die Saponine, Stuttgart, 1904. Chemisches Zentralblatt, 1905, I, 1265. 4 Berichte der Deutschen chemischen Gesellschaft 42, 238 (1909). POISONS NOT IN THE THREE MAIN GROUPS 223 and then, owing to formation of sugar, reduces Fehling's solu- tion with heat. Detection in Foaming Beverages (Beer, Wine, Effervescing Lemonade) l Treat the beverage to be tested for saponin with excess of basic magnesium carbonate, evaporate to about 100 cc. and mix with 2 volumes of 96 per cent, alcohol. Filter after 30 min- utes and evaporate the alcohol from the nitrate. Filter the residue hot and extract the cold nitrate with sufficient liquid carbolic acid 2 to leave about 5 cc. undissolved. Add ammonium sulphate to hasten the separation of the carbolic acid layer. Then shake the latter with water and a mixture of 2 volumes of ether and i volume of petroleum ether. Evaporate the aqueous solution to dryness upon the water-bath. Wash the residue with cold absolute alcohol, in case of wine, and with acetone, in case of beer. The residue fails to give the saponin reaction well, that is to say, a red color with concentrated sulphuric acid, unless treated as described. E. Schaer dissolves the residue in concentrated aqueous chloral hydrate solution and adds the latter to concentrated sulphuric acid as an upper layer. A saponin produces a yellow, then purple-red and finally mallow- blue zone. Detection of Githagin (Corn Cockle Saponin) in Flour Heat 500 grams of flour with i liter of alcohol (sp. gr. 0.8496 = 85 per cent, by volume). Filter hot, distil most of the alco- hol, add absolute alcohol as well as ether to the residue and let stand 12-24 hours. Collect the precipitate upon a filter and dry for a short time at 100 to coagulate possible protein. Dissolve in a little cold water, filter and precipitate githagin from the filtrate with absolute alcohol, best with addition of ether. Githagin thus obtained is a yellowish white powder having a sharp, harsh taste. To prove the presence of a saponin substance, agitate its 1 K. Brunner, Zeitschrift fur Untersuchung der Nahrungs- und Genussmittel 5, 1197 (1902). 2 Acidum carbolicum liquefactum of the German Pharmacopoeia. 224 DETECTION OF POISONS aqueous solution which should foam. Then heat the solution with dilute hydrochloric acid and test its reducing power with Fehling's solution. Finally, if possible, perform the physio- logical test with blood. Dilute defibrinated ox blood with 100 volumes of 0.9 per cent, sodium chloride solution and add the solution of supposed githagin in 0.9 per cent, sodium chloride solution. The blood solution at once becomes laky, if githagin is present. According to J. Brandl, 1 Agrostemma-Sapo toxin (githagin) produces haemolysis in very great dilution (1:50,000). But after previous treatment with cholesterin, even o.oi gram shows no haemolytic action whatever. Physiological Salt Solution and Haemolysis To prevent red blood-corpuscles from changing volume in experiments requiring dilution of blood, an isotonic salt solution must be used. What is an isotonic solution? If n gram- molecules of a body A are dissolved in a definite volume of solvent and n gram-molecules of a body B are dissolved in an equal volume of the same solvent, certain properties of the original solvent are changed equally in both cases. The freezing- point of the solutions is lowered and the boiling-point raised equally. The two solutions have the same vapor tension and the same osmotic pressure. In other words they are isotonic. Blood-corpuscles retain their volume unchanged, if brought into a salt solution having the same osmotic pressure as the blood serum. Such a salt solution is isotonic with blood serum. In the case of human and mammalian blood an isotonic solution of sodium chloride has a concentration of 9 per thousand = physiological salt solution. Such a solution formerly contained 0.6 per cent, of sodium chloride. Blood-corpuscles give up water to solutions of higher concentration than 0.9 per cent. NaCl (hyperisotonic solutions) until osmotic equilibrium is established. They shrivel and hence have a smaller volume. On the other hand, blood-corpuscles in salt solutions of lower concentration (hypisotonic solutions) take up water and be- come distended. In diluting blood with water, this swelling 1 Archiv fur experimentelle Pathologic und Pharmakologie, 54, 245. POISONS NOT IN THE THREE MAIN GROUPS 225 may go far enough to cause haemoglobin to separate from the stroma and pass into the aqueous solution. This process is called haemolysis. Alternate freezing and thawing of blood may produce haemolysis. Various chemical substances, which act as protoplasmic poisons, cause the same result. Such substances are ether, alcohol, chloroform, alkalies, gallic acids, solanine, etc. The saponins described above are also powerful haemolytic agents. Finally, those globulicidal substances, or. haemolysins, normally occurring in blood sera, as well as those produced in immunization, belong in this class. SOLANINE Solanine, Co^HgsNOis, at the same time an alkaloid and a glucoside (gluco- alkaloid) occurs in the potato plant (Solanum tuberosum) and in other Solanaceae as Solanum nigrum, Solanum dulcamara and Solanum lycopersicum (tomato). It has been found also in Scopoliaceae, as in Scopolia orientalis and Scopolia atropoides. Solanine is not uniformly distributed in all parts of the potato plant but is most abundant in the berry-like fruit and in the chlorophyll-free sprouts appearing in the spring upon potatoes that lie in a cellar. Schmiedeberg and Meyer found 0.024 gram of solanine per kilogram of peeled potatoes in January and February but 0.044 gram in unpeeled potatoes. Potato peelings gave 0.71 gram of solanine per kilogram and potato sprouts i cm. long even 5.0 grams. The appearance of solanine according to R. Werk is due to the life processes of Bacterium solaniferum (?). Solanine crystallizes in white needles having a bitter taste and melting at 244. Even boiling water dissolves only a little of this alkaloid (about i : 8000). It is soluble in 500 parts of cold and 125 parts of boiling alcohol; and in about 4000 parts of ether. These solutions are faintly alkaline. Hot saturated solu- tions of solanine in alcohol and amyl alcohol gelatinize upon cooling. Ether, chloroform and benzene do not extract solanine either from acid or alkaline solution. But hot amyl alcohol extracts solanine from acid solution and from solutions alkaline with sodium hydroxide or ammonia. Solanine is a weak base, readily dissolving in acids, as acetic acid, and forming crystalline salts. Dilute hydrochloric or sulphuric acid hydrolyzes solanine to solanidine, C^Hsi- N0 2 , galactose and rhamnose. Hydrolysis is very slow in the cold but rapid upon heating. The hydrochloride or sulphate of solanidine separates as a diffi- cultly soluble, crystalline powder. A good yield of solanidine is obtained, according to Wittmann, by heating solanine under a return-condenser with 10 times the quantity of 2 per cent, sulphuric acid, until the liquid is yellowish and the nitrate upon further boiling no longer deposits solanidine sulphate. Solanidine, precipitated from its sulphate with ammonia and recrystallized from ether, forms colorless, silky needles, melting at 207 and dissolving with difficulty in water but readily in ether or hot alcohol. Solanidine is a stronger base than solanine and the salts it forms with acids are usually crystalline and difficultly 15 226 DETECTION OF POISONS soluble in water. Solanine and solanidine are highly toxic substances having an action similar to that of the saponin substances (see above). Toxic Action. Solanine taken internally is usually very imperfectly ab- sorbed. As a glucoside its action is local and as a saponin-like substance strongly hsemolytic, rendering the blood laky. A solanine solution even in a dilution of i : 8300 causes complete haemolysis. Internal administration of solanine usu- ally produces emesis and larger doses cause gastro-enteritis (gastro-intestinal catarrh). The latter also follows intravenous and subcutaneous injection of doses not rapidly fatal. At the same time a hsemoglobinuria may appear. (R. Kobert, Intoxikationen.) Detection of Solanine and Solanidine Since very dilute mineral acids hydrolyze solanine, these acids cannot be used to detect this alkaloid. E. Schmidt 1 suggests the following procedure. Extract the material with cold water containing tartaric acid. Neutralize the filtered extract with calcined magnesia and evaporate to dryness upon the water- bath. Extract the residue with alcohol and filter hot. Tf the quantity of solanine is not too small, the alcoholic extract gel- atinizes upon cooling. Otherwise, evaporate the alcoholic solution and examine the residue for solanine. L. Kobert extracts solanine from alkaline solution with isobutyl alcohol. Phospho-molybdic acid is the only general reagent giving a pre- cipitate with a solanine solution and that is yellow. But sol- anidine, that is to say, a solanine solution that has been boiled with excess of hydrochloric acid, being a stronger base, gives precipitates with most of the other general reagents. Special Tests for Solanine and Solanidine 1. A solution of, solanine in selenic-sulphuric acid 2 is rasp- berry-red. Gentle heat favors the appearance of this color. Solanidine gives the same result. 2. Solutions of solanine and solanidine in vanadic-sulphuric acid 3 are orange-yellow, soon becoming red and finally blue- violet. Solanine may be dissolved first in sulphuric acid and a drop of vanadic-sulphuric acid added to this solution. 1 Pharmazeutische Chemie, Organischer Teil. 2 A mixture of 1.3 grams of sodium selenate (Na 2 SeO 4 .io H 2 O), 8 cc. of water and 6 cc. of concentrated sulphuric acid. 3 Dissolve o.i gram of ammonium vanadate (H 4 N.VO 3 ) in 100 grams of con- centrated sulphuric acid. POISONS NOT IN THE THREE MAIN GROUPS 227 3. Solutions of solanine and solanidine in ethyl sulphuric- acid 1 are red. An alcoholic solution of solanine, carefully added to concentrated sulphuric acid as an upper layer, produces a red zone where the two liquids meet (E. Schmidt). 4. A solution of solanine in concentrated sulphuric acid is orange but becomes brownish red on longer standing or gentle warming. Red streaks appear, if bromine water is added drop by drop to a solution of solanine in concentrated sulphuric acid. 5. A solution of solanine in Froehde's reagent is first yellow- ish red then evanescent cherry-red and finally red-brown. The methods for estimating solanine quantitatively in pota- toes are described in Chapter VI (see page 291). THEBAINE Thebaine, Ci 9 H 2 iNO 3 = CnHi 5 (OCH 3 ) 2 NO, constitutes about 0.15 per cent. of opium. This alkaloid crystallizes from dilute alcohol in leaflets having a jj jj silvery glitter and from absolute alcohol in C C N.CH 3 prisms melting at 193. It is nearly insoluble -/\ /\ /\ in water, rather easily soluble in hot alcohol, H f ff C ? C ? H2 ether> benzene and chloroform. It differs from CH 3 O C C C CH 2 morphine in being nearly insoluble in caustic \/ \# \/ alkalies. Its solutions are tasteless and laevo- C C CH rotatory. 1 I H I H Constitution. Thebaine is a strong tertiary \/> base, forming as a rule well crystallized salts C with acids. But excess of acid, especially mineral acid, usually decomposes these salts R Pschorr's formula wlt ^ ease - Being a tertiary base, it easily combines with methyl iodide, forming thebaine iodomethylate, Ci 9 H 2 iNO 3 .CH 3 I, crystallizing in prisms. Two of the three oxygen atoms in thebaine are methoxyl-groups ( OCHs) and the third prob- ably forms an ether-like combination, a so-called bridge-oxygen. The thebaine molecule appears not to contain hydroxyl. Heated with acetic anhydride, thebaine gives the acetyl derivative of the phenol thebaol, Ci6H 14 O 3 , and a nitrogenous product, methyl-oxy-ethylamine, CH 3 .NH.CH 2 .CH 2 .OH. R. Pschorr has synthesized thebaol, or the methyl ether of thebaol, and shown by this synthesis that thebaol is 3,6-dimethoxy- 4-oxy-phenanthrene (see below). Pschorr assigns to thebaine the structural formula given above which is analogous to that of apomorphine and of morphine (see pages 127 and 131). Thebaol has the following structural formula: 1 Add 6 cc. of concentrated sulphuric acid to 9 cc. of absolute alcohol. 228 DETECTION OF POISONS /\ /\ HC C CH C C CH (4) HO 1 Hi L Y OCH 3 (6) Detection of Thebaine Ether and chloroform extract thebaine from an alkaline aqueous solution and consequently this alkaloid appears in ether extract B, if the Stas-Otto procedure is followed. The general reagents, phospho-tungstic acid, iodo-potassium iodide, potassium mercuric iodide and potassium bismuthous iodide precipitate thebaine even from very dilute solutions. Thebaine gives the following color reactions : 1. Concentrated Sulphuric Acid gives a deep red color with thebaine and the solution gradually becomes yellowish red. Froehde's reagent gives the same result. 2. Concentrated Nitric Acid dissolves thebaine with a yellow color. With Erdmann's reagent the color varies from dark red to orange. 3. Chlorine Water dissolves thebaine and ammonia turns the solution an intense red-brown. Toxalbumins Toxalbumins are toxic, protein-like substances either already formed in the plant or animal organism, or produced in the metabolism of pathogenic micro- organisms. These substances as yet have not been isolated pure as individual chemical compounds. The chemical and physiological properties of such vegetable toxalbumins as abrin, ricin, robin and crotin are given as a matter of fact by substances obtained from some particular part of the plant by a definite method. The vegetable toxalbumins mentioned possess the common property of clumping, agglutinating and precipitating red blood-corpuscles. Therefore R. Robert classifies them as "vegetable agglutinines." A trace of one of these agglutinines, added to defibrinated blood in a test-tube, causes clumping into a mass resembling sealing-wax. Abrin, ricin and crotin also cause coagulation of milk. POISONS NOT IN THE THREE MAIN GROUPS 229 Abrin This toxalbumin occurs in jequirity seeds from Abrus precatorius. Remove the seed envelopes and extract the finely divided seeds with 4 per cent, sodium chloride solution. Concentrate the filtered liquid in vacua and acidify with acetic acid. Precipitate abrin from this solution by addition of sodium chloride and finally purify by dialysis. Abrin is an amorphous, highly toxic powder not entirely free from ash. Though abrin and ricin are alike in some respects, they are not identical. Ricin This intensely toxic toxalbumin constitutes 2.8-3 P er cent, of the castor bean. Remove the seed envelopes and subject the seeds to powerful pressure to remove as much oil as possible. Then extract with 10 per cent, sodium chloride solution. Saturate the filtered extract at the same time with magnesium and sodium sul- phate and keep for some time in the cold at room temperature. Place the pre- cipitate, which contains ricin, in a parchment paper dialyzing tube and dialyze for several days. Finally dry the residual ricin in vacuo over sulphuric acid. Ricin is an amorphous, highly toxic powder containing ash and easily soluble in 10 per cent, sodium chloride solution. This- toxalbumin, dissolved in sodium chloride solution, gives the protein reactions. Ricin possesses in high degree the power of agglutinating blood-corpuscles. Use defibrinated blood for this test-tube experi- ment, not diluted blood or blood mixed with physiological salt solution. Ricin, according to Elfstrand, agglutinates the red blood-corpuscles of the guinea-pig even in a dilution of i ; 600,000. Ricin agglutinates the blood of all mammals but not to the same degree. Removing serum from the blood and substituting physiological salt solution strengthens rather than weakens the agglutinating action of ricin. The inference is that serum must have a certain anti-agglu- tinating action. Separation of red blood-corpuscles into stroma and haemo- globin 1 shows that ricin has not changed haemoglobin in the least. But the stromata have been altered just as the blood-corpuscles have been. To detect ricin in castor bean press-cake, or in feeds containing castor beans, extract the finely divided material with physiological salt solution at room tem- perature, filter and make the agglutination test in a test-tube with undiluted, de- fibrinated blood and with blood diluted with physiological salt solution. Crotin Crotin is a substance obtained from the seeds of Croton Tiglium. Remove the seed envelopes, express the oil and treat as described for abrin and ricin. Chem- ically crotin is very similar to ricin. Abrin and ricin agglutinate the blood-cor- puscles of all warm-blooded animals thus far tested but crotin does not behave the same with all kinds of blood. (See R. Robert, Intoxikationen.) Coagulation of Blood and Defibrinated Blood Blood is a transparent fluid, the blood-plasma, suspended in which is a very large number of solid particles, the red and white blood-corpuscles. Outside 1 The two principal components of blood-corpuscles are the stroma, which con- stitutes the true protoplasm, and the intraglobular contents, the chief constituent of which is haemoglobin. 230 DETECTION OF POISONS the organism blood coagulates even in a few minutes after being drawn. In the clotting of blood a very difficultly soluble protein, called fibrin, separates. If the blood is still, the clot is a solid mass which gradually contracts and exudes a clear liquid, usually yellow, the blood-serum. The coagulum, thus formed and en- veloping the blood-corpuscles, is called the crassamentum (Placenta sanguinis). But if the blood is whipped during coagulation, fibrin separates in threads. The fluid separated from the latter is defibrinated blood which consists of blood-cor- puscles and blood-serum. To obtain defibrinated blood, whip with twigs the fresh blood removed from a vein and fibrin will separate on these. Or run the fresh blood into an Erlenmeyer flask, containing iron filings, and shake vigorously for several minutes. Fibrin is precipitated on the filings. There are several ways to retard coagulation of blood, among which the follow- ing may be mentioned: 1. Cool blood suddenly to low temperature. 2. Draw blood direct from the vein into a neutral salt solution, for example, magnesium sulphate solution (i volume of salt solution and 3 volumes of blood) and stir. This mixture of blood and salt will not coagulate for a day. 3. Add blood to sufficient dilute potassium oxalate solution to give a mixture containing o.i per cent, of oxalate. The soluble calcium salts of the blood are precipitated by the oxalate and the blood loses its power of coagulating. 4. To prepare a non-coagulating blood-plasma, pour blood into sodium fluoride solution until it contains 0.3 per cent, of NaF. CHAPTER V SPECIAL QUALITATIVE AND QUANTITATIVE METHODS Quantitative Estimation of Phosphorus in Phosphorated Oils i. W. Straub's Method. Straub has found that his test 1 with dilute copper sulphate solution, recommended for the qualitative detection of phosphorus, may also be used to deter- mine phosphorus in a phosphorated oil. If such an oil is shaken with 3 per cent, copper sulphate solution, there is first a brown- ish black emulsion in which each individual oil drop is coated with a film of copper phosphide, PCu 3 (?). After 4-5 hours shaking, this brownish black color disappears and the mixture separates into two layers. All the phosphorus in the oil is now in the aqueous solution as phosphoric acid. This method has the further advantage that the decolorization of the emulsion serves as an indicator of the completion of the oxidation. Procedure. Put 25 cc. of 3 per cent, copper sulphate solution (taken as CuSO 4 .5H 2 O) in a separatory funnel. Add 5 cc. of the phosphorated oil 2 and agitate the mixture vigorously for a long time. If a shaking machine is available, place the mixture in a thick-walled glass bottle with a tight glass stopper and shake 3-5 hours, or until the original brown emulsion has dis- appeared and become clear and bright blue. Separate the aqueous solution in a separatory funnel and precipitate phos- phoric acid at once by the molybdate method and finally weigh as magnesium pyrophosphate, 1 Zeitschrift fur anorganische Chemie 35, 460 (1903). 2 To prepare a phosphorated oil suitable for such determinations, dissolve about o. i gram of yellow phosphorus in the smallest possible quantity of warm carbon disulphide and dilute this solution to 100 cc. with olive oil. Although carbon disulphide does not affect the determination of phosphorus, it may be removed by warming the phosphorated oil on the water-bath. 231 232 DETECTION OF POISONS Remarks. The accuracy of this method is shown by the results of Straub's determinations. Instead of 0.005 gram of phosphorus, dissolved in 5 cc. of oil, he found 0.0047 and 0.00468 gram. Even very considerable dilutions of the phosphorated oil do not affect the accuracy of the determination. In the case of the more concentrated phosphorated oils, shaking with copper sulphate solution must be kept up much longer. 2. A. Frankel's 1 and C. Stich's 2 Method. Dissolve the oil in acetone and precipitate phosphorus with hot alcoholic silver nitrate solution. Oxidize the phosphorus in the precipitate to phosphoric acid and finally determine the latter in the usual way. Procedure. Dissolve 20-50 cc. of the phosphorated oil, as phosphorated cod liver oil, in 100 cc. of acetone or ether and completely precipitate with hot alcoholic silver nitrate solu- tion. 3 First wash the precipitate of silver phosphide with ether-acetone mixture and then with alcohol. Treat next with hot 25 per cent, nitric acid, containing a little fuming acid. Expel excess of nitric acid from the filtrate on the water-bath and precipitate silver with hydrochloric acid. Finally filter from silver chloride and determine phosphoric acid in the filtrate. Remarks. Since sodium hypophosphite and phosphite are soluble in acetone and also precipitated by acetone-silver nitrate, it is advisable first to extract a test portion of the phosphorated oil with water and then test the aqueous extract for these first oxidation products of phosphorus, hypophosphorous and phosphor- ous acids. If they are present, all the phosphorated oil should first be extracted with water in the same manner. Phosphorus in phosphorated oils, especially phosphorated cod liver oil slowly disappears. C. Stich found that a phosphorated cod liver oil, containing 0.05 per cent, of phosphorus, with the usual daily removal of 5 grams, lost in 3 weeks OQ ly 3~5 milligrams of phosphorus. Such a decrease in the amount of phosphorus in phosphorated oils is only of slight significance. Dilute oily solutions of phos- phorus (i : 1000), when kept in tightly stoppered bottles and protected from light, are constant as regards their phosphorus content for a long time, even 5-6 months. Moreover, phosphorus much diluted as vapor or in solution, is oxi- dized with corresponding difficulty. The same is also true of phosphorus in the animal organism. Therefore it is possible sometimes to detect free phosphorus in the excretory organs, as the liver, even several weeks after phosphorus poisoning. The distillation method is inapplicable in the quantitative estimation of phos- 1 Pharmazeutische Post 34, 117. 2 Pharmazeutische Zeitung 37, 500 (1902). 3 Silver nitrate dissolves in about 10 parts of alcohol. SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 233 phorus in oils, as cod liver oil, since only about 40 per cent, of the phosphorus present is found in the receiver, even when the strongest oxidizing agent and the best absorbent for phosphorus are used. To place the phosphorus-content of the cod liver oil residue at the amount of the distilled phosphorus is not admissible, because cod liver oil as such contains about 0.02 per cent, of combined phosphorus. Special Methods for the Detection of Arsenic Isolation of Arsenic as Arsenic Trichloride 1 . This depends upon the volatility of arsenic as chloride, AsCl 3 , in concentrated hydrochloric acid solution and in presence of ferrous chloride. The latter serves (a) to reduce any arsenic acid possibly present in the material to arsenious acid which with concentrated hydrochloric acid then forms arsenic trichloride 03): () H 3 AsO 4 + 2HC1 + 2 FeCl 2 = H 3 AsO 3 + H 2 O + 2FeCl 3 , (0) H 3 As0 3 + 3 HC1 = AsCl 3 + 3 H 2 O. Procedure. Comminute the material and mix with very concentrated hydrochloric acid (about 40 per cent.) until rather thin. Then add 5 grams of 20 per cent, arsenic-free ferrous chloride solution or saturated ferrous sulphate solution and put the mixture into a capacious retort, the neck of which is directed obliquely upward and connected with a Liebig cooler by an obtuse angle tube, and carefully distil. Distil about a third to a half of the original mixture. Dilute the distillate with water and test for arsenic in the Marsh apparatus, using hydrochloric acid for the evolution of hydrogen. If a tubulated retort is used for the distillation, hydrochloric acid gas can be passed in during distillation so that the liquid is kept saturated with this acid. Electrolytic Detection of Arsenic To detect arsenic electrolytically, put the liquid, as the sul- phuric acid solution obtained according to the general procedure which contains arsenic as arsenic acid (see page 156), or urine or stomach contents, in a sufficiency wide U-tube with platinum electrodes (Fig. 18). Pass the current through the liquid acidified with sulphuric acid, and arsine, AsH 3 , together with 1 H. Beckurts, Archiv der Pharmazie 222, 653 (1884). 234 DETECTION OF POISONS hydrogen will appear at the cathode, if the liquid contains ar- senic. First test the hydrogen for arsenic by the Gutzeit arsenic test (see page 1 63) . If a yellow spot appears on the paper moist- ened with saturated silver nitrate solution, arsenic is present. That this is actually arsenic may be shown by connecting the U-tube as shown in the sketch with a chloride of calcium tube and a Marsh reduction-tube ; an arsenic mirror then appears in PIG. 1 8. Apparatus for the Electrolytic Detection of Arsenic. the latter when heated to redness. Use a current having an electromotive force of 7-8 volts. The electrolytic method is especially adapted for the detection of arsenic in inorganic com- pounds present in secretions, as the urine, but not for arsenic in organic combination as cacodyl compounds and arrhenal. An exception among these organic compounds of arsenic is atoxyl, or the anilid of meta-arsenic acid, AsC^.NH.CeHs. The arsenic being rather loosely bound is broken up by the electric current with formation of arsine. Destruction of Organic Matter and Detection of Arsenic (According to A. Gautier 1 and G. Lockemann 2 ) The purpose of this method is to increase the delicacy of the Marsh-Berzelius test for arsenic, and to exclude as far as possible sources of error connected with 1 Bulletin de la Societe chimique de Paris, 29, 639 (1903). 2 Zeitschrif t fur angewandte Chemie 18, 416, 491 (1905); also 19, 1362 (1906). SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 235 the destruction of organic matter, the precipitation of arsenic with hydrogen sulphide and the evolution and drying of hydrogen gas. Organic matter is destroyed without the use of hydrochloric acid, and arsenic is detected with- out precipitation as arsenic sulphide. Lockemann recommends the following procedure and uses finely divided meat as a test experiment: Place 20 grams of finely chopped meat in a porcelain dish and add a few cc. of a mixture of 10 parts of fuming nitric acid and i part of concentrated sulphuric acid. Warm upon the water-bath. , The action of the acid mixture is so vigorous that, even after the addition of about 5 cc., the entire mass, which puffs up con- siderably at first, changes to a yellowish, homogeneous, thick, oily liquid. If too much acid is added at once during warming upon the water-bath, the action may be violent enough to cause sudden charring of the whole mass with copious evolu- tion of smoke. Such an occurrence may result in loss of arsenic. Consequently, it is advisable to add the acid mixture, amounting in all to about 20 cc., to the meat in 1-2 cc. portions, not adding a fresh portion of acid until brown fumes cease coming off. The mass is dark yellow and finally becomes brown after long heating upon the water-bath. Stir with a concentrated aqueous solution of 20 grams of a mixture of potassium and sodium nitrate (i -f- r ) and evaporate upon the water-bath. There remains a yellow, crystalline residue which still contains organic matter. Gradually introduce this mixture in small portions into a platinum crucible containing 10 grams of fused potassium and sodium nitrate (i + i). Having added all the mixture, heat the crucible for a short time over a free flame. Dissolve the cold nielt in water, add sulphuric acid and heat upon the water-bath until nitrous fumes have been expelled. Test a cold solu- tion of the residue for arsenic in the Marsh apparatus. Lockemann formerly precipitated arsenic with aluminium hydroxide, Al(OH)j. Add 10 cc. of a 12 per cent, solution of crystallized aluminium sulphate, AU- (SO4)3i8H2O to the solution of the melt free from carbon dioxide and nitrous acid. Render the solution alkaline with ammonia and heat about 30 minutes upon the water-bath. Collect the precipitate upon a paper, wash with water containing ammonia and dissolve in about 30 cc. of 10 per cent, sulphuric acid. Heat the solution in a porcelain dish upon the water-bath until it no longer gives a test for nitric acid with diphenylamine-sulphuric acid. 1 Then examine this solution for arsenic in the modified Marsh apparatus 2 devised by Lockemann (Fig. 19). Lockemann 's latest results have shown that ferric hydroxide is much more effective than aluminium hydroxide as a precipitant of small quantities of arsenic. Render the water solution of the melt (see above) slightly acid with sulphuric acid, add a few cc. of iron alum solution, then in the cold, best after cooling with ice, add just enough ammonia to precipitate all the iron. Filter after 30 minutes, wash -the precipitate with cold water to remove nitrates completely, then dissolve in dilute sulphuric acid and test the solution for arsenic in the Marsh apparatus. Iron salts do not interfere with the delicacy of the Marsh test for arsenic. 1 Dissolve i gram of diphenylamine in 100 grams of concentrated sulphuric acid. A drop of the liquid with a drop of this diphenylamine solution in a por- celain dish should not give a blue color. 2 O. Pressler, 30 Bruederstrasse, Leipzig, Germany, supplies this apparatus and also the ignition-tubes. 236 DETECTION OF POISONS Zinc in sticks 1 and sulphuric acid are used in the preparation of hydrogen. Copper is the best activator of zinc in the Marsh apparatus. Break the zinc sticks into pieces weighing about 1.2-1.8 grams, place for a minute in 0.5 percent, copper sulphate solution, wash with water, dry with filter paper and preserve carefully in a closed bottle. This procedure does not interfere with the forma- tion of the mirror, whereas addition of copper sulphate to the reduction flask causes retention of arsenic. Copper sulphate used for this purpose should be carefully purified by several recrystallizations. The basic properties of fused and granulated calcium chloride, which are not entirely removed even by hydro- gen chloride and carbon dioxide, make this an unsuitable drying agent for hydro- PIG. 19. Marsh Apparatus Modified by Lockemann. gen. Lockemann found that potassium carbonate, phosphorus pentoxide and concentrated sulphuric acid cause a noticeable decomposition of arsine, and the same is true of glass wool and cotton. Crystallized calcium chloride in pieces about i cc. in volume is the best drying agent, because it is entirely indifferent to arsine. Lockemann's special drying tube (see sketch) is adapted for the use of this substance. Bohemian glass, having a wall thickness of i mm. and an internal diameter of 4 mm., is used for ignition-tubes. These are drawn out in two places to a length of 4 cm. The outer diameter of the constriction is 1.5 mm. and the inner about 0.5 mm. The reduction flask contains 4-6 pieces of coppered zinc and about 15 cc. of 15 per cent, sulphuric acid are added from the dropping funnel. After hydrogen has been passing through the apparatus for 30 minutes, heat is applied in front of the first constriction of the ignition-tube. If the materials are arsenic-free after 1.5-2 hours heating, place the flame in 1 Lockemann has found Kahlbaum's stick zinc always arsenic-free. The . same may be said of Bertha spelter from the New Jersey Zinc Company. SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 237 front of the second constriction of the ignition- tube. The solution of the iron hydroxide precipitate, prepared as described above, is added to the reduction flask from the dropping funnel which is washed with a little water or dilute sul- phuric acid. In testing for very small quantities of arsenic, it is advisable to cool the place where the mirror is deposited by keeping the cotton thread wet (see sketch). By means of the apparatus described Lockemann has detected even o.oooi mg. of arsenic distinctly. Moist air gradually oxidizes the arsenic mirror, but in an absolutely dry at- mosphere even when exposed to light there is no change. In a closed tube con- taining a little phosphorus pentoxide arsenic mirrors may be kept unchanged even for months. Glass wool, or cotton, noticeably decomposes arsine. The decomposition of arsine in aqueous solution is also hastened by the presence of fine filamentary bodies. This reaction is probably catalytic in character. Electrolytic Estimation of Minute Quantities of Arsenic (C. Mai and H. Hurt 1 ) By this method minute amounts of arsenic (fractions of a milligram) are separated quantitatively at the cathode from an arsenical electrolyte as arsine. The latter then reacts quanti- tatively with silver nitrate as follows: AsH 3 + 3H 2 O + 6AgNO 3 =H 3 AsO 3 + 6HNO 3 + 6Ag. The advantages of the electrolytic detection of arsenic are first the avoidance of traces of arsenic that sometimes come from zinc in the Marsh test and second that destruction of or- ganic matter is often unnecessary. T. E. Thorpe 2 has shown the latter to be the case in the examination of beer worts and malt extracts for arsenic. To reduce arsenic acid and its salts, a few drops of zinc sulphate solution should be added to the sul- phuric acid acting as the electrolyte. The cathode is said to have a higher tension and the hydrogen to be very active. Apparatus and Procedure. The apparatus used by Mai and Hurt is shown in Fig. 20. A is the reduction- tube and B a bulb-tube with 5-6 bulbs con- taining o.oi n-silver nitrate solution. A and B are connected 1 Zeitschrift fur Untersuchung der Nahrungs- und Genussmittel 9, 193 (1905) and also Pharmazeutische Zeitung, 1905. 2 Proceedings of the Chemical Society 19, 183 (1903). 238 DETECTION OF POISONS by a small tube g containing pieces of pumice stone saturated with an alkaline lead solution, or glass wool, to retain any traces of hydrogen sulphide. Anode a and cathode e are lead strips about 1-2 mm. thick. Their upper ends about 5 mm. thick are luted into glass tubes b which pass through the stopper of the U-tube and are tight. The dropping funnel d holds about 25 cc. and its capillary end dips about 2 cm. into the solution to be electrolyzed. Tube c for the escape of oxygen from the anode chamber contains a little water. PIG. 20. Apparatus for the Electrolytic Estimation of Arsenic. Fill U-tube A up to the mark with 12 per cent, arsenic-free sulphuric acid and bulb-tube B with 10 cc. of o.oi n-silver nitrate solution. Turn on the current and keep at 2-3 amperes. If the silver nitrate solution remains unchanged after hydrogen has been running i hour, the lead cathode and sulphuric acid are arsenic-free. Without stopping the current, introduce from the dropping funnel the solution to be 'tested for arsenic, the quantity of which should not be more than 10 cc. Add this solution as slowly as possible and wash the last traces in with a little water. If the solution contains arsenic, or arsenic acid, SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 239 the silver nitrate solution will become dark in a few minutes and the reaction will be at an end in 3 hours. Pour the contents of the bulb-tube through a small asbestos filter, wash with 3-4 cc. of water and titrate excess of o.oi n-silver nitrate with o.oi n-potassium sulphocyanate according to Volhard's method. Calculation. The reaction above shows that 6 molecules of silver nitrate correspond to i atom of arsenic (= 75). There- fore i gram-molecule of silver nitrate = - gram-atom of arsenic = = 12.5 grams of arsenic and 1000 cc. of o.oi n-silver nitrate = 0.125 gram of arsenic. Notes. Electrodes of platinum (foil or gauze) cannot be used in the electro- lytic separation of arsenic as arsine, because either solid arsine or elementary arsenic is formed. Mai and Hurt also found that gold, silver and tin cathodes gave unsatisfactory results and carbon electrodes were not much better. Pure lead alone meets all the requirements as a material for the electrodes. Oxygen compounds of arsenic are quickly and completely reduced to gaseous arsine only upon cathodes of absolutely pure lead. The attachment of a platinum wire to a lead electrode was sufficient to cause incomplete reduction of arsenic compounds. For this reason the electrodes consist of one piece of lead 1 without soldering on wire of another metal. The best electrolyte is 12 per cent, sulphuric acid. A stronger acid easily causes the formation of hydrogen sulphide and a weaker acid has the disadvantage of lower conductivity and lower specific gravity. The electrolyte should be specifically heavier than the solution to be tested to keep the latter from passing at once to the bottom of the reduction-tube. Mai and Hurt found the following amounts of As: As taken As found As 2 Os o.25mg. o.223mg. As 2 O 3 o.iomg. o.opgmg. As2Os o.iomg. o.io5mg. For qualitative tests -the bulb-tube may be replaced by the drying and ignition-tubes of the Marsh apparatus. According to Mai and Hurt the statements of Thorpe and Trotmann, that every solution can be electrolyzed without pre- viously destroying organic matter, do not always hold. In the examination of beer containing arsenic the results were fairly satisfactory, but in the case of urine the results were far too high. 1 Kahlbaum's purest lead. 240 DETECTION OF POISONS Quantitative Estimation of Arsenic and Antimony by the Gutzelt Method Using a special apparatus and paper sensitized with mercuric chloride, Sanger and Black 1 have found that the Gutzeit test can be employed to determine small amounts of arsenic quanti- tatively. The process is very simple and requires only a short time for completion. Sanger and Riegel 2 have extended this method to the estimation of antimony. ______^ Sensitized Paper. Paper strips 3 uniformly 4 mm. wide are sensitized by being soaked in 5 per cent, solu- tion of recrystallized mercuric chlor- ide. These are dried, cut into 7 cm. lengths and protected from light and moisture in a stoppered bottle containing calcium chloride, or soda lime, covered with cotton. Apparatus. A 30 cc. bottle (Fig. 21) for the reduction is closed by a glass stopper provided both with a thistle-tube, constricted to 2 mm. at the end and extending nearly to the bottom of the bottle, and with an ^SZJiSSS^ about *5 mm. just above the stopper. Connected with this exit- tube by a ground joint and at a right angle is a tube exactly 4 mm. inside diameter and approximately 9 mm. in length from the -bend. Procedure. Place 3 grams of uniformly granulated zinc 5 in the bottle and a strip of sensitized paper in the 4 mm. deposition 1 Proceedings of the American Academy of Arts and Sciences 43, 297-3 24 (1907) . 2 Ibid., 45, 21-27 (1909)- 3 A cold pressed paper made by Whatman has been found to give the best results. 4 This all glass apparatus, suggested by Mr. W. A. Boughton, is now in use in the Harvard laboratory and is a modification of Sanger's original apparatus. 5 Bertha spelter from the New Jersey Zinc Company, New York, has been proved free from arsenic. * I l J 5 1 15 20 25 30 35 40 50 60 70 FIG. 22. Standard Arsenic Bands in Micromilligrams of As 2 O 3 (Initial). 5 10 15 20 25 30 35 40 50 60 70 FIG. 22a. Standard Arsenic Bands in Micromilligrams of As2O 3 (Hydrochloric Acid Development). i 111 5 10 15 20 25 30 35 40 50 60 70 FIG. 22b. Standard Arsenic Bands in Micromilligrams of As 2 O 3 (Ammonia Development). (Facing Page 240) SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 241 tube. In estimating arsenic, place in the enlargement of the exit-tube a loose plug of lean absorbent cotton that has been kept over sulphuric acid; an hour's preliminary run is necessary to mo : sten the cotton partially. In the case of antimony sub- stitute for cotton a disc of filter paper that has been moistened with normal lead acetate, dried and kept in a well stoppered bottle. Before inserting this disc moisten it with a drop of water. Next add 15 cc. of diluted hydrochloric acid 1 (i : 6) and let the hydrogen run 10 minutes to make sure the reagents cause no stain. Then add the whole, or an aliquot part of the solution to be tested. Arsenic will produce a color on the paper in a few minutes which will reach a maximum within 30 minutes. Antimony produces no visible effect on the sensitized paper, unless the amount is above 70 mmgr. (= 0.070 mg.) when a gray color may appear. If there is any color, another trial should be made with a smaller portion of solution. In the determination of arsenic a disc of lead acetate paper should be inserted beneath the cotton as a precaution against the possible formation of hydrogen sulphide. Standard Bands. (a) Arsenic. Dissolve i gram of resub- limed arsenious oxide in a little arsenic-free sodium hydroxide, acidify with sulphuric acid and make up to a liter with recently boiled water. Dilute 10 cc. of this solution (I) to a liter with freshly boiled water which gives a solution (II) containing o.oi mg. of arsenious oxide per cc. Using definite volumes of solution II, measured from a burette, prepare a series of color bands (Fig. 22), taking a fresh charge of zinc and acid for each portion. The color ranges from lemon-yellow through orange- yellow to reddish brown. (b) Antimony . Dissolve 2.3060 grams of pure, recrystallized tartar emetic in a liter of water. This solution (I) contains i .o mg. of antimonious oxide per cc. By dilution of (I) solutions containing 0.01 mg. (II) and o.ooi mg. (Ill) are prepared and used in making sensitized bands. 1 Synthetic hydrochloric acid, made from electrolytic hydrogen and chlorine by the Hooker Electrochemical Company, New York, is said to be entirely free from arsenic. Tr. 16 242 DETECTION OF POISONS These bands will eventually fade but they may be preserved longer by being sealed in glass tubes in the bottom of which is phosphorus pentoxide covered with cotton. The color of the arsenic bands may be developed (i) by placing the band in hy- drochloric acid (i : i) for 2 minutes at a temperature not over 60, washing thoroughly, drying and sealing as before; (2) by treating for a few minutes with ammonium hydroxide, which gives a dense, coal-black color, washing, drying and sealing in a tube over quicklime. To develop the antimony band; let it stand in a test-tube covered with normal ammonium hydroxide 5 minutes. A black band is slowly developed. These bands may be protected as described, or placed between glass plates cemented together and bound with passepartout .paper. The more dilute standard solutions must be freshly made up within a few hours of use. Notes. Solutions should be as free as possible from sulphur compounds yielding hydrogen sulphide; interfering organic matter; and metals retarding formation of arsine and stibine. The cotton in the exit-tube should be replaced after 10-12 runs, and the lead acetate disc after each run. If the solution contains arseniate, reduce with 10 cc. of arsenic-free sulphurous acid and expel the excess. The absolute delicacy of the method is set at 0.00008 mg. of arsenious oxide and 0.0005 m g- of antimonious oxide. The practical delicacy, using a band 4 mm. wide, is o.ooi mg. of arsenious oxide and 0.002 or 0.003 m S- f anti- monious oxide. By using, however, a band 2 mm. wide in a correspondingly narrow exit- tube, a practical delicacy of 0.0005 mg. of arsenious oxide and o.ooi mg. of antimonious oxide is obtainable. In length of band and density of developed color, the effect of arsine on the sensitized paper is from 2-3 times as great as that of stibine. The authors do not claim a greater accuracy for the method than within 10 per cent. Biological Detection of Arsenic by Penicillium Brevicaule B. Gosio 1 was the first to show that certain moulds, grown upon media containing minute quantities of arsenic, produce volatile arsenic compounds characterized by a garlic-like odor. Seven species of moulds were found to have this power. Penicillium brevicaule, however, which Gosio isolated from ^'Azione di alcune muffe sui composti fissi d'arsenico," Rivista d'igiene e sanita publica, 1892, 201. SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 243 air, and which was first found upon decaying paper, possessed this property in the highest degree. Gosio states that we are justified in regarding Penicillium brevicaule as a living reagent for arsenic. Even o.ooooi gram of arsenic can be recognized with certainty by this biological test. The test is so delicate that it should be of great value in toxicological analysis in the preliminary examination for arsenic. A. Maasen 1 states that a temperature of 28 to 32 is most favorable to the growth of the mould. Crumbs of wheat bread were found to make an especially good culture-medium. When this material is used, a vigorous growth of mould is visible even in 48 hours. Sometimes a test for arsenic can be finished in a few hours, and always in 2 or 3 days. The char- acteristic garlic odor from weak, arsenical cultures can be dis- tinctly recognized even after several months. That these "arsenic moulds" do not produce gases having a garlic odor from sulphur, phosphorus, antimony, boron and bismuth com- pounds, is an important fact. But Penicillium brevicaule possesses in high degree the power of converting solid selenium and tellurium compounds into volatile substances having a pe- culiar odor. The odor, especially from tellurium cultures, is like that produced by arsenic cultures, namely, distinctly like garlic! The odor from selenium cultures, however, differs fiom that arising from arsenic cultures. It is more of a mer- captan odor. Biginelli 2 found that the gases, generated from arsenic cultures by Penicillium brevicaule, are completely absorbed by mercuric chloride solution. Colorless crystals, having the composition (AsH(C 2 H 5 ) 2 .2HgCl 2 ), are formed. This is a double compound of mercuric chloride and diethyl arsine. This compound can easily be decomposed. It then diffuses an intense garlic odor. On the other hand, Klason 3 has recently shown as a result of an investigation of the gas given off when Penicillium brevicaule subsists upon an arsenical medium that 1 Arbeiten aus dem Kaiserlichen Gesundheitsamt, 1902, 478. 2 Chemisches Centralblatt (1900), II, 1067, and also (1900), II, noo. 3 Berichte der Deutschen chemischen Gesellschaft 47, 2634 (1914). 244 DETECTION OF POISONS a double compound of ethyl-cacodylic oxide and mercuric chloride is formed, the formula of which is (C 2 H 5 ) 2 As-O-As- (C 2 H 5 ) 2 + 4HgCl 2 . R. Abel and J. Buttenberg 1 state that a mould to be of use in the biological detection of arsenic must satisfy the fol- lowing conditions: "It must grow rapidly, and not generate any odors during growth, except the garlic odor produced from an arsenical medium. It must not be restricted as to culture medium. It must grow in presence of large, or very small quantities of arsenic. Finally, it must demonstrate its specific action in presence of metallic arsenic and all kinds of arsenical compounds." The best material for these experiments is white or Graham bread, either of which is a favorite culture medium for moulds. The crust is the only part of bread having a specific aromatic odor. When this has been removed, the crumbs may be said to be practically odorless. Procedure. When the material examined is liquid, absorb it completely by adding bread crumbs, and scatter a small quantity of dry bread over the surface. Solid material should be finely ground, or cut into as small pieces as possible, and placed in not too small a flask. Add at least the same quantity of bread crumbs, thoroughly mix the two substances by shaking, and moisten the mass with a little water. Close the flask with a cotton plug, and sterilize in steam. Sterilization must kill all micro-organisms in the flask. Therefore, heat the flask in an autoclave 10 to 30 minutes under a pressure of i to 1.5 atmos- pheres. There is no danger of volatilizing arsenic during ster- ilization. Then inoculate the sterilized material when cold. Place in a flask a sh'ce of potato, superficially coated with mould in the spore-forming stage, and agitate it with bouillon (peptone), salt solution or sterilized water, until it is finely disintegrated. Observe all necessary precautions, and add the mould, suspended in water, in sufficient quantity to impregnate the entire surface of the material suspected of containing arsenic. Theie should not be more liquid, how- 1 Zeitschrift fur Hygiene, 32, 440 (1899). SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 245 ever, than the culture medium will absorb. Too much mois- ture retards the growth of the mould. Finally, draw a tight rubber cap over the mouth of the flask and cotton plug. Flasks thus closed may stand in the room, but it is better to keep them at a higher temperature, for example, in an incubator at 37, since these conditions are most favorable to the growth of the mould. As soon as a growth of mould is distinctly visible to the naked eye upon the medium, the first indication is given that a test of the culture for volatile arsenic compounds may prove successful. In a very favorable case, this is possible in 24 hours. There is always a luxuriant growth of mould in 48 to 72 hours, so that a decision can be reached. If there is no odor, the flask is closed, and the test is repeated once or twice daily on the following days. Sulphuric, hydrochloric and other strong mineral acids prevent the growth of the mould. This preventive action may be overcome by neutralization with calcium carbonate, which may be present in excess without ill effect. Alkalies also interfere with the growth of the mould. They may be removed by neutralization with tartaric or citric acid, either of which may be present in excess. The great advantage of the biological over the purely chemical method lies in the fact that less time is required to get a result. The tedious and un- avoidable destruction of organic matter in the material is rendered unnecessary Moreover, a number of tests for arsenic may be made at the same tune. Abel and Buttenberg (loc. cit.) speak as follows, regarding this method: "The biological method of detecting arsenic has so many advantages, that it deserves to be recommended for the most varied purposes. Its application is very general, and the method of procedure is simple. The culture of the mould can be kept a long time, even a year or more, without being revived. The test is very delicate, and the odor is readily recognized. The generation of the odor, in the case of cultures containing only o.oooi gram of arsenic, can be demonstrated for a week." Besides being practically unlimited in application, the biological method is extraordinarily delicate. In this respect, it exceeds the best known chemical methods for detecting arsenic. It is, for example, considerably more delicate than Bettendorff's test, and it might equal in delicacy the Marsh and Gutzeit tests. Detection of Arsenic in Organic Arsenic Compounds Cacodylic Acid, Arrhenal, Atoxyl 1 The ordinary reagents usually fail to show arsenic in an or- ganic arsenic compound dissolved in water. Several of these 1 C. E. Carlson, Zeitschrift fur physiplogische Chemie 49. 4*o (1906). 246 DETECTION OF POISONS compounds persistently resist the most powerful oxidizing and reducing agents. Cacodylic Acid, (CH 3 ) 2 AsO-OH, and its salts have been used of late as drugs. A 2 per cent, solution of sodium cacodylate, (CH3) 2 AsO-ONa.3H 2 O, conducts the electric current very feebly but no arsine appears at the cathode. BettendorfFs reagent (stannous chloride-hydrochloric acid) does not cause separation of arsenic from cacodylic acid even after evaporation with hydrochloric acid and potassium chlorate. If heated with stannous chloride-hydrochloric acid, cacodylic acid is reduced to the foul-smelling cacodylic oxide, [(CH 3 ) 2 As] 2 0, recognized by its odor. Distillation of sodium cacodylate by Schneider's method with the strongest hydrochloric acid gives no arsenic trichloride in the distillate. The arsenic changes to another form, not precipitable by hydrogen sulphide. Evaporation of the distillate upon the water-bath with nitric acid leaves solid, non-volatile cacodylic acid in which arsenic may be detected by reduction with sodium carbonate-potassium cyanide mixture. Even fuming nitric acid does not oxidize cacodylic acid to ar- senious or arsenic acid. Arrhenal, Sodium Methyl-Arseniate (CH 3 )AsO(ONa) 2 .5H 2 0, forms white crystals very soluble in water. Possibly owing to partial hydrolysis, an aqueous solution of this compound is alkaline and conducts the electric current feebly. Only traces of arsine appear at the cathode after electrolysis in presence of a good conductor. In arrhenal the arsenic is not held as strongly as in the cacodyl compounds. Hydrogen sulphide precipitates yellow arsenic trisulphide. Distillation with strong hydro- chloric acid gives arsenic trichloride in the distillate. Betten- dorff's reagent gives a red-biown precipitate, if considerable arrhenal is present. Atoxyl, the Anilide of Metarsenic Acid, AsO 2 .NH.C 6 H 6 , forms white, odorless crystals readily soluble in water and hav- ing a faint, saline taste. As compared with cacodylic acid, arsenic in atoxyl is less firmly bound. Electrolysis gives arsine abundantly at the cathode. Hydrogen sulphide precipitates sulphide of arsenic. Arsenic trichloride passes over, upon dis- SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 247 tillation with concentrated hydrochloric acid. Bettendorffs reagent gives a lemon-yellow precipitate. Urine. In suspected arsenic poisoning first examine the urine, since arsenic is very slowly eliminated by this channel. Carlson in experiments upon him- self was able to detect arsenic direct in the urine by the electrolytic method and also by the Gutzeit and Marsh tests. He took 10 drops of Fowler's solution 1 daily. Five days after the last dose Carlson could still get a distinct test for arsenic in concentrated urine. The urine was not wholly free from arsenic until 14 days had passed. He then experimented with sodium cacodylate, taking daily 29 drops of a i per cent, solution. He could not detect a trace of arsenic in the urine by the electrolytic method. Therefore the salt of cacodylic acid had passed through the organism unaltered. But cacodylic acid can be de- tected easily in the urine, upon treating the latter with hypophosphorous acid (sp. gr. i.is). 2 Cacodylic oxide is formed and can be recognized by its odor. Sometimes the mixture must stand several hours in a closed test-tube. Arrhenal, in daily doses of about 30 drops of i per cent, solution, behaved like the cacodyl compound. Arsenic could not be detected in the urine by electrolysis. Con- sequently neither arsenious nor arsenic acid had been formed within the organism. Hypophosphorous acid immediately precipitated arsenic from arrhenal and gave the cacodyl odor. To detect cacodylic acid in urine, phosphorous acid, as well as zinc or tin and hydrochloric acid, may be used instead of hypophosphorous acid. Frequently it is advisable to oxidize most of the organic matter in the urine beforehand. Boil 25 cc. of urine with 25 cc. of water, 5 per cent, potassium permanganate solution and 10 cc. of 25 per cent, sodium hydroxide solution, until the filtrate is odorless and nearly colorless. Excess of hydrochloric acid (sp. gr. 1.19) and zinc filings, added to this nitrate, produce with heat the odor of cacodyl, if the urine contains cacodylic acid. Arsenic from atoxyl can be isolated at the cathode in the form of arsine by electrolysis. Therefore arsenic can be detected in the urine electrolytically after administration of atoxyl. Quantitative Estimation of Minute Amounts of Arsenic (Karl Th. Morner 3 ) This method is said to be useful in estimating arsenic quantitatively in various kinds of fabrics and in urine in cases of poisoning. It is a titration method de- vised especially for quantities of arsenic not exceeding 0.5 mg. Arsenic is first precipitated as trisulphide with thioacetic acid, CH 3 .CO.SH. Under the conditions arsenious as well as arsenic acid is thus precipitated. In alkaline 1 Fowler's solution contains i per cent, of As 2 0j as potassium arsenite. 2 Instead of free hypophosphorous acid, prepare Engel and Bernard's arsenic reagent (Comptes rend, de 1'Acad. des sciences 122, 399), or J. Bougault's (I. Pharm. Chim. (6), 15, 527). Dissolve 20 grams of sodium hypophosphite in 20 cc. of water and add 200 cc. of hydrochloric acid (sp. gr. 1.17). Filter through a cotton plug to remove NaCl and use the nitrate. 3 Zeitschrift fur analytische Chemie 41, 397 (i9 2 )' 248 DETECTION OF POISONS solution potassium permanganate readily oxidizes arsenic trisulphide completely to arsenic acid and sulphuric acid: As 2 S 3 + 140 = As 2 5 + 3SO 3 . Potassium permanganate solution, added to an alkaline solution of arsenic trisulphide, immediately loses its color, being decomposed in the proportion of 9 molecules to i molecule of arsenic trisulphide. 1 Since 2 molecules of potassium permanganate in sulphuric acid solution yield 5 atoms of oxygen for oxidation, 9 molecules 'according to the proportion 2 :$ = 9 :x (x = 22.5) should give 22.5 atoms of oxygen. But according to the reaction above, only 14 atoms of oxygen are used to oxidize i molecule of arsenic trisulphide, whereas the remaining 8.5 atoms are stored up in the precipitate as hydrated manganese dioxide (MnO 2 .H 2 O). But if the reaction mixture is heated with oxalic acid in presence of dilute sulphuric acid, these oxygen atoms become active: (MniO O) COOH + I i = MnSO 4 + H 2 + 2 C0 2 + H,O. so 4 iHsi cob ;H Since 2 molecules of KMnO4 yield 5 atoms of oxygen and since 14 atoms of oxygen are necessary for i molecule of As 2 S 3 , according to the following pro- portion Atoms : Mols.KMnO 4 5 : 2 = 14 : x (x = 5.6) 5.6 molecules of potassium permanganate are required for i molecule of As 2 Sj (= 214), or 2 atoms of arsenic (= 150). 1000 cc. of o.oi n-potassium permanganate (= 0.3162 gram KMnO4) contain in solution = 0.002 gram-molecule of KMnO 4 which according 10 X 10 X 10 to the proportion Gram-mols.KMnO 4 : Grm. As 5.6 : 150 = 0.002 : x (x = 0.0536) represents 0.0536 gram of arsenic. Hence 1000 cc. of o.oi n-potassium per- manganate solution correspond to 0.0536 gram of arsenic. Procedure. Dissolve arsenic trisulphide in 0.5 per cent, potassium hydroxide solution 2 and run this solution into a small flask containing 25 cc. of o.oi n-potas- sium permanganate solution. Mix the contents and add 5 cc. of 5 per cent, sul- phuric acid, as well as the quantity of o.oi n-oxalic acid solution found necessary by special titration. Warm until the color is discharged and finally titrate with o.oi n-potassium permanganate solution. 1 Since 2 mols. KMnO 4 give in alkaline solution 3 atoms of available oxygen (2KMnO 4 = 2MnO 2 + 30 + K 2 O), i mol. of As 2 S 3 , according to the pro- portion: 3:2 = i4:x (x = 9.33), requires not 9 mols, but more exactly 9.33 mols. of KMnO 4 . 2 Ammonium Hydroxide cannot be substituted for potassium or sodium hydroxide solution. SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 249 Prelimnary Titration. Add the same quantity .of 0.5 per cent, potassium hydroxide solution used to dissolve arsenic trisulphide, as well as 5 cc. of 5 per cent, sulphuric acid, to 25 cc. of o.oi n-potassium permanganate solution. Heat the mixture to boiling and add oxalic acid solution in slight excess so that the liquid becomes colorless. Titrate back with o.oi n-potassium permanganate. This titration shows how much oxalic acid solution, in conjunction with traces of reducing substances that may be present in the potassium hydroxide solution or sulphuric acid, is needed in a regular titration for the exact reduction of 25 cc. of o.oi n-potassium permanganate. Example. Suppose that 25.5 cc. of oxalic acid solution were required to decolorize the boiling liquid. Titration required 0.3 cc. of o.oi n-potassium per- manganate solution. Therefore 25 + 0.3 = 25.3 cc. of o.oi n-potassium per- manganate correspond to 25.5 cc. of oxalic acid solution and 25 cc. of the former correspond to 25.2 cc. of the latter solution. Consequently in a regular titration 25-2.cc. of o.oi n-oxalic acid solution must be used. The amount of potassium permanganate in 25 cc. of o.oi normal solution is sufficient for all amounts of arsenic up to 0.5 mg. Morner's method of deter- mining arsenic gives very reliable results, if arsenic is in the form of the trisul- phide and free from every other substance soluble in 0.5 per cent, potassium hydroxide solution and capable of reducing permanganate. Using the strongest hydrochloric acid, Morner first distils arsenic as arsenic trichloride by the Schneider-Fife method. About 200 sq. cm. of carpet, 100 sq. cm. of other woven and paper materials and 15 grams of sealing wax, stearine of wax candles and dried apples were used for each determination of arsenic by this method. According to Morner, the distillate from such materials by the Schneider-Fife method always contains organic matter, even when caught in dilute nitric acid. To remove this organic matter before precipitating arsenic with thio-acetic acid, collect the distillate in a receiver containing dilute nitric acid and evaporate to dryness in a porcelain dish. Add to the small residue in the dish upon the water-bath successively 2 cc. of potassium hydroxide solution (0.5 per cent. KOH) heating i minute, then 2 cc. of potassium permanganate solution (5 per cent. KMnO 4 ) heating about 3 minutes, and finally i cc. of tartaric acid solution (20 per cent. Hi.C&tOs) 1 heating until the color is dis- charged. Filter into a porcelain dish, wash the filter with a little water and set the dish upon a boiling water-bath. Add after i minute i cc. of thio-acetic acid (5 per cent. CH 3 . COSH) 2 and warm the mixture 3 minutes. Arsenic is precipi- tated as arsenic trisulphide. After cooling for 5 minutes, collect the precipitate upon a filter and wash first 5 'times with 2 cc. portions of 0.5 per cent, sulphuric acid and then 3 times with 2 cc. portions of water. Place under the funnel a 1 Tartaric acid readily dissolves the precipitate of manganese peroxide. To reduce the latter, Morner used oxalic and lactic acids, sodium sulphite and also thio-acetic acid. But tartaric acid proved to be bettter than any of these substances. 2 Prepare thio-acetic acid solution by shaking 5 cc. of thio-acetic acid with 100 cc. of water. Filter and keep this solution in a dark flask. This solution gradually decomposes with evolution of hydrogen sulphide: CH 3 .COSH+H 2 =CH,.COOH+H,S. 250 DETECTION OF POISONS small flask containing 25 cc,of o.oi n-potassium permanganate solution and pour over the filter 3 portions of 0.5 per cent, potassium hydroxide solution, using 2 cc. each time. The alkaline solution of arsenic trisulphide thus drops directly into the permanganate solution. Otherwise, proceed as described. Subtract 0.3 cc. of o.oi n-permanganate solution from the volume of this solution used. This correction is necessary because even the finer qualities of filter paper contain traces of substances which dissolve in 0.5 per cent, potassium hydroxide solu- tion and reduce permanganate. 1 Note. The procedure described separates arsenic trisulphide from every other substance soluble in 0.5 per cent, potassium hydroxide solution and capable of reducing potassium permanganate. This method is accurate to 0.02 mg. of arsenic. Detection of Salicylic Acid in Foods and Beverages Wine. 2 Place 50 cc. of wine in a cylindrical separating funnel with 50 cc. of a mixture of equal parts of ether and petroleum ether. Shake frequently, taking care not to form an emulsion but yet to mix the liquids thoroughly. Remove the ether- petroleum ether layer, pour through a dry filter, evaporate upon the water-bath and add a few drops of ferric chloride solu- tion to the residue which becomes red-violet if salicylic acid is present. But if the color is black or dark brown, add a few drops of hydrochloric acid, dissolve in water, extract with ether-petroleum ether and proceed with the extract as just described. Meat and Meat Products. 3 For experimental purposes add about o.oi gram of salicylic acid to some chopped meat. Ex- tract the finely divided material with 50 per cent, alcohol and add some milk of lime to the filtered alcoholic solution. Evapo- rate to dryness upon the water-bath and stir the residue with a slight excess of dilute sulphuric acid. Shake with ether without filtering, pass the ether extract through a dry filter and evapo- 1 After passing through the entire process in several blank experiments, Morner never obtained higher results for permanganate used. Consequently the method of washing described completely removes tartaric and thio-acetic acids. 2 "Official Directions for the Chemical Examination of Wine" of June 25th, 1896. (German.) 3 "Agreements with regard to uniformity in inspecting and testing foods, household supplies and other articles used in the German Empire" Heft I, 36. SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 251 rate. Dissolve the residue in hot water and test the filtered solution for salicylic acid with very dilute ferric chloride solution. Milk. 1 Mix 100 cc. of milk with 100 cc. of water at 60. Precipitate with acetic acid and mercuric nitrate solution, using 8 drops of each, shake and filter. Extract the filtrate with 50 cc. of ether, evaporate the ether, dissolve the residue in 5 cc. of hot water and test the filtered solution for salicylic acid with dilute ferric chloride solution (sp. gr. i.oo5~i.oib). Maltol Maltol, CeHeOs, 2 is formed in the preparation of caramel from malt, possibly from maltose or isomaltose. Ether or chloroform extracts this substance from the condensed vapors given off during caramelization and also from beer-wort. Maltol crystallizes in monoclinic prisms and plates from a cold saturated solution in 50 per cent, alcohol (Osann). Chloroform gives denser crystals. This sub- stance dissolves with difficulty in cold water or benzene; more readily in hot water, alcohol, ether or chloroform; and is insoluble in petroleum ether. It dis- solves in caustic alkaline solutions but is reprecipitated by carbon dioxide. Maltol sublimes in shining leaflets and is volatile with water vapor. It reduces silver solution in the cold and Fehling's solution with heat. An aqueous maltol solution resembles salicylic acid in becoming intense violet with ferric chloride solution, but differs from carbolic and salicylic acids in not turning red with Millon's reagent. Maltol, shaken with benzoyl chloride and sodium hydroxide solution, gives a mono-benzoyl derivative and consequently must contain one hydroxyl group. Aqueous Chloral Hydrate Solution as a Solvent for Alkaloids, Glucosides and Bitter Principles and Its Use in lexicological Analysis Richard Mauch (Communication from Professor E. Schaer's Institute, Strassburg) One part of water at 17.5 dissolves 4 parts of chloral hydrate, forming a very mobile solution which is easily filtered and capable of being kept for a long time without decomposition. This 80 per cent, solution of chloral hydrate easily dissolves relatively large quantities of alkaloids and glucosides without altering them chemically. At 17.5 one part of each of the following substances re- quires for solution the number of parts of solvent stated in the table: 1 Method of Ch. Girard, Zeitschrift fiir analytische Chemie 22, 277 (1883) and the above "Agreements" Heft I, 62. 2 J. Brand, Berichte der Deutschen chemischen Gesellschaft 27,806 (1894), H.Kiliani and M. Bazlen, Ibidem 27, 3115 (1894). 252 DETECTION OF POISONS Chloral Hydr Solution (80%) ate Water 600 20OO 700 500O 5OOO 6600 Ether 5 freely soluble freely soluble 1250 125 1300 Chloroform 3-5 2 freely soluble 100 4 6 6 Cocaine ... 5 Morphine Santonin Strychnine 5 4 6.5 6 5 Veratrine. . . 7. ? Caffeine is the only alkaloid which forms with chloral hydrate a molecular compound soluble in water. If a chloral hydrate solution of an alkaloid, which has been freshly prepared in the cold, is diluted with considerable water, the unchanged alkaloid is precipitated almost quantitatively, for instance, morphine, strychnine and quinine. Substances like picrotoxin, santonin and acetanilide behave similarly. But when such solutions stand for a long time at ordinary temperatures, or are heated for 1-2 hours, chloral hydrate is decomposed by the vegetable base into chloroform and formic acid. Since the alkaloidal salts of formic acid are soluble in water, dilution with this solvent does not precipitate the alkaloids. R. Mauch has shown clearly that atropine, brucine, quinine, cocaine, morphine, narcotine, strychnine and veratrine behave as just described. In the tests ordinarily made with the ether or chloroform residue, R. Mauch recommends dissolving the residue in 80 or 60 per cent, chloral hydrate solution. The "chloral solution" should prove of great value in color tests which depend upon the use of pure sulphuric acid or sulphuric acid containing iron or molybdic acid. These solutions contain so little water that it cannot modify the action of sulphuric acid upon the substance in solution. Such a "chloral solution" is also well adapted for zone tests. An aqueous solution forms an upper layer with the "chloral solution," and the latter forms an upper layer with concentrated sulphuric acid. Specific gravity of 80 per cent, chloral hydrate solution = 1.514. Specific gravity of 60 per cent, chloral hydrate solution = 1.3535. In the tests ordinarily performed in test-tubes, it is best to use small tubes (6 or 7 cm. high; i cm, in diameter) holding 6 cc. They should not be made of too thin glass. The chloral solution cannot be used in detecting picrotoxin, be- cause chloral hydrate itself produces the same reduction changes caused by picrotoxin. The same is true of the test for strychnine, where sulphuric acid and potassium dichromate, or any other oxidizing agent, are used. Coniine and nicotine also belong to the class of alkaloids which cannot be detected in chloral hydrate solution. Concentrated chloral hydrate solutions cannot be used di- rectly in making tests with general alkaloidal reagents, because precipitates do not appear until the solutions have been diluted with 6-8 volumes of very dilute hydrochloric or sulphuric acid. In using the "chloral hydrate method'' in toxicological analysis, the ether, chloroform or amyl alcohol extract should be evaporated with gentle heat upon a SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 253 watch-glass of medium size (about 5 cm. diameter) and not too flat. Add to the residue, depending upon the quantity, about 3 cc. of 75 per cent, chloral hydrate solution. Cover the glass and let it stand for some time. Occasionally tilt the glass and bring the solution thoroughly in contact with the residue. Pass the solution through a very small filter, if necessary, and wash both watch-glass and filter with a few drops of pure chloral hydrate solution. Use this chloral hydrate solution for the individual tests. In testing for strychnine, evaporate a part of the chloral solution to dryness upon the water-bath. Warm, until the residue does not smell of chloral, and then test for strychnine with sulphuric acid and potassium dichromate. To recover from the chloral hydrate solution most of the alkaloids and sub- stances like picrotoxin, acetanilide and phenacetine, add excess of sodium hy- droxide solution and extract thoroughly with a little chloroform. The "chloral hydrate method" is conducive to very neat work and this is a great advantage. The use of metallic utensils like knives and spatulas is entirely unnecessary. ESTIMATION OF ALKALOIDS i. Picrolonate Method of H. Matthes 1 Knorr 2 gave the name picrolonic acid to i-p-nitrophenyl-3- methyl-4-isonitro-5-pyrazolone. This compound is formed by the action of nitric acid upon methyl-phenyl-pyrazolone. Picrolonic acid resembles picric acid in its properties and is characterized by forming crystalline salts with many organic bases, as the alkaloids. As a rule these salts dissolve with dimculty and are yellow or red. Heat causes their decomposi- tion. Picrolonic acid is frequently of service in characterizing bases. Hydrochloric acid precipitates this compound from a solution of its sodium salt as a yellow, mealy powder, melting when rapidly heated at about 128, becoming dark in color and undergoing decomposition with rapid evolution of gas. Knorr first gave picrolonic acid formula I but formula II 3 is preferred: I. N0 2 .C 6 H 4 .N II. NO 2 .CeH4.N I/\.OH N CO H. Matthes has estimated many alkaloids quantitatively by 1 H. Matthes and O. Rammstedt, Zeitschrift fur analytische Chemie 46, 5^5 (1907) and Archiv der Pharmazie 245, 112 (1907). 2 Berichte der Deutschen chemischen Gesellschaft 30, 914 (1897)- 3 R. Zeine, Inaugural Dissertation, Jena, 1906. 254 DETECTION OF POISONS means of picrolonic acid. Collect the precipitated alkaloidal picrolonate in a weighed Gooch crucible, wash, dry and weigh. Estimation of alkaloids is possible by this method, because the picrolonates are constant in composition. Morphine, hydras- tine, codeine, strychnine, brucine, pilocarpine and stypticine 1 can be quantitatively estimated by this method. Estimation of Morphine, Codeine and Stypticine in Solutions, Tablets and Sugar Triturations Dissolve the weighed trituration or tablet in the smallest quantity of water possible and add picrolonic acid solution (about o.i n-solution in alcohol) in slight excess. The picro- lonate separates at once, or very soon, as yellow crystals or a crystalline meal. Cool 15-30 minutes in ice water and collect the precipitate in a weighed Gooch crucible. Wash with a little ice water, dry 30 minutes at 110 and weigh. Morphine picrolonate usually separates in 10-30 minutes. Cooling aids precipitation. Formula Mol. Wt. Decomposition-point Morphine picrolonate: CnHigNOs.CioHsNAi. 549 200-210 Codeine picrolonate: CisHziNOs.CioEUN^. 563 about 225 Cotarnine picrolonate: C^HisNO^CioHsNAi. 501 205-210 Notes. Practice Analyses: Morphine powder: 0.01-0.02 gram morphine hy- drochloride, CnHigNOs.HCl.sHkO + 0.5 gram sugar. 0.2-0.5 g ram codeine phosphate, Cigl^iNOs.HaPO^HzO + 0.5 gram sugar. Stypticine tablets E. Merck. Do not use too dilute solutions of the alkaloids in these determinations and do not wash the picrolonate precipitates with too much water. Dissolve the pow- dered morphine and sugar mixture in about 5-10 cc. of water. Matthes and Rammstedt in examining the morphine powder obtained the following results: Weights taken: 0.019 morphine hydrochloride + 0.5 gram sugar. Results obtained: I. 0.0273 -gram morphine picrolonate = 0.0187 gram morphine hydrochloride. II. 0.02 74 gram morphine picrolonate = 0.0187 gram morphine hydrochloride. In a second experiment every 10 cc. of an aqueous solution contained 0.0104 gram of morphine hydrochloride. Results obtained: I. 0.0147 gram morphine picrolonate = o.oioi gram morphine hydrochloride. II. 0.0146 gram morphine picrolonate = 0.0099 gram morphine hydro- chloride. 2 1 Stypticine = cotarnine hydrochloride, Ci2HiBNO 4 .HCl.H 2 O. 2 Professor Matthes has kindly stated that this picrolonate method gives less satisfactory results with smaller quantities of morphine (0.005 and less;. SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 255 The application of the picrolonate method to the estimation of hydrastine in hydrastis root and extract, of nux vomica alkaloids in nux vomica and extract and of pilocarpine in jaborandum leaves is described in Chapter VI (see pages 282, 296 and 286. 2. Estimation of Alkaloids by Means of Potassium Bismuthous Iodide (H. Thorns 1 ) Dissolve the particular alkaloid in sulphuric acid and pre- cipitate completely with potassium bismuthous iodide prepared as described by Kraut. 2 Decompose the precipitate with a mixture of sodium carbonate and hydroxide, extract the free alkaloid with ether and weigh. By this method Thorns has recovered atropine, hyoscyamine, scopolamine, strychnine, quinine, caffeine and antipyrine from their potassium bismuthous iodide precipitates unaltered and nearly quan- titatively. He has also used this method with success in estimating quantitatively the alkaloids in belladonna extract. Procedure. Dissolve the alkaloidal salt, or 2 grams of bella- donna extract, in 50 cc. of water. Add first locc. of 10 per cent, sulphuric acid, stir and precipitate with 5 cc. of potassium bis- muthous iodide solution. Collect the precipitate upon a dry filter and wash twice with 5 cc. portions of 10 per cent, sulphuric acid. Transfer the thoroughly drained precipitate and paper to a wide-mouth extraction cylinder having a tight glass stopper. Add 0.3 gram of sodium sulphite, then 30 cc. of 15 per cent, so- dium hydroxide solution and shake. Add quickly 15 grams of sodium chloride and 100 cc. of ether. Shake frequently and let stand for 3 hours. The ether contains the alkaloid and settles well. Remove with a pipette 50 cc. of the ether solution ( = half the solution of the alkaloidal salt, or i gram of belladonna extract) and titrate this ether solution in a flask with o.oi n-hydrochloric acid, using iodeosine as indicator. After titrating the belladonna alkaloids, use in the calculation the equivalent weight of atropine-hyoscyamine, Ci7H 23 NO3= 1 Berichte der Deutschen pharmazeutischen Gesellschaft 13, 240 (1903); iS> 85 (1905); 16, 130 (1906) (D. Jonescu). 'Annalen der Chemie und Pharmazie 210, 310 (1882). See "Preparation of Reagents," page 318. 256 DETECTION OF POISONS 289. 1000 cc. of o.oi n-hydrochloric acid correspond to 2.89 grams of atropine-hyoscyamine. Atropine and hyoscyamine being isomeric, monacid bases, their formula weight and equiva- lent weight are the same. Quinine, caffeine and antipyrine were also recovered un- altered from potassium bismuthous iodide precipitates. After decomposition of the precipitates, they were obtained almost quantitatively but were estimated gravimetrically. Dissolve 2 grams of quinine 1 in 50 cc. of water acidified with sulphuric acid and precipitate with potassium bismuthous iodide. Filter the precipitate with suction and wash with 5 per cent, sulphuric acid. Transfer precipitate and paper to an extraction cylinder and shake thoroughly with a mixture of 20 grams of crystallized sodium carbonate and 40 cc. of 10 per cent, sodium hydroxide solution. The yellowish red precipitate gradually becomes white. Add 30 cc. of ether and shake well for 30 minutes. Pipette off 25 cc. of the clear ether solution, evaporate in a weighed glass dish, dry the residue at 100 and weigh. The weight of quinine was 0.9405 instead of i gram. Caffeine was estimated in the same way, except that this alkaloid was extracted with chloroform after decomposition of the potassium bismuthous iodide precipitate with alkaline hydroxide and carbonate. The weight of caffeine was 0.9546 instead of i gram. The precipitate obtained by adding potassium bismuthous iodide to a solution of antipyrine in sulphuric acid (10 per cent. H^SO.*) is not decomposed as easily as are those of quinine and caffeine. The precipitate from 2 grams of antipyrine must be shaken i hour with 20 grams of sodium carbonate and 60 cc. of ip per cent, sodium hydroxide solution. Antipyrine must be extracted with chloroform. The weight of antipyrine was 0.9273 instead of i gram. Notes. Potassium bismuthous iodide precipitates fixed and volatile alkaloids but not ammonium salts. If the estimation of volatile bases is unnecessary, as in the examination of belladonna extract, evaporate the 50 cc. of ether extract (see above) upon the water-bath. Warm the residue and in a few minutes 1 According to experiments of D Jones cu (loc. ciL}. SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 257 the strong narcotic odor of volatile bases will disappear. Dissolve the residue in a little acid-free alcohol and dilute with ether. Before using a flask for titrations carefully test it beforehand for alkalinity. If a positive test is ob- tained, alkalinity must be removed. An odor like iodoform, probably due to the action of sodium hypo-iodite upon the alkaloid, has been observed when sodi- um hydroxide solution acts upon potassium bismuthous iodide precipitates. Addition of sodium sulphite may prevent this action. After addition of sodium chloride ether takes up the alkaloid more readily. But vigorous shaking is always needed to cause complete transfer of alkaloid to the ether. 3. Estimation of Alkaloids by H. M. Gordin 1 Gordin has found that periodides of the alkaloids, whatever be their composi- tion, when precipitated from aqueous solution by iodo-potassium iodide in pres- ence of acids, always contain one equivalent of combined acid for every molecule of monacid alkaloid. These periodides have the general formula (Alkaloid, HI) m I n . Iodo-potassium iodide, added to a solution of a monacid alkaloid acidified with hydrochloric acid, first gives an alkaloid hydrochloride, changed by potassium iodide to alkaloid hydriodide and finally precipitated as insoluble periodide by taking up iodine: (a) Alkaloid + HC1 = Alkaloid.HCl, (0) Alkaloid.HCl + KI = Alkaloid.HI + KC1, (7) m (Alkaloid.HI) + In = (Alkaloid.HI) mln = Precipitate. In the precipitation of an alkaloid in acid solution with iodo-potassium iodide, one equivalent of acid goes with the precipitate and disappears from solution. In many cases potassium mercuric iodide may be substituted to advantage for iodo-potassium iodide. Gordin has found that the composition of the precipi- tate changes only as regards mercuric iodide and not as far as acid is concerned, for in this case also the precipitate contains one equivalent of acid for a monacid alkaloid. Use in the titration 0.05 n-hydrochloric acid and 0.05 n-potassium hydroxide solution. Prepare a solution containing a weighed quantity of pure alkaloid, for example, pure morphine. Dissolve about 0.2 gram of chemically pure morphine, previously completely dehydrated at 120, in 30 cc. of 0.05 n-hydrochloric acid in a 100 cc. volumetric flask. Shake and add gradually iodo-potassium iodide to this solution, until precipitation ceases and the supernatant liquid is dark red. Dilute to the 100 cc. mark with water and shake vigorously until the liquid above the precipitate is entirely clear. Pass 50 cc. of solution through a dry filter, de- colorize the filtrate with a few drops of sodium thiosulphate solution and titrate excess of 0.05 n-hydrochloric acid with 0.05 n-potassium hydroxide solution, using phenolphthalein as indicator. Calculate from the result how many grams of morphine have been neutralized by i cc. of the acid. Comparison of the equiva- lent weight of morphine with that of any other monacid alkaloid gives the corre- sponding factor to be used in the calculation. For example, Gordin found in his experiments that i cc. of approximately 0.05 n-hydrochloric acid neutralized 1 Berichte der Deutschen chemischen Gesellschaft 32, 2871 (1899). Archiv der Pharmazie 238, 335 (1900). Gordin and A. B. Prescott, Archiv der Pharmazie 237, 380 (1899). 17 258 ' DETECTION OF POISONS 0.0137 gram of anhydrous morphine, CnHigNOs. The factor (x) for strychnine, C2iH22N 2 O2 (= 334), which is also a monacid base, is as follows: Morphine : Strychnine 285 : 334 = 0.0137 : x (x = 0.0160) and that for the monacid base cocaine, Ci7H 21 NO 4 (= 303), according to the proportion is: Morphine : Cocaine 285 : 303 = 0.0137 : x (x = 0.0146). If potassium mercuric iodide is used to precipitate an alkaloid, the method is the same as with iodo-potassium iodide except that there is no need of treating the 50 cc. of filtered solution with thiosulphate. Berberine and colchicin cannot be estimated by Gordin's method. Quantitative Estimation of Strychnine and Quinine Together E. F. Harrison and D. Gair 1 Occasionally a small amount of strychnine must be estimated in presence of a relatively large quantity of quinine, as in certain pharmaceutical preparations. 2 Separation of the two alkaloids is possible by means of Rochelle salt. Quinine tartrate, (C 2 oH24N 2 O 2 )2.C4H6O 6 .2H 2 O, being difficultly soluble in water, forms a white crystalline precipitate, whereas strychnine tartrate remains in solution. Procedure. Render the solution of the mixed alkaloids in about 40 .cc. of water faintly acid with sulphuric acid. Add enough ammonia to cause a slight turbidity, then 15 grams of solid Rochelle salt and more ammonia, still leaving the liquid acid to litmus paper. Heat for 15 minutes upon the water-bath and then set aside for 2 hours until entirely cold. Filter precipitated quinine tartrate by suction and wash with aqueous Rochelle salt solution (15 grams of salt in 45 cc. of water) containing 1-2 drops of dilute sulphuric acid. To determine strychnine, add sodium hydroxide solution to the combined nitrate and wash water from quinine tartrate until the reaction is alkaline. Extract 2-3 times with chloroform, pour the chloroform extract through a dry filter and distil in a weighed flask to about 4 cc. Add 10 cc. of absolute alcohol and evaporate to dryness upon the water-bath. To remove quinine still adhering to the strych- nine, extrast the dry residue 2-3 times with i cc. portions of ether, 3 dry at 100 and weigh. This residue consists of pure, quinine-free strychnine. Estimation of Toxicity of Chemical Compounds by Blood Haemolysis (A. J. J. Vandevelde 4 ) Vandevelde originally used living cells of a variety of Allium cepa (red Bruns- wick onion), the cell membrane of which is rich in anthocyan. The presence of this substance obviated the necessity of using a special coloring matter in determining plasmolytically the toxicity of alcohols, ethereal oils and other substances. 6 Vandevelde has recently recommended determining the toxicity of chemical compounds by blood haemolysis, using for this purpose defibrinated ox 1 Pharmaz. Journ. (4) 17, 165. 2 Compound Syrup of Hypophosphites, U. S. P. 3 More ether dissolves a weighable quantity of strychnine. 4 Chemiker Zeitung 29, 565 (1905). 6 Bulletin de 1' Association Belgee de Chimie 17, 253. SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 259 blood (see pages 229 and 230). To establish the toxicity of different alcohols, the concentration at which haemolysis just ceases is determined. A solution, in which blood-corpuscles are not hydrolyzed after a definite time but are hydrolyzed upon addition of the slightest trace of the substance being examined, is a non- toxic solution for blood-corpuscles, called by Vandevelde a "critical solution." The estimation requires: 1. A solution of 0.9 per cent, sodium chloride in 50 per cent, alcohol by volume. 1 2. An aqueous 0.9 per cent, sodium chloride solution. 3. A suspension of 5 per cent, defibrinated ox blood in 0.9 per cent, aqueous sodium chloride solution. Experiments are made in test-tubes. Place first in each of several tubes 2.5 cc. of the mixtures (of different concentration) of alcoholic and aqueous sodium chloride solution and then 2.5 cc. of the suspended blood. The end-point for the appearance of haemolysis was set at three hours. Vandevelde's experiments with ethyl alcohol gave the following results: Alcoholic con- Cc. of alcoholic Cc. of aqueous centration of After 3 Cc. of sus- NaCl solu- NaCl solution mixture in vol. hours pended blood tion per cent. 2-5 2.20 0.30 22. O Haemolysis 5 2-15 0-35 21-5 Haemolysis 5 2. 10 0.40 21.0 Haemolysis 5 2.05 o.4S 20.5 Haemolysis 5 2.00 0.50 2O. O Haemolysis 5 i-9S o-SS 19-5 No haemolysis 5 1.90 0.60 19.0 No haemolysis Consequently the critical solution of ethyl alcohol is one containing 19.5 cc. of absolute alcohol (C 2 HO) in 100 cc., or 15.489 grams of C 2 H 6 O in 100 cc. The specific gravity of absolute alcohol being 0.7943, 19.5 cc. weigh 19.5 X 0.7943 15-489. According to Vandevelde's experiments addition of methyl alcohol diminished the toxicity of ethyl alcohol, whereas the higher alcohols were found to be more toxic than the latter. If the toxicity of 100 parts by weight of ethyl alcohol is taken as 100, then 47 parts by weight of isopropyl, 29 parts of isobutyl and 12.5 parts of amyl alcohol are isotoxic with that quantity of ethyl alcohol. Using the plasmolytic method with onion cells, Vandevelde obtained the following results with the same series: 100, 36.8, 21.2, 12.6. The hzemolytic method is easily performed in test-tubes and does not require the use of the microscope. The form of the tube, especially its diameter, is quite important in these experiments. The speed of haemolysis increases with the diameter of the tube. The quantity of the blood-corpuscles is of slight influence except in narrow tubes and at the beginning of the reaction. Vandevelde applies the -term "critical coefficient" to the number giving the concentration of a sub- stance necessary to kill the cells. 1 The specific gravity of such an alcohol at 15 is 0.9348. CHAPTER VI QUANTITATIVE ESTIMATION OF ALKALOIDS AND OTHER PRINCIPLES Estimation of Alkaloids in Drugs and Pharmaceutical Preparations (German Pharmacopoeia) Alkaloids are nitrogenous bases occurring in plants. The term "plant base" is synonymous with alkaloid. The plant families especially rich in alkaloids are berberideae, cinchonaceae, papaveracese, solanaceae and strychnaceae. Alkaloids as a rule are not uniformly distributed in all parts of plants. They occur most often in roots, fruits and seeds. If the plant is a tree, there is often more alkaloid in the bark than in other parts. The particular part of the plant usually contains only a few per cent, of alkaloid. Quinine bark is an exception, the quantity of alkaloid being 5-10 per cent, and sometimes more. Plants as a rule do not contain free alkaloids but their salts. They are combined not only with the mineral acids, sulphuric, hydro- chloric and possibly phosphoric, but with organic acids, as malic, aconitic, tannic, citric, quinic and meconic. Most free plant bases dissolve only slightly in water but readily in ether and chloroform. The German Pharmacopoeia prescribes a mixture of ether and chloroform for the extraction of free alkaloids. The finely powdered drug should first be treated with a solution of sodium hydroxide, ammonia or sodium carbonate to liberate alkaloids from their salts: C 20 H 24 N 2 O 2 C 6 H 7 (OH) 4 COOH + NaOH = C 20 H 24 N O 2 + C 6 H 7 (OH)4COONa + HzO. Quinine quinate 1 Quinine Sodium quinate The ether-chloroform mixture removes not only alkaloids from the drug but varying amounts of other substances, as fat, 1 Cinchona bark contains quinine in the form of this salt. 260 QUANTITATIVE ESTIMATION OF ALKALOIDS 261 resin, wax and pigments. To free the alkaloid from such im- purities, shake the ether-chloroform extract with a measured excess of o.i or o.oi n-hydrochloric acid. The alkaloid passes into aqueous solution as hydrochloride : C 2 oH 24 N 2 2 + HC1 = C 2 pH 24 N 2 2 .HCl Quinine Quinine hydrochloride But the impurities remain in the ether-chloroform mixture. Finally, determine excess of hydrochloric acid by titration with o.i or o.oi n-potassium hydroxide solution, employing usually iodeosine as indicator. Calculate the amount of alkaloid in the drug from the difference between the original quantity of acid and the excess. The estimation of alkaloids in drugs and pharmaceutical preparations, according to directions given by the German Pharmacopoeia, requires the following steps: 1. Liberation of alkaloids from salts by means of stronger bases, as potassium and sodium hydroxides, ammonia and sodium carbonate. 2. Extraction of free alkaloids with ether-chloroform mixture. 3. Transference of alkaloids from ether-chloroform to aque- ous o.i or o.oi n-hydrochloric acid solution. 4. Determination of excess of hydrochloric acid in an aliquot volume, usually 50 cc. of the hydrochloric acid solution of the alkaloid diluted to 100 cc., by titration with o.i or o.oi n-potas- sium hydroxide solution. Alkaloids in Aconite Root Officinal aconite root is the root of Aconitum Napellus col- lected at the end of flowering. Two alkaloids are present, namely, aconitine, Cs^rNOn, and picraconitine, C 3 iH 4 7NOio (?), characterized by its very bitter taste. Both alkaloids are combined with aconitic acid, CH.COOH C.COOH CH 2! COOH 262 DETECTION OF POISONS Boiled with water, or alcoholic potassium hydroxide solution, aconitine yields a new base, aconine, benzoic and acetic acids: 1 On + 2 H 2 O = CzsH^NOs + C H 4 O 2 + C 6 H 6 .COOH. Aconitine Aconine Acetic Benzoic acid acid This reaction presents aconitine as acetyl-benzoyl-aconine, /COCHa C 26 H 3 9NO9< X COCH 6 . Since aconitine has been shown to contain four methoxyl groups, the formula of this alkaloid may be written: /COCH, C 2 iH 27 (OCH 3 )4N0 6 < \COC 6 H 6 . Aconine, therefore, is CaiHarCOCHsMOH^NOs, and picra- conitine must be regarded as benzoyl-aconine, having the for- mula C 2 iH 21 (OCH 3 ) 4 (OH)NO4(COC6H 5 ). Estimation of Aconitine (German Pharmacopoeia) Place 12 grams of rather finely powdered aconite root dried at 100 in an Erlenmeyer flask and add 90 grams of ether and 30 grams of chloroform. Shake well and add 10 cc. of a mixture of 2 parts of sodium hydroxide solution and i part of water. Let the mixture stand 3 hours, shaking vigorously at frequent intervals. Then add 10 cc. of water, or enough to cause the powder to gather into balls after vigorous shaking and leave the supernatant ether-chloroform solution perfectly clear. After an hour pass 100 grams of the clear ether- chloroform solution through a dry filter kept well covered and receive the filtrate in a small flask. Distil about half the solvent and pour the remainder into a separating funnel. Wash the flask with three 5 cc. portions of a mixture of 3 parts of ether and i part of chloroform, and thoroughly shake the combined solutions with 25 cc. of o.oi n-hydrochloric acid. When the liquids have separated perfectly clear, add enough ether to bring the ether-chloroform solu- tion to the surface. Pass the acid solution through a small filter moistened with water and collect the filtrate in a 100 cc. flask. Make three more extractions of the ether-chloroform solution with 10 cc. portions of water, and pass these extracts through the same filter. Wash the latter with water and dilute the total solution with water to TOO cc. Place 50 cc. of this solution in a 200 cc. flask with 50 cc of water and enough ether to make a layer about i cm. thick. Add 5 drops of iodeosine solution and run in, while shaking, enough o.oi n-potassium hydroxide solution to turn the aqueous solution pale red. ' x Freund and Beck, Berichte der Deutschen chemischen Gesellschaft 27, 433. 720 (1894); 28, 192, 2537 (1895). QUANTITATIVE ESTIMATION OF ALKALOIDS 263 Calculation. Dissolve the aconite alkaloids set free from their salts by sodium hydroxide solution in 120 grams of ether-chloroform. Weigh 100 grams of this solution (= alkaloids from 10 grams of aconite root). Dissolve the alkaloids with 25 cc. of o.oi n-hydrochloric acid, bringing the volume to roo cc. Determine excess of acid in 50 cc. of this solution (= alkaloids from 5 grams of root). If, for example, this requires 8.5 cc. of o.oi n-potassium hydroxide solution, then the alkaloids have combined with 12.5 - 8.5 =4 cc. of o.oi n-acid. Since the equivalent weight of aconitine CsJ^NOu = 645, 100 cc. of o.oi n-hydrochloric acid unite with 6.45 grams of aconitine. The proportion 1000 : 6.45 = 4 : x (x = 0.0258) shows that 5 grams of aconite root contain 0.0258 gram o* alkaloids, correspond- ing to 0.51 per cent. The German Pharmacopoeia demands this quantity of aconitine in aconite root as a minimum. Using a different method, the United States Pharmacopoeia has the same limit. Estimation of Cantharidin in Spanish Flies (German Pharmacopoeia) Place 25 grams of Spanish flies ground mediumly fine in an Erlenmeyer flask and add 100 grams of chloroform and 2 cc. of hydrochloric acid. 1 Shake the mixture frequently during 24 hours. Then pour 52 grams of the chloroform solu- tion through a dry filter kept well covered, and collect the nitrate in a weighed flask holding 80-100 cc. Distil the chloroform, and add 5 cc. of petroleum ether to the residue. Stopper the flask, and let the mixture stand 12 hours, with oc- casional agitation. Dry at 100 and weigh a filter (5 cm. in diameter). Pass the liquid through this filter, having first moistened it with petroleum ether. Treat the undissolved residue twice with petroleum ether, each time using 10 cc. and shaking. Pass this solvent through the same filter, and disregard crystals adhering to the side of the flask. Dry the filter and flask, and wash both with a little water, containing a drop of ammonium carbonate solution to every 10 cc., until this solvent is only faintly yellow. Finally, wash once with 5 cc. of water, and dry both flask and filter. Place filter and contents in the flask, and dry at 100 to constant weight. The crystalline residue should weigh at least o.i gram. Notes. Additional information about cantharidin is given on page 203. Spanish flies contain cantharidin partly free and partly as an alkali salt of cantharidic acid (cantharidate) . Hydrochloric acid sets cantharidic acid free and the latter then passes at once into cantharidin, its internal anhydride. Conse- quently hydrochloric acid is essential to the determination of that cantharidin present in Spanish flies as cantharidate. Chloroform not only dissolves cantharidin but fatty_substances 1 Specific gravity 1.124 = 2 S P er cent. HC1. 264 DETECTION OF POISONS in the flies. To isolate pure cantharidin from these impurities, distil the chloroform and let the residue stand for 1 2 hours in the cold with petroleum benzene. Fat readily dissolves but can- tharidin is as good as insoluble in this solvent. The German Pharmacopoeia finally directs weighing the cantharidin from 12.5 grams of powdered Spanish flies. The quantity should be at least o.i gram, corresponding to 0.8 per cent, of cantharidin as a minimum. With sufficient care, white crystalline can- tharidin may be isolated from Spanish flies. Baudin obtained from good flies 1.06 per cent, of cantharidin, of which 0.72 per cent, was free and 0.34 per cent, combined as cantharidate. Dieterich found only 0.3 per cent, of free cantharidin. Estimation of Cinchona Alkaloids (German Pharmacopoeia) i. In Cinchona Bark. To determine total alkaloids, pour 90 grams of ether and 30 grams of chloroform upon 12 grams of finely ground cinchona bark, dried at 100 and placed in an Erlenmeyer flask. Add 10 cc. of sodium hydroxide solution. Shake vigorously at frequent intervals during 3 hours. Then add 10 cc. of water, or enough to cause the powdered cinchona to gather into lumps after vigorous shaking, thus leaving the supernatant ether-chloroform solution perfectly clear. Let the ether-chloroform solution ?tand an hour, and then pass 100 grams through a dry filter, kept well covered. Collect the filtrate in a flask, and distil half the solvent. Pour the remaining ether-chloroform solution into a separating funnel, and wash the flask three times with 5 cc. portions of a mixture of 3 parts of ether and i part of chloroform. Thoroughly extract the total ether-chloroform solution with 25 cc. of o.i n-hydrochloric acid. When the contents of the separating funnel are perfectly clear, add enough ether to bring the ether-chloroform solution to the surface. Pass the acid solution through a small filter moistened with water, and receive the filtrate in a 100 cc. flask. Make three more extractions of the ether-chloroform solution with 10 cc. portions of water, and pass these extracts through the same filter. Wash the filter with water, and bring the volume of the filtrate to 100 cc. Finally, measure 50 cc. of this solution with a pipette, and add freshly prepared hsematoxylm solution, made by dissolving a small particle of this substance in i cc. of alcohol. Shake and add enough o.i n-potassium hydroxide solution to give the mixture a yel- lowish color, which quickly changes after vigorous agitation to bluish violet. 1 Notes and Calculation. Both quinine and quinidine have the formula C 20 H 2 4N 2 O2 and cinchonine and cinchonidine the x The German Pharmacopoeia prescribes that not more than 4.3 cc. of o.i n-potassium hydroxide should be required. QUANTITATIVE ESTIMATION OF ALKALOIDS 265 formula Ci 9 H 22 N 2 O. These are the most important alkaloids in cinchona bark. They are present in all true cinchona barks as salts of quinic acid, CnHiaOe, and quino-tannic acid. Fuller information regarding the chemistry of quinine and cinchonine is given on page 119. Quinic acid is widespread in the vegetable kingdom. This monobasic, pentatomic acid, having the formula, C 6 H 7 (OH) 4 . COOH, is a hexahydro-tetroxy-benzoic acid. It crystallizes in large monoclinic prisms melting at 162. As far as the chemical behavior of quinic acid is concerned, either of the following formulas is possible: I. H OH II. H OH H 2 C CH.OH H 2 C CH.OH H 2 C CH.OH HO.HC CH 2 X X HO COOH HO COOH The formation of tetra-acetyl-quinic acid, C 6 H 7 COOH, and tetra-benzoyl-quinic acid, COOH, shows that quinic acid contains four alcoholic hydroxyl groups. Addition of sodium hydroxide solution to cinchona powder sets the alkaloids free from their salts: C 6 H 7 (OH) 4 COOH + NaOH = C 20 H 24 N 2 O 4 + H 2 O + C 6 H 7 (OH)4COONa. Quinine quinate Quinine Sodium qumate Only 100 grams of the original 120 grams of ether-chloroform mixture (=12 grams of cinchona powder) are in the nitrate. This solution contains the alkaloids in 10 grams of bark. These 100 grams are extracted with 25 cc. of o.i n-hydrochloric acid, the alkaloids passing into aqueous solution as hydro- chlorides, and the volume is brought to 100 cc. Finally, excess of a.i n-hydrochloric acid in 50 cc. (= alkaloids in 5 grams of bark) of this hydrochloric acid solution is determined by titration. In these determinations with very dilute hydro- 266 DETECTION OF POISONS chloric acid, cinchona alkaloids behave as monacid bases, 1 quinine forming C 2 oH 24 N 2 O 2 .HCl and cinchonine Ci9H 22 N 2 O.- HC1. The mean of the equivalent weight of quinine (324) and cinchonine (294), that is to say, (324 + 294) divided by 2 = 309, may be taken as the equivalent weight. This value agrees approximately with the actual quantities of these alkaloids in cinchona bark. Consequently 1000 cc. of o.i n-hydrochloric acid are equivalent to 30.9 grams of cinchona alkaloids. Example. Titration of 50 cc. of the hydrochloric acid solution of alkaloids, in preparing which 12.5 cc. of o.i n-hydrochloric acid were used, required 2.6 cc. of o.i n-potassium hydroxide solution, equivalent to the volume of o.i n-hydro- chloric acid in excess. 12.5-2.6 = 9.9 cc. of o.i n-hydrochloric acid have combined with the alkaloids in 5 grams of cinchona bark. The proportion Cc. o.i n-HCl:Grams of Alkaloids 1000 : 30.9 = 9.9 : x (x = 0.30591) shows that 5 grams of bark contain 0.30591 gram of alkaloids. Consequently 100 grams of bark contain 20 X 0.30591 = 6.11 grams of alkaloids. Titrate the filtered ether-chloroform solution of cinchona alkaloids at once. The solution should not be exposed for any length of time to direct sunlight. Otherwise chloroform may give free hydrochloric acid CHC1 3 + O = COC1 2 + HC1 which will neutralize alkaloids. The decomposition of 0.05 gram of chloro- form would give enough hydrochloric acid to neutralize 0.25 gram of cinchona alkaloids. Panchaud 2 has shown that such chloroform solutions of cinchona alkaloids after standing 12 hours yield only 80 per cent, of the total quantity of alkaloids originally present. Haematoxylin, CieHuOe-sHzO, occurs in logwood, the heart-wood of Haema- toxylon campechianum. It usually crystallizes in colorless, shining, quadratic prisms containing 3 molecules of water, more rarely in rhombic crystals with i molecule of water. It dissolves only slightly in cold water but freely in boiling water, alcohol or ether. In contact with air haematoxylin gradually becomes reddish. 2. In Aqueous and Alcoholic Cinchona Extracts. To determine total alkaloids in these preparations, dissolve 2 grams of the given extract in an Erlenmeyer flask, using 5 grams of water and 5 grams of absolute alcohol. Add 50 grams of 1 Quinine dihydrochloride, C2oH 2 4N2O2.2HCl, is formed by passing gaseous hydrogen chloride over quinine and also by dissolving the monohyflrochloride, C 2 oH24N 2 O2.HCl, in strong hydrochloric acid with gentle heat. An aqueous solution of the dihydrochloride has an acid reaction. 2 Schweizer Wochenschrift fur Pharmazie 44, 580. QUANTITATIVE ESTIMATION OF ALKALOIDS 267 ether and 20 grams of chloroform, and, after vigorous shaking, 10 cc. of sodium carbonate solution (1:3). Shake frequently, and let the mixture stand an hour. Then pass 50 grams of the ether-chloroform solution through a dry filter, kept well covered. Receive the filtrate in a flask, and distil half the solvent. Pour the remainder into a separating funnel, wash the flask three times with 5 cc. portions of a mixture of 3 parts of ether and i part of chloroform, and then shake the total ether-chloroform solution with 10 cc. of o.i n-hydrochloric acid. When the contents of the separating funnel are perfectly clear, add enough ether to bring the ether-chloroform solution to the surface. Pass the acid solution through a small filter moistened with water, and receive the filtrate in a 100 cc. flask. Make three more extractions of the ether-chloroform solution with 10 cc. portions of water, and pass these extracts through the same filter. Wash the filter with water and bring the volume of the filtrate to 100 cc. Finally, measure 50 cc. of this solution with a pipette, and add freshly prepared haematoxylin solution, made by dissolving a small particle of this substance in i cc. of alcohol. Shake and add enough o.i n-potassium hydroxide solution to give the mixture a yellowish color, which quickly changes upon vigorous agitation to bluish violet. Notes and Calculation. The alkaloids in the two cinchona extracts are set free from their salts by sodium carbonate: 2 | + Na 2 CO 3 = 2C 20 H 2 4N 2 O 2 + 2C 6 H 7 (OH) 4 COONa + H 2 O + CO 2 . C 6 H 7 (OH) 4 COOH Quinine Quinine Sodium quinate quinate The alkaloids from 2 grams of extract are dissolved in 75 grams of alcohol-ether-chloroform mixture. Two-thirds of this solution, or 50 grams (= alkaloids in 1.33 grams of extract) are used in the determination. The free alkaloids in this por- tion pass into aqueous solution as hydrochloride, C 20 H 24 N 2 O 2 .- HC1, upon extraction with 10 cc. of o.i n-hydrochloric acid. The excess of hydrochloric acid in half of this solution diluted to 100 cc., that is to say, in 50 cc. (= alkaloids in 0.666 gram of extract), is finally determined by titration. If 3.7 cc. of o.i n-potassium hydroxide solution are required, 5 3-7 = 1.3 cc. of o.i n-hydrochloric acid have combined with the alkaloids in 0.666 gram of cinchona extract. The mean equivalent weight of quinine and cinchonine (= 309), used in the proportion: 1000 130.9 = 1.3 :x (x = 0.04017) shows that 1.3 cc. of o.i n-hydrochloric acid correspond to 0.04017 gram of alkaloids, or 6.03 per cent. The German 268 DETECTION OF POISONS Pharmacopoeia demands this quantity as a minimum for the aqueous extract of cinchona bark. In the determination of alkaloids in alcoholic cinchona ex- tract, titration of excess of o.i n-hydrochloric acid in 50 cc. should not require more than 2.3 cc. of o.i n-potassium hy- droxide solution. Then 5 2.3 = 2.7 cc. of o.i n-hydrochloric acid represent the alkaloids in this solution. This extract at the minimum must contain 12.55 P er cent - of alkaloids. Sulphate Method of Estimating Quinine in Mixtures of Cinchona Alkaloids (J. Carles) 1 This method is especially recommended for practical purposes because of its accuracy and simplicity. Differences in the solubilities of the sulphates of cinchona alkaloids in ammonium sulphate solutions form the basis of the method. E. Schmidt has found that these sulphates have the following solubilities in water at 15: Quinine sulphate i : 800 Cinchonine sulphate i : 65 Quinidine sulphate i : 100 Cinchonidine sulphate i : 97. Guareschi has found quinine sulphate practically insoluble in an ammonium sulphate solution, a result which Hille 2 has confirmed. An addition of 0.0078 gram to the quantity of quinine sulphate obtained is necessary on account of the quinine sulphate in the 20 cc. of wash water used. i. Cinchona Bark. Place 12 grams of finely powdered cinchona bark dried at 100 in an Erlenmeyer flask and add 90 grams of ether and 30 grams of chloro- form. Add 10 cc. of sodium hydroxide solution. Shake vigorously at frequent intervals for 3 hours. Then add 10 cc. of water, or enough to cause the powdered cinchona to gather into balls after thorough shaking and leave the supernatant ether-chloroform layer perfectly clear. After i hour pass 100 grams of the ether-chloroform solution through a dry filter kept well covered. Collect the nitrate in a dry weighed flask, distil the ether-chloroform and dry the flask at 1 10 to constant weight. The increase in the weight of the flask corresponds to the total alkaloids in 10 grams of bark. 1 Zeitschrift fur analytische Chemie 9, 467 (1870). 2 W. Hille (Archiv der Pharmazie 241, 54 (1903), has reviewed critically the various methods that have appeared thus far for the estimation of quinine in pres- ence of other cinchona alkaloids. QUANTITATIVE ESTIMATION OF ALKALOIDS 269 Warm the alkaloidal residue in the flask with water and dilute sulphuric acid and niter the solution. Wash the flask 3 times with water containing sulphuric acid and pour the wash water through the same filter. Dilute the nitrate to about 50 cc., heat to boiling and exactly neutralize with ammonia. Cool and after 6 hours collect upon a weighed filter, or better in a Gooch crucible, the flocculent precipitate of quinine sulphate, wash with 20 cc. of cold water, dry at 110 and weigh. Add 0.0078 gram to the weight of quinine sulphate found and calculate the quantity of quinine in 10 grams of cinchona bark as follows: C2 H24N 2 O2.H 2 SO4:C2oH24X 2 O2 = Quinine sulphate + 0.0078 :x. (746) (648) found 2. Cinchona Extract. Dissolve 3 grams of aqueous cinchona extract in 5 grams each of water and absolute alcohol and place in a measuring cylinder. Add 50 cc. of ether, 10 cc. of chloroform and, after shaking vigorously, 10 cc. of sodium car- bonate solution (1:3). Shake at frequent intervals during 3 hours. When two layers have formed, bring the ether layer to the 75 cc. mark with more ether. Rotate the container carefully and evaporate 50 cc. of the clear ether-chloroform layer in a dry weighed flask. Dry i hour at 105 and weigh when cold. The in- crease in weight corresponds to the total alkaloids in 2 grams of cinchona extract. There should be at least 0.12 gram, or 6 per cent, of alkaloid. To determine quinine, pour very dilute sulphuric acid over the weighed alka- loidal residue in the flask, warm and filter. Rinse the flask several times with very dilute sulphuric acid, bring the volume to about 50 cc. with water and pro- ceed as directed above under cinchona bark. Collect the quinine sulphate in 2 hours upon a weighed filter, or Gooch crucible. The calculation is the same as for cinchona bark. Estimation of Colchicin in Colchicum Seed and Conns (J. Katz and G. Bredemann 1 ) Exhaust colchicum seed or corms with 60 per cent, alcohol and evaporate 50 grams of this extract to 20 cc. Add 0.5 gram of solid paraffine and 20 cc. of water. Warm until the paraffine is melted and the alcohol has been completely expelled. Cool the liquid evaporated to 10-15 cc - anc * P ass through a moist filter. Melt the paraffine cake upon the water-bath with 10 cc. of 10 per cent, acetic acid and pour the cold liquid through the same filter. Wash the latter, the paraffine cake and the dish with water. Saturate the total filtrate with sodium chlo- ride and extract first with 20 cc. of chloroform and then with 10 cc. portions until a few drops of the aqueous liquid show .scarcely any turbidity with 0.05 n-iodine solution. Pass the chloroform solution through a filter moistened with this solvent 1 Pharmazeutsche Zentral-Halle 42, 289 and Apotheker-Zeitung 18, 817- 270 DETECTION OF POISONS and evaporate. To expel chloroform retained by the colchicin, dissolve the residue in a little water and filter. Evaporate the solution in a weighed dish and dry the residue over sulphuric acid to constant weight. Note. Using this method, Bredemann obtained the following quantities of colchicin: In seed 0.46 -0.13 per cent. In conns 0.032-0.06 per cent. In fresh flowers 0.6 per cent. In dry flowers 1.8 per cent. Alkaloids in Pomegranate Bark Pomegranate bark, the bark of Punka Granatum, contains the following four alkaloids : Pelletierine, C 8 H 15 NO, Methyl-pelletierine, C 9 H 17 NO, Isopelletierine, C 8 H 15 NO, Pseudo-pelletierine, C 9 H 15 NO. According to Piccini there is still another alkaloid in the bark of pomegranate root isomeric with methyl-pelletierine and there- fore called isomethyl-pelletierine. Ciamician and Silber have determined the structure of pseudo-pelletierine which they call n-methyl-granatonine. Pseudo-pelletierine (I) is a ketone which, upon treatment with sodium amalgam or with sodium and alcohol, adds two atoms of hydrogen and passes into the corresponding secondary alcohol, n-methyl-granatoline (II). Chromic and sulphuric acids oxidize the latter to n-methyl-granatic acid (III) . Nitro- gen can be eliminated by exhaustive methylation and the final product is normal suberic acid (IV) : I. H 2 C CH CH 2 II. H 2 C CH CH 2 H 2 C N.CH 3 CO H 2 C N.CHsCH.OH H 2 C CH CH 2 ' H 2 C CH CH 2 Pseudo-pelletierine = n-methyl-granatolin n-methyl-granatonine III. H 2 C CH COOH IV. H 2 C CH 2 .COOH H 2 C N.CH 3 > H 2 C H 2 C CH CH 2 CO.OH H 2 C CH 2 .CH 2 .COOH n-methyl-granatic acid Normal suberic acid QUANTITATIVE ESTIMATION OF ALKALOIDS 271 Estimation of Alkaloid in Pomegranate Bark (German Pharmacopoeia) To determine total alkaloids, pour 90 grams of ether and 30 grams of chloro- form upon 12 grams of rather finely ground pomegranate bark, dried at 100 and placed in an Erlenmeyer flask. Shake vigorously and add 10 cc. of a mixture of 2 parts of sodium hydroxide solution and i part of water. Let the mixture stand 3 hours, shaking vigorously at frequent intervals. Then add 10 cc. of water, which will cause the powder to gather into balls after vigorous shaking, and leave the supernatant ether-chloroform solution perfectly clear. After an hour, pass 100 grams of the clear ether-chloroform solution through a dry filter, kept well covered and receive the filtrate in a separating funnel. Extract this filtrate with 50 cc. of o.oi n-hydrochloric acid and pass this acid solution, when perfectly clear, through a small filter moistened with water into a TOO cc. flask. Make three more extractions with 10 cc. portions of water, and pass these extracts through the same filter. Wash the filter with water and dilute the total solution with water to 100 cc. Place 50 cc. of this solution in a 200 cc. flask with 50 cc. of water and enough ether to make a layer about i cm. thick. Add 5 drops of iodeosine solution and enough o.oi n-potassium hydroxide solution, shaking vigorously after each addition, to give a pale red color to the lower aqueous solution. Calculation. The 100 grams of filtered ether-chloroform solution correspond to 10 grams of bark. The alkaloids are transferred from this ether-chloroform solution to 50 cc. of o.oi n-hydrochloric acid which are diluted with water to 100 cc. The excess of hydrochloric acid in 50 cc. of this solution (= alkaloids from 5 grams of bark) is determined by titration. ^If, for example, n cc. of o.oi n-potassium hydroxide solution are used, then 25 n = 14 cc. of o.oi n-hydro- chloric acid have combined with the alkaloids in 50 cc. of the solution. If the mean of the equivalent weights of pelletierine (141) and pseudo-pelletierine (153), or 147, is used in the calculation, 1000 cc. of o.oi n-hydrochloric acid neu- tralize 1.47 grams of the mixed alkaloids. According to the proportion 1000 : 1.47 = 14 : x (x = 0.02058) 5 grams of pomegranate bark contain 0.02058 gram of alkaloids which corre- sponds to an alkaloid content of 20 X 0.02058, or 0.41 per cent. The German Pharmacopoeia demands this quantity of alkaloids in pomegranate bark as a Estimation of Caffeine in Coffee, Tea, Cola Nuts and Guarana (Literature) A. Hilger and A. Juckenack. Zur Bestimmung des Kaffeins in Kaffee und Tee. Forschungsberichte iiber Lebensmittel und ihre Beziehungen zur Hy- giene 4, 40-50; C 1 1897 I, 775 and also 4, I45~ I S4 and C 1897 II, 233. H. Trillich and H. Gockel . Beitrage zur Kenntniss des Kaffees und der Kaffeesurrogate. Forschungsberichte iiber Lebensmittel und ihre Beziehungen zur Hygiene 4, 78-88 and C 1897 I, 1248. 1 C = Chemisches Zentralblatt. 272 DETECTION OF POISONS L. Graf. Ueber Zusammenhang von Kaffemgehalt und Qualitat bei chines- ischen Tee. Forschungsberichte ueber Lebensmittel und ihre Beziehungen zur Hygiene 4, 88-89, and C 1897 I, 1249. A. Forster and R. Riechelmann. Zur Bestimmung des Kaffeins im Kaffee. Zeitschrift fur 6'ffentliche Chemie 3, 129-131 and C 1897 I, 1259. C. C. Keller! Die Bestimmung des Kaffeins im Tee. Berichte der Deutschen pharmaceutischen Gesellschaft 7, 105-112 (1897) and C 1897 I, 1134. A. Forster and A. Riechelmann. Zur Bestimmung des Kaffeins im Kaffee. (Entgegnung.) Zeitschrift fiir offentliche Chemie 3, 235-236 and C 1897 II, 436. E. Tassily. Ueber ein neues Verfahren zur Bestimmung des Kaffeins im Kaffee. Bulletin de la Societe" chimique, Paris, (3) 17, 766-768 and C 1897 11, 644. K. Dieterich. Ueber die Werthbestimmung der Kolanuss und des Kolaex- traktes. Vortrag auf der Naturforscherversammlung in Braunschweig gehalten. Pharmaceutische Zeitung 42, 647-650 and C 1897 II, 977. H. Brunner and H. Leins. Ueber die Trennung und quantitative Bestim- mung des Kaffeins und Theobromins. Schweizer Wochenschrift fiir Pharmacie 36, 301-303 and C 1898 11, 512. J. Gadamer. Ueber Kaffeinbestimmungen in Tee, Kaffee und Kola. Archiv der Pharmacie 237, 58-68 and C 1899 I, 713. E. Katz. Ueber die quantitative Bestimmung des Kaffeins. Berichte der Deutschen pharmaceutischen Gesellschaft 12, 250 (1902). i. C. C. Keller's Method. Pour 120 grams of chloroform upon 6 grams of dry, unbroken tea leaves 1 in a wide-mouth separating funnel. In a few minutes, add 6 cc. of ammonium hydroxide solution (10 per cent. HaN), and at frequent intervals shake vigorously during 30 minutes. Then let the separating funnel stand at rest, until the solution is perfectly clear and the tea leaves have absorbed all the water. This may require 3-6 hours, or even longer, depending upon the variety of tea. Pass 100 grams of clear chloroform extract, representing 5 grams of tea, through a small filter moistened with chloroform. Receive the filtrate in a small, weighed flask and distil the chloro- form upon the water-bath. Pour 3-4 cc. of absolute alcohol over the residue. Heat upon the water-bath to remove alcohol and expel alcohol vapor with a hand bellows. In a few minutes 1 When a wide-mouth separating funnel cannot be obtained, triturate the tea leaves somewhat, solely to facilitate their removal from the separating funnel after extraction. Finely powdered tea is not only unnecessary, but even ob- jectionable, because the extracts have a much deeper color, and the yield of caffeine is not increased. QUANTITATIVE ESTIMATION OF ALKALOIDS 273 the caffeine will be dry and at the same time free from im- prisoned chloroform. In a measure also, this treatment with alcohol separates caffeine from extraneous chlorophyll. The lattei adheres to the bottom and side of the flask, whereas caffeine forms a white incrustation upon it. Caffeine thus obtained is usually impure from small quantities of ethereal oil, fat, vegetable-wax and principally chlorophyll. Conse- quently, it must be purified. Set the flask upon a boiling water- bath and pour a mixture of 7 cc. of water and 3 cc. of alcohol over the crude caffeine, which upon being shaken will pass into solution almost immediately. Then add 20 cc. of water, stopper the flask and shake vigorously. The chlorophyll will form lumps and the solution will filter easily. Pass the caffeine solution through a small filter moistened with water, wash flask and filter with 10 cc. of water, evaporate the total filtrate in a weighed glass dish to dryness upon the water-bath and weigh the residue of nearly pure caffeine. The weight of this residue multiplied by 20 will give the percentage of caffeine in the tea. Notes. Ammonia causes tea leaves to swell considerably, and at the same time combines with the tannic acid present. Caffeine is set free and dissolved by chloroform. The color of the chloroform extract depends upon the variety of tea. Black teas (Pekoe, Souchong and Congo) give clear, pale green to yellowish green solutions. Teas not so black, or green teas, give darker and moie brownish green solutions. In assaying those varieties of tea, which probably contain a small quantity of caffeine, take 12 grams and extract with 150 grams of chloroform. C. C. Keller has shown that the best and most expensive varieties of tea contain most caffeine. The average percentage of caffeine, based upon 50 assays of tea, was found to be 3.06. A green tea gave the smallest yield, namely, 1.78 per cent, of caffeine; and a Pekoe tea the highest yield, namely, 4.24 per cent, of caffeine. J. Gadamer states that Keller's method of estimating caffeine in tea is appliqable also to coffee and cola preparations. Keller's method is especially useful for roasted coffee. The caffeine, though somewhat brown, is aways sufficiently pure. 274 DETECTION OF POISONS 2. Hilger-Juckenack Method. Macerate 20 grams of finely ground coffee, or triturated tea, with 900 grams of water for several hours in a large beaker at room temperatures. Then boil thoroughly and replace the water lost by evaporation. Raw coffee requires 3 hours and roasted coffee and tea 1.5 hours. Cool somewhat (60 to 80) and add 75 grams of aluminum acetate solution (see note, page 289) and gradually, while stirring, 1.9 grams of acid sodium carbon- "N ate. Boil about 5 minutes and bring the total weight when cold to 1020 grams. Filter 750 grams (=15 grams of original material) and add to the clear nitrate 10 grams of precipitated and pow- dered aluminum hydroxide and some filter paper made into a magma by agitation with water. Stir frequently and evaporate upon the water-bath. Thoroughly dry the residue in an air-closet at 100, and extract for 8-10 hours with pure tetrachloromethane (CCU), using a Soxhlet apparatus (Fig. 23). Tetrachloromethane, which* is always colorless, is finally distilled and the residue of per- fectly white caffeine is dried at 100 and weighed. The results thus obtained are usually accepted without question. But if an absolutely accurate result is required, nitrogen in the crude caffeine may be determined by Kjeldahl's method. The quantity of anhydrous caffeine is calculated on the basis of this analysis. One cc. of o.i n-oxalic acid represents 0.00485 gram of anhydrous caffeine. Com- mercial tetrachloromethane is usually impure and cannot be used PIG. 23. Soxhlet Apparatus. QUANTITATIVE ESTIMATION OF ALKALOIDS 275 directly in the extraction. It should be shaken 3-4 times with sodium carbonate solution, then several times with water, dried over fused calcium chloride and distilled fractionally. It boils at 76-77. 3. Trfflich-Goeckel Modification of Hilger's Method Exhaust 10 grams of finely ground coffee with water. This will require 3 extractions with boiling water, using 200 cc. portions and heating each for 30 minutes. Combine the filtered extracts, cool and dilute to 495 cc. Add 5 cc. of basic lead ace- tate solution, shake thoroughly, filter 400 cc. and pass hydrogen sulphide through the filtrate. Dilute this filtrate to 500 cc., shake thoroughly and again filter 400 cc. This filtrate will represent 6.4 grams of coffee. Concentrate this filtrate (400 cc.) upon the water-bath, and, after addition of i gram of magnesium oxide and sand, evaporate to dryness. Triturate the residue and extract for 30 hours with acetic ether, using a Soxhlet apparatus. Evaporate the acetic ether extract in a Kjeldahl flask, or distil the solvent, and determine nitrogen in the residue by Kjeldahl's method. One gram of nitrogen rep- resents 3.4643 grams of caff erne. Crude caffeine is easily decomposed by the acid used hi the Kjeldahl process and by mer- curic oxide. Roasted coffee may easily give too much caffeine by this method, because the bases formed by roasting coffee, pyridine for example, are also extracted by acetic ether. 4. Trillich-Goeckel Modification of Socolof's Method. Put 10 grams of finely ground, dried coffee into a separating funnel, provided with a plug of glass wool for a filter, and moisten with ammonium hydroxide solution. Let the mixture stand for 30 minutes, extract for 12 hours with 200 cc. of acetic ether and shake frequently. Filter, and wash three times with 50 cc. portions of acetic ether. Distil the acetic ether upon the water-bath and boil the residue with milk of magnesia. Filter, and evaporate the filtrate to dryness upon the water-bath. Dissolve the residual caffeine in acetic ether or chloroform. Filter this solution into a weighed dish or Kjeldahl flask. Evaporate the solvent and weigh the caffeine, or calculate it from the percentage of nitrogen. The latter method is the more 276 DETECTION OF POISONS accurate. According to C. Wolff 1 the residue from the acetic ether or chloroform extract should not be accepted as pure caf- feine. Determination of nitrogen in this residue by Kjeldahl's method is the most reliable way of estimating caffeine in the extract. 5. E. Kate's Method of Estimating Caffeine. This method is based upon the fact that chloroform will extract caffeine quantitatively from 'a solution which is ammoniacal, or faintly acid with hydrochloric acid. Shake 10 grams of powdered coffee, or tea, for 30 minutes with 200 grams of chloroform and 5 grams of ammonium hy- droxide solution. When the liquid has settled, filter 150 grams of the chloroform solution through a Sander's filter which will give a perfectly bright filtrate free from water. Dis- til the chloroform completely and dissolve the residue with gentle heat in about 6 cc. of ether. Add 20 cc. of 0.5 per cent, hydrochloric acid and, in an assay of coffee, also 0.2-0.5 gram of solid paraffine. Evaporate the ether and filter the cold, aqueous solution. Wash the flask and filter paper a few times with small portions of 0.5 per cent, hydrochloric acid. Finally extract the total aqueous hydrochloric acid solution four times with 20 cc. portions of chloroform. Distil the filtered chloro- form extract, dry the residue and weigh. This residue will consist of nearly pure caffeine. J. Katz found the following percentages of caffeine : Caffeine Average Raw Coffee Beans 0.9 -1.27 per cent. 1.14 per cent. Dried Cola Nuts 1.51-1.94 per cent. 1.68 per cent. Black Tea 2.51-3.56 per cent. 3.07 per cent. Guarana 2.83-4.74 per cent. 4.08 per cent. J. Katz recommends the following method for estimating caffeine in mate or Paraguay tea: Treat finely triturated tea with ammonium hydroxide solu- tion and chloroform, as described above, and dissolve the chloro- form residue in ether. Add water to the ether and evaporate. Warm the aqueous solution 10 minutes upon the water-bath 1 Zeitschrift fur offentliche Chemie 12, 186. QUANTITATIVE ESTIMATION OF ALKALOIDS 277 with 2 cc. of lead hydroxide suspended in water (i : 20). If it is very difficult to get a clear nitrate from this liquid, add a little calcined magnesium oxide. This treatment usually gives a nitrate, which is perfectly clear when cold, and but slightly colored. Chloroform extracts quite pure caffeine from this solution. By this method mate yields 0.3-1.6 per cent, of caffeine, the average being 0.71 per cent. 6. K. Dieterich's Method of Estimating Total Alkaloids (Caffeine and Theobromine) in Cola Nuts. Moisten 10 grams of finely grated cola nuts with a little water, mix with 10 grams of granulated, unslaked lime and extract with chloroform in a Soxhlet apparatus for 45 minutes. Evaporate the extract almost to dryness and dissolve the residue with gentle heat in 20 cc. of normal hydrochloric acid. Filter and dilute to 100 cc. Add ammonium hydroxide solution in large excess to this nitrate, shake at frequent intervals during 15 minutes and extract three times with 20 cc. portions of chloroform. Evapo- rate this chloroform solution in a weighed flask and dry the residue, which usually consists of perfectly pure caffeine, at 100 to constant weight. This method may also be used in estimating caffeine in Paraguay tea. Mix the finely ground material with unslaked lime, and extract with chloroform, in a Soxhlet apparatus. Tea gives pure, white caffeine free from chlorophyll. Estimation of Alkaloids in Ipecac Ipecac has been shown 1 to contain three alkaloids : Cephaeline, C 2 8H4oN 2 O4, Emetine, Cso^NsO^ Psychotrine. The composition of the last alkaloid is unknown. This drug acts as an expectorant and emetic, because of cephaeline and emetine. Psychotrine is said not to possess these properties. Therefore, in assaying ipecac for medicinal purposes, only the percentage of the first two alkaloids need be estimated. The equivalent weights of these two alkaloids (cephaeline 234 and 1 Frerichs and de Fuentas Tapis, Archiv der Pharmacia, 1902, Heft 5 and 6. 278 DETECTION OF POISONS emetine 248) are so nearly the same, that the mean of the two (241) may be used as the factor. Procedure. Put 6 grams of finely powdered root in a dry Erlenmeyer flask and shake with 60 grams of ether. Then add 5 cc. of ammonium hydi oxide solution, or 5 cc. of sodium carbonate solution (i 13), and shake frequently during an hour. Add 10 cc. of water and, after shaking vigorously, filter 50 grams of the ether extract into a small flask. Evaporate half the ether upon the water-bath, and extract the remainder in a separating funnel with 10 cc. of o.i n-hydrochloric acid. Pass the acid solution through a small filter into a 200 cc. flask. Make two more extractions of the ether with 10 cc. portions of .water, and pass these through the same filter. Bring the volume of the acid solution to 100 cc., and then add enough ether to form a layer about i cm. thick after thorough agi- tation. Add 5 drops of iodeosine solution (i : 250), and titrate excess of hydrochloric acid with o.i n-potassium hydroxide solution. The number of cc. of o.i n-hydrochloric acid, com- bined with the alkaloids, multiplied by 0.0241 gives the quan- tity of emetine and cephaeline in 5 grams of ipecac. To estimate -these alkaloids gravimetrically, shake vigorously the ether solution of the alkaloids (50 grams = 5 grams of root) in a separating funnel with 5 cc. of dilute hydrochloric acid and 10 cc. of water. Transfer the acid solution to another separating funnel. Make two more extractions of the ether with 10 cc. portions of water and add these to the acid extract. Add 5 cc. of ammonium hydroxide solution to the acid extract and shake vigorously with 50 grams of ether. Remove the aqueous layer and filter 40 grams of the ether solution into a weighed flask. Evaporate the ether and weigh the flask after drying for an hour at 100. This will give the quantity of eme- tine and cephaeline in 4 grams of root. Test for Cephaeline. This reaction is very characteristic of this alkaloid. Froehde's reagent dissolves pure cephaeline, as the free base, almost without color. A trace of hydrochloric acid, or better sodium chloride, added to this solution produces an intense blue color. Pure emetine gives no color with QUANTITATIVE ESTIMATION OF ALKALOIDS 279 Froehde's reagent, nor when sodium chloride is added. This test for cephaeline may be made with the ether residue. The method of estimating alkaloids in ipecac, prescribed by the German Pharmacopoeia, is the same as that for cinchona bark. Use 12 grams of finely powdered root dried at 100, but in ascertaining excess of acid use iodeosine, and not hsematoxylin, as the indicator. Finally, measure with a pipette 50 cc. of the proper solution having a volume of 100 cc., place in a 200 cc. flask and add about 50 cc. of water and enough ether to make a layer i cm. thick. Add 5 drops of iodeosine solution and enough o.oi n-potassium hydroxide solution, shaking thoroughly after each addition, to give the lower aqueous layer a pale red color. This should require not more than 20 cc. of alkaline solution. Estimation of Nicotine in Tobacco i. R. Kissling's 1 Method. First remove the ribs and then cut the tobacco leaves into small pieces. Dry 1-2 hours (50-60), and then reduce to a uniform, coarse powder. Triturate 20 grams of this powder with 10 cc. of dilute, alcoholic sodium hydroxide solution (6 grams of sodium hydroxide dissolved in 40 cc. of water and 60 cc. of 95 percent, alcohol). Transfer this moist powder to a paper thimble and extract 2-3 hours with ether in a Soxhlet apparatus. Carefully distil the ether solution so that a portion of the solvent remains. Add 50 cc. of very dilute sodium hydroxide solution (4 grams of sodium hydroxide in 1000 cc. of water) to the residue- and distil with steam. Begin introducing steam after the nicotine solu- tion has been boiling several minutes. Collect about 400 cc. of distillate, that is to say, continue distilling until the distillate is no longer alkaline. Mix well, add a few drops of rosolic acid solution to the distillate, and titrate nicotine with o.i n-sul- phuric or oxalic acid until the red color has just disappeared. Calculation. Although nicotine, Ci Hi 4 N 2 (162), as a di-acid base can combine with two equivalents of acid, it behaves upon titration, with rosolic acid or iodeosine as indicator, as if^it were a monacid base with the equivalent weight 162. 1000 cc. of o.i n-acid consequently correspond to 16.2 grams of nicotine. 2. C. C. Keller's 2 Method. Pour 60 grams of ether and 60 grams of petroleum ether over 6 grams of dry tobacco in a 200 1 Zeitschrift fur analytische Chemie 34, 1731 and 21, 76. 2 Berichte der Deutschen pharmazeutischen Gesellschaft 8, 145 (1898). 280 DETECTION OF POISONS cc. Erlenmeyer flask. Add 10 cc. of 20 per cent, aqueous potassium hydroxide solution and let the mixture stand half an hour, shaking vigorously at frequent intervals. After the liquid has stood at rest 3-4 hours, pour 100 grams of ether solution through a small, plaited filter and receive the filtrate in a 200 cc. Erlenmeyer flask. Nicotine is in solution together with a little ammonia, which must be removed before titration. By means of a hand bellows and a glass tube reaching to the bottom of the flask force a current of air through the solution, so that there is considerable agitation. It requires about a minute and a half to expel all ammonia. At the same time 8-10 grams of ether evaporate. Add 10 cc. of alcohol, a drop of i per cent, iodeosine solution and 10 cc. of water to the ammonia- free solution. Stopper the flask and shake vigorously. Nicotine and iodeosine dissolve in the water which has a red color. Add a slight excess of o.i n- hydrochloric acid, enough to discharge the color, and titrate excess of acid with o.i n-potassium hydroxide solution. The quantity of nicotine in tobacco shows a wide variation and ranges from 0.6 to 4.8 per cent. 3. J. Toth's 1 Method. According to Toth two sources of error in C. C. Keller's method lead to low results. An aqueous potassium hydroxide solution retains variable quantities of nicotine and a current of air passed through an ether solution of nicotine volatilizes some of this alkaloid. Therefore Toth recommends the following procedure: Mix 6 grams of air-dried tobacco with 10 cc. of 20 per cent, sodium hydroxide solution in a porcelain dish. Add gypsum until the mixture is like powder. Extract thoroughly with 100 cc. ether-petroleum ether mixture (i : i) and after i hour pipette off as quickly as possible 25 cc. of the solvent. Add 40-50 cc. of water, i drop of iodeosine solution and an excess of o.i n-sul- phuric acid. Determine excess of acid by titration with o.i n-sodium hydroxide solution. The ether-petroleum ether mixture takes up at most 0.0005 gram of ammonia. 1 Chemisches Zentralblatt, 1901, i, 973. QUANTITATIVE ESTIMATION OF ALKALOIDS 281 Estimation of Hydrastine in Fluid Extract of Hydrastis (German Pharmacopoeia) Evaporate 15 grams of fluid extract of hydrastis to about 5 grams in a weighed dish upon the water-bath, and wash the residue into an Erlenmeyer flask with about 10 cc. of water. Add 10 grams of petroleum ether, 50 grams of ether and 5 grams of ammonium hydroxide solution. Let the mixture stand an hour, shaking vigorously at frequent intervals. Then pass 50 grams of the clear ether solution through a dry filter into a separating funnel. Add 10 cc. of a mixture, composed of i part of hydrochloric acid and 4 parts of water, and shake the solution vigorously several minutes. When the liquids have separated clear, run the acid solution kito an Erlenmeyer flask. Make two more extractions of the ether with 5 cc. portions of water containing a few drops of hydrochloric acid, and add these to the first extract. Add to the total extract excess of ammonium hydrox- ide solution and 50 grams of ether. Let the mixture stand an hour, shaking vigorously at frequent intervals. Pass 40 grams of the clear ether solution through a dry filter and collect the filtrate in a weighed, dry flask. Distil the ether, dry the residue at 100 and weigh when cold. The residue should weigh at least 0.2 gram. Notes. Additional information about hydrastine is given on page 116. Ammonia, added to an aqueous solution of the residue from hydrastis extract (15 grams), sets the alkaloids, hydrastine and berberine, free from their salts. The ether- petroleum benzine mixture dissolves hydrastine but not ber- berine, the latter being nearly insoluble in this mixed solvent. But phytosterin, which is always present in hydrastis extract, is dissolved. Only 50 grams (= hydrastine in 12.5 grams of extract) of the original 60 grams of ether-petroleum benzine mixture are used. Hydrastine is extracted from the solvent by agitation with dilute hydrochloric acid and dissolved in the acid solution as hydrochloride. The alkaloid is then precipi- tated from the acid solution by ammonia and dissolved in 50 grams of ether: C 21 H 21 N0 6 .HC1 + (H 4 N)OH = C 2 iH 21 NO 6 + H 2 O + (HCH | || >CH | || >CH ( 3 )CH 3 .N C N^ ( 3 )CH 3 .N C N^ HN = C N^ Theobromine Theophylline Paraxanthine Theophylline occurs in tea leaves and paraxanthine has been isolated from human urine. The latter is therefore called urotheobromine. Estimation of Alkaloids in Leaves of Atropa Belladonna, Hyoscyamus Niger and Datura Strammonium (E. Schmidt's Modification of Keller's Method 1 ) Shake vigorously 10 grams of finely powdered leaves, dried to constant weight over quicklime, in an Erlenmeyer flask with 90 grams of ether and 30 grams of chloroform. Add 10 cc. of 10 per cent, sodium hydroxide solution and shake vigorously and often for 3 hours. Then add 10 cc. of water, or enough to cause the powder to gather into balls when thoroughly shaken. After i hour pass 60 grams of the ether-chloroform extract (= 5 grams of leaves) through a dry filter kept well covered. Distil 60 cc. of this filtrate to half its volume to remove ammonia, and transfer the deep green solution to a separating funnel, rinsing the flask with three 5 cc. portions of ether. Shake the combined extracts well with 10 cc. of o.oi n-hydrochloric acid. Add enough ether to cause the ether- chloroform solution to rise to the top, and pass the acid solution through a moist filter into a 200 cc. glass stoppered flask. Shake the ether-chloroform solution 3 times with 10 cc. .portions of water, pouring these extracts through the same filter and washing the latter with enough water to bring the total volume to 100 cc. Add enough ether to make a layer i cm. deep and 5 drops of iodeosine solu- tion. Having determined beforehand the exact relation of acid to alkali, titrate excess of o.oi n-hydrochloric acid with o.oi n-potassium hydroxide solution. The calculation is the same as that for extract of belladonna (see page 301). Notes. Using this method, E. Schmidt obtained 0.4 per cent, of alkaloid in wild belladonna leaves but only 0.26 per cent, in cultivated leaves. The average of many determinations gave 0.4 per cent, in strammonium leaves and 0.27-0.28 per cent, in hyoscyamus leaves without stalks. Alkaloids were calculated as atropine. Sodium hydroxide solution liberates alkaloids from the acids with which they are naturally combined in the plant, for example: (Ci7H 23 NO 3 )2.H 2 S04 2 + 2NaOH = 2 Ci 7 H 23 NO3 + 2H 2 O + Na 2 S0 4 . 1 Apotheker-Zeitung 15, 13. 2 The formula of atropine sulphate used in medicine. QUANTITATIVE ESTIMATION OF ALKALOIDS 301 Estimation of Alkaloids in Extract of Belladonna (German Pharmacopoeia) Dissolve 2 grams of extract of belladonna in an Erlenmeyer flask in 5 grams of water and 5 grams of absolute alcohol, and add 50 grams of ether and 20 grams of chloroform to this solution. Shake vigorously and add 10 cc. of sodium car- bonate solution (i : 3). Let the mixture stand and agitate at frequent intervals for an hour. Then pass 50 grams of the clear ether-chloroform solution through a dry filter kept well covered, and receive the nitrate hi a flask. Distil half the solvent and pour the remainder into a separating funnel. Wash the flask three times with 5 cc. portions of ether. Thoroughly extract the total ether-chloro- form solution with 20 cc. of o.oi n-hydrochloric acid. When the liquids have separated clear, if necessary, after addition of enough ether to bring the ether- chloroform solution to the surface, pass the acid solution through a small filter moistened with water and receive the nitrate in a 200 cc. flask. Make three extractions of the ether-chloroform solution with 10 cc. portions of water, and pass these washings through the same filter. Finally, wash the filter with water and bring the entire solution to 100 cc. Add enough ether to make a layer i cm. thick and 5 drops of iodeosine solution. Run in o.oi n-potassium hydroxide solution, shaking vigorously after each addition, until the aqueous solution is pale red. Calculation. Sodium carbonate like sodium hydroxide liberates the alkaloids atropine and hyoscyamine, from their salts in belladonna leaves: (C 1 7H23N0 3 )2.H 2 SO4 + Na 2 CO 3 = 2Ci 7 H 23 N0 3 + Na 2 S04.+ CO 2 + HjO. The free alkaloids dissolve in the alcohol-ether-chloroform mixture. Fifty grams of this solution ( = alkaloids from 1.33 grams of extract) are extracted with 20 cc. of o.oi n-hydrochloric acid, the alkaloids passing into the aqueous solution as salts of hydrochloric acid (CiyH^NOs.HCl). Excess of acid in this solution is determined by titration. If 13 cc. of o.oi n-potassium hydroxide solution are used, then 20 13 = 7 cc. of o.oi n-hydrochloric acid correspond to the alka- loids in 1.33 grams of extract. The equivalent weight of the two isomeric bases, atropine and hyoscyamine, being 289, 1000 cc. of o.oi n-hydrochloric acid corre- spond to 2.89 grams of alkaloids. The proportion 1000 : 2.89 = 7 : x (x = 0.02023) shows that 1.33 grams of belladonna extract contain 0.02023 gram of alkaloids corresponding to 1.51 per cent. The German Pharmacopoeia places this per- centage as the minimum for total alkaloids in extract of belladonna. Extract of Hyoscyamus The alkaloids in 2 grams of this extract are determined in the manner described for extract of belladonna. Use 10 cc. of o.i n-hydrochloric acid instead of 20 cc. to extract alkaloids. The German Pharmacopoeia requires that not more than 6.5 cc. of o.oi n-potassium hydroxide solution shall be used in titrating the excess of hydrochloric acid. Therefore 10 - 6.5 = 3-5 cc. of o.oi n-hydrochloric acid are combined with the alkaloids in 1.3 grams of henbane extract ( = of the original extract). The proportion 1000 : 2.89 = 3.5 : x (x = o.oion) 302 DETECTION OF POISONS shows that 1.33 grams of extract contain o.oioi gram of alkaloid, corresponding to 0.76 per cent. This percentage is placed as the minimum for total alkaloids in henbane extract. Assaying Officinal Extracts (E. Merck 1 ) With a view to obviating as many sources of error as possible, E. Merck has proposed the following procedures : Extract of Belladonna. Dissolve 4 grams of extract in 6 cc. of water and wash the solution into a separating funnel with an additional 10 cc. Add 100 cc. of ether, shaking well, then 10 cc. of sodium carbonate solution (1:3) and shake at once for 5 minutes. Stopper the funnel and let the mixture stand for 20 minutes. Then pass the ether layer through a dry filter (10 cm. in diameter) into a glass- stoppered flask. To lessen evaporation of ether as much as possible, cover the funnel with a glass plate. If an emulsion keeps the ether from separating well, add a few grams of powdered tragacanth at the end of the time stated above. Shake until the tragacanth gathers into balls in the aqueous layer. After 15 minutes decant 75 cc. of the ether layer. To check results by making more than one assay, use 25 cc. of the ether solution (= i gram of extract). Test a clean glass-stoppered flask to make sure that it does not give up alkali to the water. Then introduce into such a flask 50-60 cc. of water, 5 drops of iodeosine solution and 20 cc. of ether. Shake and add o.oi n-hydrochloric acid until the aqueous layer just becomes colorless upon shaking. This procedure obviates a special determination of the alkalinity of the water, since the resulting mixture is brought to the neutral point. Now add 25 cc. of the ether solution of the alkaloid and titrate until there is no color. Multiply the number of cc. of o.oi n-hydrochloric acid used by 0.00289. 2 The product is the quantity of alkaloid, calculated as atropine, in i gram of belladonna extract. Upon the average this preparation contains 1.8 per cent, of alkaloid. Extract of Cinchona. Haematoxylin is frequently an unsatisfactory indicator in the titration of cinchona alkaloids, because the color change is slow enough to make it difficult to fix the end-point exactly. Therefore E. Merck makes a gravimetric and volumetric determination at the same time by the following method: Dissolve 3 grams of aqueous cinchona extract in 10 cc. of water in a porcelain dish. Pour the solution into a 250 cc. shaking flask, rinsing it in with 10 cc. of water. Add 150 cc. of ether and 10 cc. of sodium carbonate solution (1:3) to this mixture and shake vigorously for TO minutes. Cork the flask and let the mixture stand at rest for 30 minutes. This extract frequently forms an emulsion. In that case add a few grams of tragacanth powder which has no effect upon the result. Pour the ether solution of cinchona alkaloids as rapidly as possible through a dry creased filter. Use 30 cc. ( = i gram of cinchona extract) for each determination. Distil the solvent from the 50 cc. in a weighed 100 cc. flask and 1 Zeitschrift fur analytische Chemie 41, 584 (1902) and also Merck's Bericht iiber das Jahr; 1900. 2 289 = the equivalent weight of the two isomeric bases, atropine and hyoscy- amine, CnH^sNOs. QUANTITATIVE ESTIMATION OF ALKALOIDS 303 dry the residue in an air bath at 100-110 to constant weight. The alkaloids obtained are nearly colorless or faintly yellow. Having ascertained the weight of the alkaloids, proceed with the titration. Dissolve the residue in the flask in 10 cc. of alcohol, adding 50 cc. of water, which partially precipitates the alkaloids, and then alcoholic haematoxylin solution. 1 Run in o.i n-hydrochloric acid until the alkaloids again dissolve and the red color of the solution passes through reddish yellow into a pure yellow. The mean equivalent weight of the cinchona alkaloids is 309. Therefore i cc. of o.i n-hydrochloric acid = 0.0309 gram of alkaloid. Upon the average, officinal aqueous extract of cinchona contains 9 per cent, of alkaloid. Extract of Nux Vomica. Dissolve o.i gram of this extract in a flask in 5 grams of absolute alcohol and 10 grams of water. Add 95 grams of ether and shake well. Then add 10 cc. of sodium carbonate solution (1:3) and shake vigorously at once for about 10 minutes. After 15 minutes pour the ether solu- tion as rapidly as possible through a creased filter. Weigh in a flask 50 grams of this solution (= 0.05 gram of the original extract), having previously placed in this flask a neutral mixture of 50 cc. of water, 20 cc. of ether and 5 drops of iod- eosine solution. Add 20 cc. of o.oi n-hydrochloric acid and titrate with o.or n-potassium hydroxide solution until the aqueous layer is just red. Calculation. Since strychnine and brucine are present in nux vomica in nearly equal parts, the mean equivalent weight of such a mixture of bases (334 + 394) : 2 = 364. Hence 0.00364 gram of the mixed alkaloids neutralizes i cc. of o.oi n-hydrochloric acid. The officinal extract of nux vomica contains 18 per cent, of alkaloid. 1 E. Merck advises keeping on hand an alcoholic solution of haematoxylin, because a freshly prepared solution usually gives a blue-violet instead of a red color change. CHAPTER VII DETECTION OF CARBON MONOXIDE BLOOD, BLOOD STAINS AND HUMAN BLOOD i. Carbon Monoxide Blood Carbon monoxide (CO) has a direct toxic action upon the blood. This gas passed into blood displaces loosely bound oxygen from oxyhaemoglobin forming the more stable carboxy- hsemoglobin. The latter compound is cherry-red, not dichroic and entirely resistant to putrefaction if air is excluded. In carbon monoxide poisoning the cherry-red color of the blood is usually noticed at once. Detection of Carbon Monoxide Blood 1. Boiling Test. Blood containing carbon monoxide gives a brick -red coagulum, if boiled or warmed upon the water-bath. Ordinary blood gives a grayish brown or brownish black precipitate. 2. Sodium Hydroxide Test. Carbon monoxide blood shaken with 1-2 volumes of sodium hydroxide solution (sp. gr. 1.3 = 26.8 per cent.) remains red and in a thin layer is the color of red lead or vermilion. Normal blood similarly treated is al- most black and in a thin coating upon a porcelain plate is dark greenish brown. A procedure recommended consists in diluting the blood with 6-10 times its volume of water and using about 5 drops of sodium hydroxide solution to 10 cc. of diluted blood. Even gentle warming with sodium hydroxide solution (10 per cent. NaOH) does not alter the red color of this carboxyhsemo- globin solution, whereas a solution of normal human blood becomes greenish to dark brown. 3. Basic Lead Acetate Test. Mix 4-5 volumes of basic lead acetate solution in a test-tube with diluted or undiluted carbon monoxide blood and shake vigorously for a minute. 304 DETECTION OF CARBON MONOXIDE BLOOD 305 Such blood remains bright red but normal blood is first brownish and then chocolate to greenish brown. 4. Potassium Ferrocyanide Test. Mix undiluted blood (15 cc.) with an equal volume of 20 per cent, potassium ferrocyanide solution and 2 cc. of diluted acetic acid. 1 Shake the mixture gently and a coagulum will gradually form. That from normal blood is dark brown but from blood containing carbon monoxide bright red. This difference disappears slowly but not entirely for weeks. 5. Tannin Test. Mix an aqueous blood solution 2 with 3 times its volume of i per cent, tannin solution and shake thor- oughly. A difference in color between normal and carbon monoxide blood can be recognized after several hours, most distinctly after 24 hours. Normal blood is gray but carbon monoxide blood is crimson-red. This difference is apparent even after several months. Ten per cent, of carboxyhaemo- globin can be detected in blood by tests 4 and 5. 6. Copper Sulphate Test. A drop of saturated copper sul- phate solution added to 2 cc. of carbon monoxide blood mixed with the same volume of water gives a brick-red precipitate. The deposit from normal blood is greenish brown. In all these precipitation tests (4, 5 and 6) the less easily decomposed carbon monoxide blood remains bright red but the more easily decomposed normal blood in presence of the precipitants used and others is off color or dark. 7. Ammonium Sulphide Test. Mix 0.2 cc. of ammonium sulphide solution and 0.2-0.3 cc. of 30 per cent, acetic acid with 10 cc. of 2 per cent, aqueous blood solution. Carbon monoxide blood gives a fine rose color but normal blood is greenish gray. The former within 24 hours gives a red flocculent precipitate. 8. Palladous Chloride Test. Carbon monoxide precipitates black metallic palladium from a neutral aqueous palladous chloride solution: CO + H 2 O + PdCl 2 = C0 2 + 2HC1 + Pd. 1 Mix i volume of glacial acetic acid with 2 volumes of water. This acid con- tains about 30 per cent, acetic acid. 2 Use i part of blood to 4 parts of water. 306 DETECTION OF POISONS Mix a few drops of potassium hydroxide solution with the blood and warm gently upon the water-bath. By means of a suction pump draw through the solution air that has been washed until pure. Pass the gas evolved first through lead acetate solution to remove possible hydrogen sulphide, then through sulphuric acid to absorb ammonia and finally through a neutral light red palladous chloride solution (i: 500). 9. Spectroscopic Examination. The detection of carboxy- haemoglobin with the spectroscope is comparatively easy. The Oxyhaemoglobin. Haemoglobin. Methaemoglobin. Carboxyhaemoglobin. Hsematoporphyrin, very dilute, acid. Haematoporphyrin, not so dilute, alkaline. FIG. 24. Absorption-Spectra. two absorption-bands of this compound are quite similar to those of Oxyhaemoglobin but they lie somewhat nearer together and more toward the violet. The main difference, however, between the absorption-bands of these compounds is that those of carboxyhsemoglobin are not extinguished by reducing agents. To prepare the blood solution for spectroscopic examination, dilute 1-1.5 parts of blood with 100 parts of water and make the DETECTION OF CARBON MONOXIDE BLOOD 307 observations through a layer i cm. thick. To reduce i per cent, blood solution, mix thoroughly with a few drops of am- monium sulphide solution and add 4-6 drops more of the same reagent as a surface layer to exclude air. Reduction begins in about 6-8 minutes. A solution of tartaric acid and ferrous sulphate in presence of an excess of ammonium hydroxide solution will also reduce oxyhaemoglobin. Oxyhaemoglobin under these conditions is changed to reduced hemoglobin. The two absorption-bands characteristic of the former disappear and a broad diffuse absorption-band occupies the previous bright space between the two bands. The spec- trum of carboxyhaemoglobin remains unchanged only when 27 per cent, at least of the haemoglobin is saturated with carbon monoxide. If allowed to stand in an open vessel, blood will lose carbon monoxide within 8 days. But carbon monoxide blood sealed in glass tubes is said to keep for years. Carbon monoxide has been detected in blood of a cadaver after 18 months. Tollens 1 recommends adding some formaldehyde to the blood solution. This reagent has not the slightest effect upon the two oxyhaemoglobin bands. Warm- ing the mixture very gently with ammonium sulphide solution develops a third and nearly as distinct black band almost midway between the original bands which gradually disappear. Finally only this band will remain. This is a far more satisfactory test than that given by the indefinite band of blood alone. If the solution is cooled and agitated with air, this third band will disappear and the two original oxyhsemoglobin bands will return. If carbon monoxide is present, formaldehyde does not have this action. 2. Detection of Blood Stains The detection of blood in dry stains upon fabrics, wood, knives, weapons, etc., 2 is more certain and less open to question, if haemin crystals (Teichmann's blood crystals) are prepared from the blood pigment. If haemin crystals are obtained, the stain in question may be regarded with certainty as due to blood. Fresh blood when dry is bright red and has a smooth surface. 1 Berichte der Deutschen chemischen Gesellschaft 34, 1426 (1901). 2 Blood mixed with iron oxide as, for example, blood upon rusty knives and weapons usually fails to give haemin crystals. 308 DETECTION OF POISONS Flakes of such blood scraped from any material are garnet-red by transmitted light. A solution of fresh blood stains in potas- sium or sodium hydroxide is dichroic, being red by transmitted and green by reflected light. Later dried blood becomes brownish red or dark brown. These color changes are due to conversion of oxyhaemoglobin into methaemoglobin and then into haematin. The first two substances are soluble in water but the last is not. But haematin is soluble in alkalies and in alcohol containing sulphuric acid. This change of the blood pigment depends not only upon the age of the stain but really upon the action of air (oxygen), light,, heat and moisture upon the blood before it is dry. If the blood is in a thin layer, haemoglobin will sometimes change into methaemoglobin even in 3-10 days. Boiling water causes immediate insolubility. The action is also very rapid in direct sunlight. Washing in alkaline solutions (boiling solutions of potassium or sodium soap, sodium carbonate solution, ammonia and sewage) also causes rapid decomposition. But acids, nitric and hydro- chloric, as well as putrefaction, act more slowly, giving the blood a laked appearance and even making it clear and colorless. If, however, the blood has once dried, these injurious agencies, even putrefaction, act less easily. Preparation of Haemin Crystals. Prepare a cold aqueous extract of the stain as free as possible from fibers and evaporate the solution upon a watch-glass away from dust. Add a trace of sodium chloride 1 to the residue, also 8-10 drops of glacial acetic acid, and stir with a glass rod. Heat just for an instant over a small flame, then evaporate the solution gradually upon a moderately warm water-bath and examine the residue with a microscope magnifying 300-500 times. If haemin 1 Strzyzowski (Chemisches Centralblatt, 1897, I, 295) advises using sodium iodide instead of sodium chloride. Place a small particle of material suspected of containing blood upon a glass slide and add a drop of sodium iodide solution (i : 500). Evaporate and cover with a cover-glass. Heat for 3-6 seconds with concentrated acetic acid which is allowed to run under the cover-glass. The test with this modification is said to be more delicate, owing to the darker color of the hsematin hydriodide crystals. The crystals are usually obtained in less time and with as small a quantity as 0.000025 gram of fresh blood. Tr. DETECTION OF CARBON MONOXIDE BLOOD 309 crystals fail to appear, repeat the evaporation several times, using in each instance 8-10 drops of glacial acetic acid, and examine the residue each time under the microscope. Hsemin crystals are brownish red to dark brown and form rhombic scales which frequently lie crossed (Fig. 25). Usually glacial acetic acid is the only solvent that will extract the pigment from old blood stains. Brucke heats the stains or scrapings to boiling in a test-tube with 10-20 drops of glacial acetic acid. The decanted or filtered solution, after addition of a trace of sodium chloride, is evaporated upon a watch-glass to dryness at 40-80 and the residue is examined FlG aSm ^^ Crystals . under the microscope. By this method it is immaterial whether the blood has coagulated or not. Cold water is without effect upon blood stains, if they have previously been treated with hot water. Protein substances in the blood are thus coagulated and rendered insoluble. In such a case treat the stain with water containing a few drops of sodium hydroxide solution. If the stains are upon wool, use very dilute sodium hydroxide solution since alkalies dissolve wool. Water containing ammonium hydroxide will extract stains and this alkali does not act upon wool. Use the alkaline aqueous extract to prepare hsemin crystals. Evaporate the solution to dryness in a watch-glass upon the water-bath and mix the residue intimately with 8-10 drops of glacial acetic acid. Add a trace of sodium chloride and again evaporate. Sometimes it is advisable, after acidifying the extract of the stain with acetic acid, to add tannic acid, or zinc acetate, and prepare Teichmann's crystals from the precipitate. Occasionally it is necessary to extract suspected stains with hot alcohol containing sulphuric acid. Haematin formed from the blood pigment dissolves. If this compound is present, the solution has a brown color. Excess of sodium hydroxide solu- tion will produce the dichroism characteristic of an alkaline 310 DETECTION OF POISONS haematin solution, namely, red by transmitted and green by reflected light. Obviously, haematin should be identified by the spectroscope both in acid and alkaline solution. Blood mixed with iron oxide (blood upon rusty weapons) usually fails to give haemin crystals but the extract with dilute sodium hydroxide solution frequently shows the dichroism of hasmatin solution. Since iron oxide or rust forms an insoluble compound with haematin, warm such stains for some time upon the water-bath with sodium hydroxide solution to dissolve any haematin present. Haematin. Warming an aqueous blood solution to about 70 decomposes the blood pigment oxyhaemoglobin into a protein substance called globin and haema- tin, a pigment containing iron. Acids, alkalies and several metallic salts decom- pose oxyhaemoglobin in the same way. If this decomposition takes place in the absence of oxygen, another pigment appears. Hoppe-Seyler gave the latter the name hsemochromogen and other experimenters have called it "reduced haema- tin." Oxygen and consequently air rapidly oxidizes this pigment to haematin. On the other hand reducing agents like ammonium sulphide convert haematin into haemochromogen. Different formulas are given for haematin. W. Kiister and others now give it the formula C34H34N 4 FeO5. Haematin is amorphous and has a dark brown or blue-black color. In water, dilute acids, alcohol, ether and chloroform it is insoluble but soluble in alcohol or ether containing acid. In even very dilute solutions of caustic alkalies it is freely soluble. Alkaline haema- tin solutions are dichroic. In rather thick layers the color appears red by trans- mitted light and greenish in thin layers. Acid solutions are always brown. Alkaline haematin solutions are precipitated by calcium or barium hydroxide solution. Haemin is the hydrochloric ester of haematin. Very prob- ably haemin has the empirical formula Cs-iHas^FeC^Cl. Note. If the blood stain is perfectly fresh, it may be recog- nized by observing blood-corpuscles with the microscope. Human blood can be differentiated from animal blood by comparing blood-corpuscles with those of animal blood as to size, only when the corpuscles are still intact. Spectroscopic Detection of Blood If the extract of a blood stain with cold water is already brown, a third fainter and narrower band will appear in ad- dition to the two oxyhaemoglobin bands. This lies in the orange between C and D and is the methaemoglobin band. Cold DETECTION OF CARBON MONOXIDE BLOOD 311 water will dissolve most of the methaemoglobin from fresh dried blood stains. Acetic acid will discharge these two bands, if the oxyhaemo- globin solution is not too dilute. At the same time the solution will become mahogany-brown from formation of haematin in acid solution. This solution has a characteristic spectrum, namely, four absorption-bands in the yellow and green. If excess of ammonium hydroxide is added to this solution, the alkaline solution contains haematin, recognizable by a broad faint absorption-band lying between the red and yellow. A few drops of ammonium sulphide solution will extinguish this band and bring out two broad bands, namely, one in the green and the other in the light blue. These bands lie farther to the right than do those of oxyhaemoglobin and are of about the same width. This is the spectrum of reduced haematin (haemo- chromogen). All these spectroscopic tests are very charac- teristic, especially the spectra of oxyhaemoglobin, haemoglobin and, in the case of old blood stains, that of reduced haematin. Up to the present time no red solution has been found which, upon abstraction and addition of oxygen, will give the same spectroscopic phenomena as blood. When the quantity of blood is very small, 01 when the blood pigment has undergone further decomposition, so that the bands of oxyhaemoglobin are no longer visible, it is advisable to extract the stains for several hours with concentrated potas- sium cyanide solution. Blood will give a light red or yellowish brown solution containing the cyano-compound of haematin. The spectrum of haematin in alkaline solution will appear as a broad, faint band. The investigations of Kratter and Hammerl have shown that charred blood, which no longer responds to any of the other blood reactions, will still 'give the haematoporphyrin spectrum upon treatment with concentrated sulphuric acid (E. v. Hof- mann, Lehrbuch der gerichtlichen Medizin, 1903). Ammoniacal carmine solution gives two absorption-bands similar to those of oxyhaemoglobin but they do not change upon addition of acetic acid or ammonium sulphide. A band 312 DETECTION OF POISONS given by fuchsine analogous to that of haemoglobin remains unchanged after agitation with air. Other Blood Tests i. Schb'nbein-Van Been Ozone Test. A mixture of ozon- ized turpentine 1 and alcoholic tincture of guaiac resin, shaken with a little blood, produce a light blue color. Separated from the turpentine, the tincture is deep blue. Though very delicate, this test is not characteristic of blood, for many inorganic and organic substances under the same conditions produce "guaiac blue." Nitrous acid, chlorine, bromine and iodine, chromic and permanganic acids, ferric and cupric salts produce blue solutions direct with guaiac resin. In examining blood stains usually it is possible to exclude these substances beforehand. But other substances like cell contents or haemo- globin, having the power of transferring ozone, may attach false significance to the guaiac-blue reaction. Enzymes (diastases), hydrolytic ferments (enzymes in the narrower sense), as well as the so-called oxidation feiments (oxidases), are organic substances of this character. They occur in different parts of plants, especially in fungi and in seeds. Saliva, ex- tracts of certain organs, contents of white blood-corpuscles and pus cells are animal products of similar nature. E. Schaer 2 states that these animal and vegetable substances differ from hydrogen dioxide in being catalytic in action and carriers of oxy- gen at the same time. And also that a temperature of 100, or contact with hydrocyanic acid, completely destroys their power of transferring oxygen, or at least greatly diminishes it, in which respect they are essentially different from haemoglobin. Neither high temperature (100) nor hydrocyanic acid has any restraining influence upon haemoglobin so far as transference of oxygen is concerned. Consequently an extract, containing one of these ferment-like substances but no blood, placed even for a 1 Turpentine always contains ozone, if exposed to light for a long time in a loosely stoppered bottle. 2 Forschungsberichte iiber Nahrungsmittel, etc., 3, i (1896) and Archiv der Pharmazie 236, 571 (1898). DETECTION OF CARBON MONOXIDE BLOOD 313 short time in a hot water-bath, loses the power of giving the "guaiac blue" test. In absence of blood, the result will also be negative, if the extract of the suspected stain is treated with hydrocyanic acid. For these reasons great care is necessary in interpreting a positive guaiac test given by the extract of a supposed stain. The guaiac test is certainly very useful as a delicate preliminary test and in many instances as a check upon blood. The three forms of the blood pigment entering into such an examination, namely, haemoglobin, methaemoglobin and haematin, are alike in the guaiac test, at least qualitatively, as far as transference of oxygen is concerned. The examination and extraction of the stain may, therefore, be conducted in neutral, acid or alkaline solution, depending upon the nature of the substance, and either hot or cold. Render an alkaline extract faintly acid with acetic acid before adding guaiac tincture. In many instances it is advisable to extract the blood stain with hot alcohol containing sulphuric acid. Treat such an acid, alcoholic hasmatin solution with guaiac tincture direct. Addition of water will precipitate the resin with the adherent blood pigment. (a) Vitali's Procedure. Extract the stain with water con- taining carbon dioxide, or old stains with very dilute sodium hydroxide 1 solution free from nitrite and nitrate. F Iter the extract and add a little alcoholic guaiac tincture to a portion of the nitrate after acidification with acetic acid, if necessary. If the milky liquid is not blue in 15 minutes, interfering oxidiz- ing agents are absent. Then add a few drops of old turpentine and shake. The milky liquid will turn blue at once, or in a short time, if blood pigment is present. Very .gentle wanning upon the water-bath increases the delicacy of the reaction. Even putrid blood 2 months old is said to give a positive test. (6) E. Schaer's Procedure. Blood stains upon linen, though quite old, dissolve completely when treated for some time with 70 per cent, chloral hydrate solution. Moistening the stains beforehand with glacial acetic acid aids solution. Also pre- pare an extract of guaiac resin in 70 per cent, chloral hydrate 1 In this test use sodium hydroxide prepared from metallic sodium. 314 DETECTION OF POISONS solution. Mix the extract of the stain with an equal volume of the latter solution. In absence of nitrites, the color of this mixture is brownish yellow to light brown. If preferred, a contact test for blood may be made by this method. Add to the mixture of blood and guaiac Hiinefeld's 1 turpentine solu- tion, or hydrogen peroxide, as a surface-layer. An intense blue zone will appear where the two solutions meet. Guaia- conic acid in guaiac resin produces ' ' guaiac blue. ' ' O . Dobner has suggested substituting a dilute solution of guaiaconic acid for guaiac resin. Blood, or blood pigment, behaves like a ferment and activates the ozonized turpentine or hydrogen peroxide, either of which by itself will not turn the solution of guaiac resin blue. 2. Schaer's Aloin Test. The same conditions, producing "guaiac blue" from guaiaconic acid, give rise to "aloin red" from aloin. This substance has a stronger coloring power and lasts longer than "guaiac blue." Use the same solution of blood in 70-75 per cent, chloral hydrate solution mixed with a weak chloral hydrate solution of aloin. Add Hiinefeld's hydro- gen peroxide solution as a surface layer. After some time a violet-red zone will appear and a red color of equal intensity will gradually extend throughout the aloin solution. Another method of making this test consists in first extracting the blood stain with pure water, acetic acid, chloral hydrate solution or alkaline salt solution. Neutralize this solution and add dilute alcoholic aloin solution and hydrogen peroxide. If the sus- pected stain contains blood pigment, a red color will appear or once and persist for a long time. 3. Biological Detection of Human Blood 2 Injection of bacteria produces specific, bacteriolytic bodies and similarly injection of the blood of one animal species into 1 See page 321 for the preparation of this reagent. 2 This subject has been introduced for the sake of completeness. If such an investigation is for forensic purposes, the chemist will either decline to undertake it, or conduct the experiment with an associate who has had bacteriological and pathological experience. DETECTION OF CARBON MONOXIDE BLOOD 315 an animal of a different species gives rise to specific, hamiolytic and agglutinating bodies. Rabbit's blood, for example, in- jected repeatedly into a guinea pig, develops in the serum of such a guinea pig substances capable of agglutinating and dis- solving red corpuscles of the rabbit, setting hemoglobin free and rendering the blood laky. Blood serum from an animal, into which denbrinated blood, or blood serum from a different animal species has been injected intravenously, subcutaneously or intraperitoneally, that is to say, into the peritoneal cavity, has the peculiar property of causing precipitation only in blood serum of this particular animal species. Uhlenhuth, 1 Wasser- mann and Schiitze, 2 and others have made independent experi- ments of this kind with blood serum to find for forensic purposes a test, based upon this biological method, which shall differ- entiate human blood from the blood of every other animal species. Repeated injection of 10 cc. of defibrinated human blood, or human blood serum free from cells, into a rabbit, either intraperitoneally or subcutaneously, yields a serum producing a heavy, cloudy precipitate in an aqueous solution of human blood. This coagulin is specific in action, producing a precipi- tate only in presence of human blood. Wassermann and Schiitze tested the blood of 23 different animals, among which were mammals, birds and fishes, and obtained negative results with blood solutions from these very different animal species. By use of blood serum it is possible to differentiate even old human blood, dried for many weeks, from the blood of other animals. To demonstrate the use of this method, A. Dieudonne 3 prepares i per cent, blood solutions, placing 2 cc. of the clear filtered solution in small test-tubes and adding an equal volume of double physiological salt solution (=1.8 per cent. NaCl). Then add 6 drops of serum to each portion and place the tubes in an incubator at 37. The serum of the rabbit, treated with 1 Deutsche medizinische Wochenschrift, 1901, No. 6; und Zeitschrift fUr Medizinalbeamte, 1903, Heft 5 and 6. ^Berliner klinische Wochenschrift, 1901, No. 7. 3 Miinchener medizinische Wochenschrift, 1901, page 533- 316 DETECTION OF POISONS human blood serum, added to an aqueous solution of 'human blood, produced in a few minutes a distinct flocculent precipi- tate which gradually became more and more marked. As a check, test also with normal rabbit's serum which will cause no precipitate in a solution of human blood. Dieudonne found also that rabbit's serum, obtained after injecting human blood serum, causes precipitates not only in human blood solutions but in human urine containing albumin, with an exudate from human pleura and with peritoneal exudate. But precipitation in the case of human blood was much more marked than in these other tests. In his experiments Dieudonne used blood ex- pressed from the placenta, repeatedly injecting it subcutane- ously into rabbits in separate doses of 10 cc. and at intervals of 3-4 days. The animals were bled several days after the last injection and the blood was kept upon ice. The antiserum used in detecting blood should above every- thing else be perfectly clear. To prepare such serum, use a sterile Berkefeld filter attached to a water pump. The anti- serum should be active in very dilute solution. Distinct tur- bidity should appear immediately in a solution diluted i : 1000, or in 1-2 minutes at latest. Sera must be of this high efficiency for practical use. Uhlenhuth has shown that the biological method of detecting blood is specific for human albumin. A necessary consequence of this fact is that the material should first of all be shown to be blood. The first question for the expert to answer in such an investigation must always be: "Is there any blood at all present?" If the answer is affirma- tive, the next question is: "Is it human or animal blood?" Consequently the material should first be examined for blood stains by van Deen's ozone test, Teichmann's haemin test and by the spectroscope. If the suspected stains are upon a hard surface, as a knife, hatchet, gun-barrel, wood, stone, etc., they should be scraped off for the biological blood test and extracted for several hours in a test-tube with physiological salt solution (=0.9 per cent. NaCl). First, filter the extract through paper. If the filtrate is not clear, next use a Berkefeld filter. APPENDIX PREPARATION OF REAGENTS General Alkaloidal Reagents. A class of reagents, known as general alkaloidal reagents, added to solutions of most of the alkaloids or of their salts, produce precipitates characterized by their color, their amorphous or crystalline appearance and their insolubility or sparing solubility in water. But these reagents do not precipitate alkaloids exclusively. Several members of this class, for example, the chlorides of gold, platinum and mercury, phospho-molybdic and phospho-tungstic acids, react similarly with ammonia and many ammonium derivatives. An explanation of this similarity in behavior is found in the fact that most of the alkaloids, being secondary or tertiary bases, are themselves ammonium derivatives. Nearly all the general alkaloidal reagents also precipitate proteins, albumoses, pep- tones, creatinine and the nuclein bases, adenine, guanine, hypoxanthine and xanthine. The general alkaloidal reagents are especially useful in detecting the presence, or absence, of alkaloids and other basic compounds. If there is only a slight residue from the ether extract of the alkaline solution in the Stas-Otto method, test first with the general alkaloidal reagents and then, if necessary, for individual alkaloids. To perform these tests, dissolve the given residue in very dilute hydrochloric or sulphuric acid, distribute the filtered solution upon several watch-glasses and add to each portion a drop of the more sensitive reagents. If an alkaloid or any other basic substance is present, distinct precipitates or at least decided cloudiness will appear in all or in nearly all of the tests. The most important general alkaloidal reagents are the following : Gold Chloride dissolved in water (i : 30) produces white, yel- low or brown precipitates which are amorphous or crystal- 317 318 DETECTION OF POISONS line. These precipitates decompose to some extent with separation of metallic gold. Platinum Chloride dissolved in water (i : 20) produces yel- lowish white to yellow precipitates which are usually granular and crystalline. These precipitates are usually analogous in composition to ammonium chloroplatinate, (EUN^PtCle- Mercuric Chloride dissolved in water (i : 2,0) produces white to yellowish precipitates which are usually amorphous but gradually become crystalline. lodo-potassium Iodide, prepared by dissolving 5 parts of iodine and 10 parts of potassium iodide in 100 parts of water, produces brown precipitates which are usually flocculent. Potassium Cadmium Iodide, prepared by dissolving 20 grams of potassium iodide in 20 cc. of boiling water, adding 10 grams of cadmium iodide and diluting to 100 cc., produces white or yellowish precipitates with sulphuric acid solutions of most of the alkaloids, even when these solutions are very dilute. These precipitates, at first amorphous but later crystalline, dissolve in an excess of the reagent and also in alcohol. Potassium Bismuthous Iodide may be prepared according to Kraut 1 by dissolving 80 grams of bismuth subnitrate in 200 grams of nitric acid (sp. gr. 1.18 = 30 per cent. HNO 3 ) and pour- ing this solution into a concentrated solution of 272 grams of potassium iodide in water. Allow the potassium nitrate to crystallize and dilute the solution with water to 1000 cc. This reagent produces orange-red precipitates with sulphuric acid solutions of many alkaloids. By shaking these precipitates with sodium hydroxide and carbonate solution, it is often pos- sible to recover the alkaloids unchanged and sometimes almost quantitatively. Potassium Mercuric Iodide, prepared by dissolving 1.35 grams of mercuric chloride and 5 grams of potassium iodide in 100 cc. of water, produces white or yellowish precipitates with hydrochloric acid solutions of most of the alkaloids. These precipitates, at first amorphous, gradually become crystalline. 1 Annalen der Chemie und Pharmazie, 210, 310 (1882) und Archiv der Phar- mazie, 235, 152 (1897). PREPARATION OF REAGENTS 319 Potassium Zinc Iodide is prepared by dissolving 10 grams of zinc iodide and 20 grams of potassium iodide in 100 cc. of water. Phospho-molybdic Acid may be prepared by either of the following methods : (a) Saturate sodium carbonate solution with pure molybdic acid, add i part of crystallized disodium phosphate (Na 2 HPO 4 .- i2H 2 O) to 5 parts of the acid and evaporate to dryness. Fuse the residue in a porcelain crucible and dissolve the cold melt in water. Prepare 10 parts of solution from i part of this residue. Add enough nitric acid to the filtered solution to produce a golden yellow color. (b) If molybdic acid is not at hand, completely precipitate at 40 with excess of sodium phosphate solution the nitric acid solution of ammonium molybdate used in testing for phos- phoric acid. Thoroughly wash the yellow precipitate, add water and dissolve in warm concentrated sodium carbonate solution. Evaporate this solution to dryness and fuse the resi- due until ammonia is completely expelled. If there is any reduction (blue or black color), moisten the residue with nitric acid and fuse again. Dissolve this residue in hot water and add nitric acid in large excess. Prepare 10 parts of solution from i part of residue. The golden yellow solution should be protected from ammonia vapor. Phospho-molybdic acid produces yellowish, amorphous pre- cipitates with sulphuric acid solutions of most of the alkaloids. After a while these precipitates are frequently greenish or bluish from reduction of molybdic acid to molybdic oxide. Phospho-tungstic Acid, prepared by adding a little 20 per cent, phosphoric acid to an aqueous solution of sodium tungs- tate, produces precipitates similar to those given by phospho- molybdic acid. Tannic Acid is a 5 per cent aqueous solution of tannin. This reagent produces whitish or yellowish, flocculent precipitates partially soluble in hydrochloric acid. Alkaloids may be recovered in part from these precipitates by treating them with lead or zinc carbonate, evaporating to dryness and extracting the residue with ether, alcohol or chloroform. 320 DETECTION OF POISONS Picric Acid is a concentrated aqueous solution of picric acid which produces yellow crystalline precipitates, or amorphous precipitates which soon become crystalline. Picrolonic Acid is used as o.i normal alcoholic solution by dissolving 26.4 grams of solid picrolonic acid (CioH 8 N 4 05) in a liter of alcohol. With most of the alkaloids this solution pro- duces salts called picrolonates which are crystalline, difficultly soluble and yellow to red in color. Picrolonic acid behaves to- ward bases like a monobasic acid. 1 B. Other Reagents and Solutions Erdmann's Reagent. Sulphuric acid containing nitric acid, prepared by adding to 20 cc. of pure concentrated sulphuric acid 10 drops of a solution of 6 drops of concentrated nitric acid in 100 cc. of water. Froehde's Reagent. A solution of molybdic acid in sulphuric acid, prepared by dissolving 5 mg. of molybdic acid, or sodium molybdate', in i cc. of hot, pure concentrated sulphuric acid. This solution, which should be colorless, does not keep long. Fehling's Solution. The two following solutions, which should be kept separate, are used in preparing this reagent: 1. Copper Sulphate Solution. Dissolve 34.64 grams of pure crystallized copper sulphate (CuSO 4 .5H 2 O) in sufficient water to make 500 cc. 2. Alkaline Rochelle Salt Solution. Dissolve 173 grams of Rochelle salt (K.Na^H^Oe^H^O) and 50 grams of sodium hydroxide in hot water and dilute this solution when cold to 500 cc. These two solutions, mixed volume for volume, constitute Fehling's solution which should be prepared just before being used. Fehling's solution, which has been made up and kept, should always be tested before being used. The solution should not be used, if it gives a red precipitate of cuprous oxide when warmed by itself. 1 L. Knorr, Berichte der Deutschen chemischen Gesellschaft, 30, 914 (1897); H. Matthes and O. Rammstedt, Zeitschrift fiir analytische Chemie 46, 565 and Archiv der Pharmazie 245, 112 (1907). PREPARATION OF REAGENTS 321 Formaldehyde-sulphuric Acid. Add 2-3 drops of aqueous formaldehyde solution (formalin) to 3 cc. of pure concentrated sulphuric acid just before using. Giinzfcurg's Reagent. 1 Dissolve i part of phloroglucinol and i part of vanilline in 30 parts of alcohol. This reagent is used to detect free mineral acid, especially hydrochloric acid, but it does not react with free organic acids. Hiinef eld's Solution. Add 25 cc. of alcohol, 5 cc. of chloro- form and 1.5 cc. of glacial acetic acid to 15 cc. of old turpentine which has been exposed for some time to air and light. The turpentine used should not produce a blue color with guaiac tincture direct nor with 15 cc. of 3-5 per cent, hydrogen peroxide free from acid. This solution is used in the detection of blood. lodic Acid Solution. Prepare a 10 per cent, aqueous solution of iodic acid (HI0 3 ). Magnesia Mixture. Dissolve 1 1 grams of crystallized mag- nesium chloiide (MgCl 2 .6H 2 0) and 14 grams of ammonium chloride in 130 cc. of water and add 70 grams ol ammonium hydroxide solution (sp. gr. 0.96 = 10 per cent, of NH 3 ). This mixture should be clear. It is used to detect arsenic and phosphoric acids. Mandelin's Reagent. Dissolve i part of ammonium meta- vanadate (H 4 N.VO 3 ) in 200 parts of pure concentrated sulphuric acid. Millon's Reagent. Dissolve i part of mercury in i part of cold fuming nitric acid. Dilute with twice the volume of water and decant the clear solution after several hours. Nessler's Reagent. Dissolve separately in the cold 3.5 grams of potassium iodide in 10 cc. of water and 1.7 grams of mercuric chloride in 30 cc. of water. Add mercuric chloride solution to potassium iodide solution until there is a permanent precipitate. Dilute with 20 per cent, sodium hydroxide solu- tion until the volume is 100 cc. Add mercuric chloride solution, until there is again a permanent precipitate and let the solution i It is advisable to prepare this reagent as required. Keep two, separate alco- holic solutions (i : 15) of phloroglucinol and vanilline and mix volun as needed. Tr. 21 322 DETECTION OF POISONS settle. Decant the clear solution and keep in small bottles in the dark. This reagent improves upon standing. Mecke's Reagent. 1 Dissolve 0.5 gram of selenious acid in 10 grams of pure concentrated sulphuric acid. Stannous Chloride Solution. Mix 5 parts of crystallized stannous chloride with i part of hydrochloric acid and com- pletely saturate with dry hydrochloric acid gas. Let this solution settle and filter through asbestos. It is a pale, yellowish, refractive liquid (sp. gr. at least 1.9). This solution is used to detect arsenic (Bettendorffs Arsenic Test). C. The Indicator lodeosine lodeosme, or erythrosine, Cz<>H.&LiQo, is a tetra-iodo-fluoresceine, formed by treating fluoresceine with iodine, and has the formula: / CeHI 2 (OH)\ CfC 6 HI 2 (OH)/ U I \C 6 H 4 .CO.O I I The commercial preparation usually contains as impurities small quantities of substances almost insoluble in ether. To obtain a pure product, 2 dissolve com- mercial iodeosine in aqueous ether and extract iodeosine from the filtered ether solution by means of dilute sodium hydroxide solution. Strong sodium hydrox- ide solution, added to this aqueous alkaline solution, precipitates the sodium salt of iodeosine. Filter, wash with cold alcohol and crystallize from hot alcohol. Well formed, almost rectangular plates having a green color on the surface are obtained. Hydrochloric acid precipitates pure iodeosine from the aqueous solu- tion of the sodium salt. Pure iodeosine dried at 1 20 is markedly lighter than the commercial preparation. It is almost insoluble in absolute ether, benzene and chloroform; more easily soluble in acetone, alcohol and aqueous ether. The tone of the purified pigment dissolved in aqueous alkali is yellower than that of the crude product. lodeosine is a scarlet crystalline powder which dissolves in alco- hol with a deep red and in ether with a yellowish red color. lodeosine is said to be insoluble in water containing a trace of hydrochloric acid. To prepare iodeosine solution for use as an indicator, dissolve i gram of the pigment in 500 grams of alcohol. 1 Zeitschrift fiir offentliche Chemie 5, 350 (1899). 2 Fr. Mylius and F. Foerster, Berichte der Deutschen chemischen Gesellschaft 24, 1482 (1891). INDEX OF NAMES Abel, R., Biological arsenic test, 244. Mould for same, 245. Bischoff, C., Carbolic acid in the body, 27. Oxalic acid in the body, 190. Potassium chlorate in putrefac- tion, 197. Archangelsky, Chloral hydrate estima- Black, Gutzeit estimation of arsenic, 240. Bloemendal, Normal arsenic, 176. tion, 41. Acetone on dogs, 55. Autenrieth, W., Phenacetine test, 76. Morphine in putrefaction, 137. Blondlot, Phosphorus test, 8. Destruction of organic matter, Bodlander, Tin poisoning, 181. 151. Copper in organs, 178. B v. Babo, Destruction of organic matter, 148. Detection of arsenic, 161. Barger, Ergotoxine, 209. Baudin, Cantharidin in Spanish flies, 264. Baumann, E., Carbolic acid in putre- faction, 27. Baumert, Nitric acid detection, 185. Bazlen, M., Maltol, 251. Beck, Aconine, 262. Beckurts, H., Carbolic acid estimated, 31. Arsenic isolated as trichlo- ride, 233. Theobromine and caf- feine in cacao and chocolate, 298. Beissenhirtz, Chromic acid test for aniline, 45. Benedikt, R., Compound of carbolic acid and bromine, 29. Bernard, Arsenic reagent, 247. Berthelot, M., Ethyl alcohol test, 50. Bertrand, Normal arsenic, 175. Berzelius, Arsenic test, 156. Borsche, Composition of isopurpuric acid, 71. Bougault, J., Arsenic reagent, 247. Boughton, W. A., Apparatus for Gut- zeit test, 240. Brand, J., Maltol, 251. Brandl, J., Haemolysis by githagin, 224. Bredemann, Colchicin estimation, 269. Brucke, Acetic acid for blood stains, 39- Bruger, P., Composition of picrotoxin, 67. Brunner, H., Caffeine and theobro- mine estimation, 272. Brunner, K., Saponin in beverages, 223. Biidinger, Chloroform in mucus, 35. Buttenberg, Biological arsenic test, 244. Mould for same, 245. Caesar, Estimation of pilocarpine, 286. Caillot, Piperine in pepper, 288 Carles, J., Quinine estimation, 268. Bettendorff, Arsenic test, 162. Prepa- Carlson, C. E., Arsenic in organic corn- ration of reagent, 322. pounds, 245. Beuttel, F., Carbolic acid determina- Carr, F. H., Ergotoxine, 209. tion, 31. Biggs, Methyl alcohol test, 53. Cazeneuve, Piperine in pepper, 288. Cerny, Normal arsenic, 176. Biginelli, Gas from biological arsenic Chancel, Solubility of carbon disul- test, 243. phide, 46. 323 324 INDEX OF NAMES Chittenden, Arsenic in the brain, 174. Ciamician, Structure of pseudo-pellet- ierine, 270. Ckindi, Solubility of carbon disul- phide, 46. Couerbe, Narcotine test, 115. Dale, H. H., Ergotinine, 210. DenigSs, Methyl alcohol test, 54. Cocaine test, 109. Dieterich, K., Cantharidin in Spanish flies, 264. Analysis of cola nuts and extracts, 272. Dieudonne, A., Biological blood test, 3iS. Dilthey, A., Veronal preparation, 80. Dobner, O., Guaiaconic acid in blood test, 314. Doepmann, Morphine in putrefaction, 137- Dragendorff, G., Phosphorus in cada- vers, 16. Narcotine test, 115. Detection of cantharidin, 205. Dusart, Phosphorus test, 8. Eichwede, Compound of carbolic acid and bromine, 29. Elfstrand, Blood agglutinated by ricin, 229. Elvers, Phosphorus in cadavers, 16. Engel, Arsenic reagent, 247. v. Engelhardt, R., Aniline black in the organism, 44. Erdmann, Narcotine test, 115. Nar- ceine test, 139. Thebaine test, 228. Reagent prepared, 320. Faust, Morphine in the organism, 137. Fehling, Reagent prepared, 320. Fihlene, Nitrobenzene absorption- band, 42. Fischer, A., Interference with phos- phorus test, 8. Fischer, B., Chloroform estimation, 38. Distribution of same in cadavers, 38. Distribution of ethyl alcohol in organism, 49. Fischer, E., Veronal preparation, 80. Excretion of same, 81. Recovery from urine/ 82. Murexide reac- tion, 85. Fleury, G., Morphine test, 135. Ni- tric acid detection, 185. Fluckiger, F. A., Carbolic acid test, 29. Foerster, F., Preparation of pure iodeosine, 322. Forster, A., Estimation of caffeine in coffee, 272. Frankel, A., Estimation of phosphorus in phosphorated oils, 232. Frerichs, Alkaloids in ipecac, 277. Frerichs, G. and H., Toxicological examination for veronal, 81. Fresenius, C. R., Phosphorus test, 10. Destruction of organic matter, 148. Detection of arsenic, 161. Freund, M., Structure of veratrine al- kaloids, 95. Narcotine structure, 1 14. Identity of pseudo-narceine and narceine, 138. Aconine, 262. Froehde, Test for codeine, 112. For narcotine, 115. For hydrastine, 118. For apomorphine, 128. For morphine, 134. For papa- verine, 217. For saponins, 222. For solanine and solanidine, 227. For thebaine, 228. For cephae- line, 278. Preparation of re- agent, 320. Fromme, G., Pilocarpine in jaboran- dum, 286. Theobromine and caf- feine in cacao and chocolate, 299. de Fuentas Tapis, Alkaloids in ipecac, 277. Fiihner, H., Thalleioquin reaction, 121. Fujiwara, Chloroform test, 37. Gadamer, J., Cantharidin structure, 203. Structure of cantharic acid and acetyl-hydrato-cantharic an- INDEX OF NAMES 325 hydride, 204. Caffeine in tea, coffee and cola, 272. Gair, D., Estimation of strychnine and quinine together, 258. Gautier, A., Normal arsenic, 1 74. De- struction of organic matter, 234. Girard, Ch., Detection of salicylic acid in milk, 251. Goeckel, H., Caffeine, 275. Goldschmiedt, G., Structure of papa- verine, 216. Gordin, H. M., Estimation of alka- loids, 257. Of strychnine, 298. Gosio, B., Moulds on arsenical media, 242. Graf, L., Caffeine in tea, 272. Grandeau, Veratrine test, 96. Guareschi, Solubility of quinine sul- phate, 268. Guglialmelli, Pyramidone test, 125. Gunning, Acetone differentiated from ethyl alcohol, 56. Giinzburg, Mineral acid test, 182. Reagent for mineral acid, 321. Gutzeit, Arsenic test, 163. Hinsberg, Phenacetine test, 76. Hirschsohn, Quinine test, 122. Hodlmoser, Normal arsenic, 176. Hofmann, A. W., Sensitiveness of phenylisocyanide test, 36. Ex- haustive methylation, 117. v. Hofmann, E., Haematoporphyrin spectrum, 311. Hofmeister, Lead in sheep, 177. Holmes, Isolation of methyl alcohol, S3- Hoppe-Seyler, F., Haemochromogen, 310- Huchard, Toxic action of nicotine, 91. Hunefeld, Preparation of blood re- agent, 321. Hurt, H., Electrolytic estimation of arsenic, 237. Electrodes for same, 239. Organic matter in ar- senic determinations, 239. Husemann, Morphine test, 133. Ipsen, Resistance of atropine to putre- faction, 103. Halasz, Z., Modification of Blondlot- Dusart phosphorus test, 13. Or- ganic phosphorus compounds, 13. Hammerl, Examination of charred blood, 311. Harmsen, Carbon disulphide on li- poids, 46. Harrison, E. F., Estimation of quinine and strychnine. 258. Helch, H., Pilocarpine test, 219. Hildebrandt, H., Chlorate in urine, 197. Hilger, Phosphorus test, n. Reduc- tion of phosphorous acid to phos- phine, 14. Apparatus to esti- mate phosphorus, 15. Sensitive- ness of Mitscherlich test, 16. Caffeine in coffee and tea, 271. Hille, W., Solubility of quinine sul- phate, 268. Hinkel, Methyl alcohol test, 54. Jaffe, Rubazonic acid, 124. Santonin in dogs, 199. Jaquet, A., Toxic action of ergotinine and hydro-ergotinine, 210. Jaworowski, Chloral hydrate differ- entiated from chloroform, 39. Jeserich, Destruction of organic mat- ter, 151. JoneScu, D., Antipyrine in the organ- ism, 123. Alkaloids estimated by potassium bismuthous iodide, 255. Jowett, Pilocarpine, 217. Formula of same, 218. Juckenack, Caffeine in coffee and tea, Katz, E., Estimation of caffeine, 272, 276. Katz, J., Estimation of colchicin, 269. Caffeine in plant products, 276. Santonin in wormseed, 290. 326 INDEX OF NAMES Keller, C. C., Ergotinine, 209. Detec- tion and estimation of same, 211. Caffeine in tea, 272. Nicotine in tobacco, 279. Alkaloids in nux vomica, 293. Kiliani, H., Digitalinum verum crys- tallisatum, 207. Hydrolysis of digitonin, 207. Test for digi- toxin, 208. Digitalin, 208. Di- gitalose, 208. Maltol, 251. Kippenberger,*C., Extraction of mor- phine, 130. Kissling, R., Nicotine in tobacco, 279. Klason, Arsenic and mercuric chloride compound in biological test, 243. Kleist, Excretion of veronal, 81. Re- covery from urine, 82. Knocke, Solubility of antimony spot, 160. Knorr, L., Picrolonic acid, 253, 320. Kobert, L., Solanine extracted with isobutyl alcohol, 226. Kobert, R., Toxic action of hydro- cyanic acid, 20. Chloroform con- verted in organism to chloride, 35. Chloral hydrate in brain, 41. Physiological action of nitroben- zene, 43. Urine in aniline poi- soning, 44. Carbon disulphide on lipoids, 46. Ethyl alcohol in blood, 49. Acetone in urine, 55. Picric acid in urine, 70. Acetani- lide poisoning, 73. Strychnine in the body, 97. Cocaine physio- logical test, 109. Metals and red blood corpuscles, 173. Toxicity of uranium, 180. Sulphuric acid poisoning, 187. Toxicity of ox- alic acid, 190. Potassium chlo- rate in blood, 194. Cytisine in urine, 206. Digitalis glucosides and urine, 209. Toxic ergot resin, 209. Saponins on blood, 221. Saponins solvents of cholesterin, 222. Toxicity of saponins, 222. Extraction of solanine, 226. Toxic action of solanine, 226. Vegetable agglutinines, 228. Crotin, 229. Toxic action of piperine, 287. Koenig, J., Piperine in pepper, 288. Koenigs, W., Structure of quinine, 119. de Konink, Detection of potassium, 193- Koppeschaar, Estimation of carbolic acid, 31. Kossler, Estimation of cafbolic acid, 33- Kraft, F., Ergotinine, 209. Kratter, Examination of charred blood, 311. Krauss, L., Iodine on potassium xanthogenate, 49. Kraut, Preparation of potassium bis- muthous iodide reagent, 255, 318. Kreis, H., Cholesterine and phyto- sterine with Melzer's reagent, 68. Kiister, W., Formula of haematin, 310. Kunkel, Normal arsenic, 175. Chro- mium poisoning, 177. Tin poi- soning, 181. Langley, Picrotoxin test, 68. Lautenschlaeger, Morphine test, 136. Laves, E., Furfural test for veratrine, 96. Leach, Estimation of methyl alcohol, 55- Legal, Acetone test, 56. Lehmann, V., Determination of silver in organs, 179. Zinc in the dog, 1 80. Toxic effect of sulphur di- oxide in air, 188. Leins, H., Separation and estimation of caffeine and theobromine, 272. Le Nobel, Modification of Legal's ace- tone test, 56. Lieben, lodoform test for ethyl alcohol, 50. Same for acetone, 56. Link, Limit of delicacy of Prussian blue test for hydrocyanic acid, 22. Lloyd, J. U., Morphine test, 134. Lockemann, G., Destruction of or- ganic matter, 234. Precautions in Marsh test, 236. Delicacy of Marsh test, 237. INDEX OF NAMES 327 Loretz, Estimation of pilocarpine, 286. Ludwig, Estimation of chloroform, 38. Lustgarten, Naphthol test for chloro- form, 3^. lodoform test, 42. Lythgoe, Estimation of methyl alco- hoi, 55- Maassen, A., Biological arsenic test, 243- Madsen, Solution of cholesterin in saponin solutions, 222. Magnin, Alkaloids extracted from animal matter, 62. Mai, C., Destruction of organic matter, 152. Electrolytic estimation of arsenic, 237. Electrodes in ar- senic determinations, 239. De- struction of organic matter in arsenic determinations, 239. Mandelin, Test for strychnine, 99. For hydrastine, 118. For pilo- carpine, 219. Preparation of re- agent, 321. Mann, Lead in urine and faeces, 176. Marquis, Morphine test, 134. Mor- phine in the organism, 137. Marsh, Arsenic test, 156. Matthes, H., Estimation of alkaloids as picrolonates, 253. Hydrastine, 282. Pilocarpine, 286. Nuxvom- ica alkaloids, 296. Mauch, R., Chloral hydrate in alkaloid tests, 88. In brucine test, 102. In codeine test, 112. In toxi- cological analysis, 251. Mecke, Codeine test, 113. Reagent for opium alkaloids, 214. Prepa- ration of reagent, 214, 322. Meine, W., Berberine, 282. Melzer, H., Carbolic acid test, 30. Picrotoxin, 67. Nicotine, 92. Mensching, J., Interference with phos- phorus test, 6. Merck, E., Assaying officinal extracts, 302. v. Mering, J., Conjugated chloral hy- drate in urine, 41. Excretion. of veronal, 81. Isolation of same from urine, 82. Messinger, Estimation of carbolic acid, 33- Meyer, G., Solanine in potatoes, 291. Meyer, H., Chloroform iu the body, 35. Action of chloral hydrate, 40. Meyer, R., Composition of picrotorin, 67. Percentage of solanine in potatoes, 225. Miller, W. V., Structure of quinine, 1 20. Millon, Carbolic acid test, 28. Sali- cylic acid test, 78. Preparation of reagent, 321. Mitscherlich, Phosphorus test, 5. Estimation of phosphorus, 15. Mockel, Limit of delicacy of Prussian blue test for hydrocyanic acid, 22. Molle, B., Excretion of veronal, 81. Isolation from urine, 82. v. Morgenstern, F., Solanine in pota- toes, 292. Morner, Karl Th., Estimation of minute amounts of arsenic, 247. Moufang, Structure of brucine, 101. Mulliken, Methyl alcohol test, 53. Musculus, Conjugated chloral hydrate in urine, 41. Mylius, Fr., Preparation of pure iodeo- sine, 322. Nattermann, Phosphorus test, n. Reduction of phosphorous acid to phosphine, 14. Apparatus for es- timating phosphorus, 15. Sensi- tiveness of Mitscherlich test, 16. Nessler, Chloral hydrate test, 39- Preparation of reagent, 321. Neubauer, Phosphorus test, 10. Neumann, Detection of phosphorus in cadavers, 16. Nietzki, Composition of isopurpuric acid, 71. Noguchi, Solution of cholesterin in saponin solutions, 222. 328 INDEX OF NAMES Oliver, Lead in the organism, 177. Osann, Maltol, 251. Page, Solubility of carbon disulphide, 46. Pagel, Normal arsenic, 175. Pagenstecher, Hydrocyanic acid test, 21. Palet, Pyramidone test, 126. Apo- morphine test, 129. Palm, R., Detection of ergot in flour, 211. Panchaud, Estimation of cinchona al- kaloids, 266. Parmentier, Solubility of carbon disul- phide, 46. Partheil, A., Identity of cytisine and ulexine, 205. P61igot, Solubility of carbon disul- phide, 46. Pellagri, Codeine test, 112. Apo- morphine, 128. Morphine, 134. Penny, Estimation of carbolic acid, 33. Penzoldt, Acetone test, 57. Petri, Composition of isopurpuric acid, 7i. Piccard, J., Ortho-xylene from can- tharidin, 204. Piccini, Isomethyl-pelletierine, 270. Pictet, A., Nicotine synthesis, 91. Pinner, Nicotine formula, 91. Nitro- gen in pilocarpine and isopilo- carpine, 218. Pisani, Cocaine test, 109. Pohl, Chloroform in blood and in the body, 35. Polacci, E., Procedure in thalleioquin test, 121. Poleck, Arsenic test, 163. Polstorff, K., Interference with phos- phorus test, 6. Pouchet, Antimony in the body, 176. Prescott, A. B., Estimation of alka- loids, 257. Proells, Cocaine in the cadaver, 107. Pschorr, R., Apomorphine structure, 127. Morphol synthesis, 132. Morphine structure, 133. The- baine structure, 227. Thebaol synthesis, 227. R Radulescu, D., Morphine test, 136. Rammstedt, O., Estimation of alka- loids as picrolonates, 253. Hy- drastine, 282. Pilocarpine, 286. Nux vomica alkaloids, 296. Ramverda, Cytisine test, 206. Ransam, Influence on saponins of cholesterin in blood, 222. Reichard, C., Cocaine test, 109. Reynolds, Acetone test, 57. Riechelmann, Estimation of caffeine in coffee, 272. Riegel, Estimation of antimony by Gutzeit method, 240. Roser, Narcotine structure, 114. Pseudo-narceine from narcotine, 138. Rossi, Conversion of nitrobenzene in organism to aniline, 43. Roussin, Nicotine test, 92. Riigheimer, Piperine synthesis, 287. Rupp, E., Iodine on xanthogenate, 49. Russanow, A., Formula of compound from phenol and benzaldehyde, Sanger, C. R., Estimation of ar- senic and antimony by Gutzeit method, 240. Schaefer, Normal arsenic, 175. Schaer, E., Saponin test, 223. Animal and vegetable catalysts and oxy- gen carriers, 312. Blood stain test, 313. Aloin blood test, 314. Schaffer, Conjugated sulphuric acid in urine in carbolic acid poisoning, 27. Scherer, Phosphorus test, 3. Estima- tion of same, 15. Schiff , Reagent for aldehydes, 54. Schindelmeiser, Nicotine test, 93. INDEX OF NAMES 329 Schmidt, E., Crystalline veratrine, 94. Apomorphine tests, 129. Detec- tion of cantharidin, 205. Iso- lation of schlererythrin, 210. Detection of solanine and solarii- dine, 226. Test for solanine and solanidine, 227. Solubilities of sulphates of cinchona alkaloids, 268. Estimation of berberine, 282. Estimation of alkaloids in belladonna, etc., 300. Schmiedeberg, O., Percentage of sola- nine in potatoes, 225. Solanine in potatoes, 291. Schmitt, R., Preparation of salicylic acid, 77. Schneider, Sodium cacodylate not de- composed by hydrochloric acid, 246. Schonbein, Hydrocyanic acid test, 21. Ozone test for blood, 312. Scholtz, M., Detection of potassium chlorate, 196. Schiitze, Biological blood test, 315. Schwarz, Resorcinol test for chloro- form, 36. Scudder, Methyl alcohol test, 53. Selmi, Organic phosphorus compounds in phosphorus test, 13. Pto- maine resembling morphine, 220. Silber, Structure of pseudo-pelletier- ine, 270. Simmonds, Methyl alcohol test, 54- Skraup, Quinine structure, 120. Socoloff, Caffeine in coffee, 275. Sonnenschein, Destruction of organic matter, 151. Stas-Otto, Process for extracting alka- loids, 63. Stich, C., Estimation of phosphorus in phosphorated oils, 232. Strassmann, Ethyl alcohol eliminated by lungs, 49. Straub, W., Test for phosphorus in phosphorated oils, 14. Estima- tion of phosphorus in same, 231. Accuracy of method, 232. Strzyzowski, Sodium iodide in haemin test, 308. Tafel, Strychnine structure, 97. Bi- chromate test for same explained, 99. Structure of brucine, loi. Tanret, Ergotinine, 209. Tassily, E., Caffeine in coffee, 272. Teichmann, Haemin blood test, 309. Thater, K., Santonin test, 200. San- tonin in wormseed, 289. Thiele, Compound of bromine and phenol, 29. Thorns, H., Separation of quinine from mixtures, 122. Destruction of organic matter, 150. Alkaloids estimated by potassium bismu- thous iodide, 255. Thorpe, T. E., Isolation of methyl al- cohol, 53. Arsenic in beer worts, 237- Tollens, B., Formaldehyde in blood test, 307. Toth, J., Nicotine in tobacco, 280. Trillich, H., Caffeine, 275. Trotmann, Arsenic determined in beer worts, 237. Tiimmel, Arsenic test, 163. U Uhlenhuth, Biological blood test, 315. Ulenberger, Lead in sheep, 17 7- Ungar, Tin poisoning, 181. Van Deen, Ozone blood test, 31.1. Van der Moer, Cytisine test, 206. Vandevelde, Toxicity estimated by blood haemolysis, 258. Vaubel, Solubility of antimony spot, 160. Vitali, Carbon disulphide test, 47 Ethyl alcohol test, 51. Vera- trine test, 96. Atropine test, 104. Detection of caustic alkalies, 193 Blood stain test, 313. 330 INDEX OF NAMES Volhard, Estimation of hydrochloric acid, 184. Von Pohl, Methyl alcohol in the or- ganism, 52. Vortmann, Hydrocyanic acid test, 23. Estimation of carbolic acid, 33. W Wangerin, Narcotine test, 115. Apo- morphine test, 128. Lloyd's mor- phine test, 135. Narceine test, 140. Warren, L. E., Papaverine test, 217. Warren, W. H., 'Alkaloids purified by picrolonic acid, 89. Normal ar- senic, 176. Wassermann, Biological blood test, 24. Weehuizen, Hydrocyanic acid test, 24. Weiss, R. S., Alkaloids purified by pic- rolonic acid, 89. Weppen, Veratrine test, 95. Werk, R., Cause of solanine in pota toeS, 22.S. White, Tin poisoning, 181. Wieser, Normal arsenic, 176. Willstatter, Cocaine structure, 106. Windhaus, Digitonin, 207. Saponin cholesterides, 222. Wittmann, Solanidine from solanine, 225. Witz, Determination of mercury in the body, 179. Wolff, C., Caffeine in coffee, 276. Zappi, Extraction of alkaloids from animal matter, 62. Zeine, Formula of picrolonic acid, 253. Zeisel, Colchicin test, 69. Deter- mination of methoxyl groups in alkaloids, 101. Ziemke, Normal arsenic, 1 76. INDEX OF SUBJECTS Abortifacients, Ergot, 209. Nitro- benzene, 42. Abrin, 229. Absorption-spectra, blood, 306, 310. Carmine, 311. Codeine, 112. Ergot, 211. Fuchsine, 312. Nitrobenzene, 42. Physos- tigmine, no. Acetanilide, 66, 73, 141. Acetic ether (see Ethyl acetate). Acetone, 55. Acetonuria, 55. Acid acetic, Aconitine, 262. aconitic, 261. amino-acetic, 58. a-amino-isobutyl-acetic, 17. angelic, 94. arsenic, 150. atropic, 103. benzoic, 58. Aconitine, 262. Cocaine, 106. brucic, 101. cacodylic, 246. Electrolysis, 234. cantharic, 204. cantharidic, 203, 263. carbolic, 26. Aniline, 34. Esti- mation, 31. Urine, 34. chloric, Organic matter, 151. Re- duction, 196. chloroplatinic, 318. Potassium, 193- chromic, Alcohol, 51. Aniline, 45. Cocaine, 108. cincholoiponic, 119. cinchonic, 119. ethyl-sulphonic, 202. ethyl-sulphuric, Solanine and solanidine, 227. Acid formic, Chloral hydrate, 39. Phosphorus, 4. glycuronic, 41, 44. Aceto-p- amino-phenol, 74. Mor- phine, 137. Organism, 123. guaiaconic, 314. hippuric, 58. hydrastic, 117. hydrochloric, 183. Colchicin, 69. Digitalin, 209. Electrolytic, 241. Organic matter, 151. Vera trine, 95. hydrocyanic, 19. Blood, 312. Estimation, 25. Oil of bitter almonds, 58. Potassium ferrocyanide, 25. Prelimi- nary test, 21. hydro-p-cumaric, 17, 28. hypophosphorous, 8. iodic, 321. Morphine, 134. isopurpuric, 71. lactic, Hydrocyanic acid, 20. loiponic, 119. mandelic, 105. meconic, 213. meconinic, 214. n-methyl-granatic, 270. Acid nicotinic, 91. nitric, 184. Antipyrine, 83. Apomorphine, 128. Co- caine, 107. Codeine, in. Colchicin, 69. Cytisine, 207. Morphine, 133. Narceine, 139. Papaverine, 217. Phenacerine, 76- Physostig- mine,no. Pyramidone, 125. Thebaine, 228. Veratrine, 95- nitrous, Antipyrine, 83. nor-meta-hemipinic, 117- 331 332 INDEX Acid opianic, Hydrastine, 117. Nar- cotine, 114. oxalic, 189. p-amino-phenyl-sulphuric, 44. p-hydroxy-benzoic, 28. p-oxy-phenyl-acetic, 17, 28. p - oxy - phenyl -a- amino - pro- pionic, 17. p-oxy-phenyl-propionic, 17, 28. phenyl-glycuronic, 26. phospho-molybdic, 319. phosphoric, Reduction, 8. phosphorous, 8, 14. Mercury, 166. phospho-tungstic, 319. picramic, Organism, 70. Picric acid, 24, 72. picric, 66, 70, 141. Hydrocyanic acid, 24. Reagent, 320. picrolonic, 320. Alkaloids esti- mated, 253; purified, 89. Hydrastine, 282. Nux vo- mica, 296. Pilocarpine, 286. piperic, 287. quinic, 119, 265. quino-tannic, 265. rubazonic, 124. salicylic, 66, 77, 142. Foods and beverages, 250. Phenols, 28, 78. _ salicyluric, 79. santonic, 198, 290. sarcolactic, 17. sclerotic, 209 selenious, 113. sphacelic/209. strychnic, 97. suberic, 270. sulphanilic, 136. sulphocyanic, 19. sulphuric, 186. Apomorphine, 128. Cocaine, 107. Code- ine, in. Colchicin, 69. Conjugations, 74. Digitalin, 208. Hydrastine, 118. Me- conine, 214. Narceine, 139. Narcotine, 115. Papaverine, 217. Physostigmine, no. Preformed and conjugate, 26. Santonin, 200. Saponins, 223. Solanine, 227. The- baine, 228. Veratrine, 95. sulphurous, 1 88. tannic, 319. Antipyrine, 83. Blood, 305. Caffeine, 85. Colchicin, 69. Narceine, 140. thioacetic, 249. tiglic, 94. trichlor-ethyl-glycuronic, 41. trimethyl-colchicinic, 69. tropic, 103, 1 06. uric, 84. urochloralic (see Trichlor-ethyl- glycuronic). vanadic-sulphuric, 226. veratric, 216. Acid xanthoproteic, 185. Acids, mineral, 182. Aconine, 262. Aconitine, 261. Agglutinines, 228. Air, Carbon disulphide, 48. Sul- phur dioxide, 188. Albuminates, metallic, 151, 172. Alcohol, ethyl, 49. Differentiation, 57. Purification, 61. Tox- icity, 259. methyl, 52. Cocaine, 106. Col- chicin, 68. Estimation, 55. Toxicity, 259. trichlor-ethyl, 41. Alkalies, 192. Alkaloidal reagents (see Reagents, alkaloidal). Alkaloids, Aconite, 261. Alkaloidal reagents, 87. Belladonna, 301. Cinchona, 264. Esti- mation, 253, 255, 257, 296. Extraction, 86. Extracts, 266. Hyoscyamus, 301. Ipecac, 277. Nux vomica, 293-295. Physiological test, 88. Pomegranate, 270. Puri- fication, 88. Selenious-sul- phuric acid, 215. Viscera, 62. INDEX 333 Aloes, Quinine test, 122. Aloin, Blood test, 314. Aluminium acetate, 289. Ammonia, 192. Fapaverine, 217. Physostigmine, no. Ammoniacal copper, Picric acid, 72. Ammoniacal silver, Morphine, 135. Picrotoxin, 67. Ammonium magnesium phosphate, 7. molybdate, 7. persulphate, 152. sulphide, Blood, 305. Sulphides, i54- Anhydro-ecgonine, 106. Aniline, 44, 93, 143. Carbolic acid, 34- Antimony, 155. Detection, 163, 165. Distribution, 175. Estima- tion, 240. Differentiation of spot, 1 60. Antipyrine, 66, 82, 87, 123, 142, 145, 147. Extract, 87. Thalleio- quin, 121. Urine, 83, 123. Antipyryl-urea, 125. Apomorphine, 127, 146. Selenious- sulphuric acid, 215. Argyria, 179. Arrhenal, 246. Electrolysis, 234. Urine, 247. Arsenic, 155. Beer, 237. Betten- dorff, 162. Biological, 242. Bougault's reagent, 247. Bulb-tube, 162. Differentia- tion, 1 60. Distribution, 173. Electrolysis, 233, 237. Eli- mination, 173. Fresenius-v. Babo, 161. Gutzeit, 163, 240. Isolation, 233. Locke- mann, 234. Marsh-Berze- lius, 156. Morner, 247. Normal, 174. Organic com- pounds, 245. Spot, 157. Urine, 247. Arsenic trichloride, 150. Arsenic iso- lated, 233. Arseno-tungstic reagent, 125. Arseno-tungsto-molybdic reagent, 125. Arsine, 156. Asparagine, 292. Atoxyl, 246. Electrolysis, 234. Urine, 247. Atropa belladonna, Alkaloids, 30x3. Atropine, 87, 102, 144. Estimation, 300, 302. Extract, 87. General reagents, 105. Physiological test, 104. Putrefaction, 103. Barbital, 79 Barium, 170. Baumert's nitric acid test, 185. Beer, Arsenic, 237. Picrotoxin, 68. Saponins, 223. Benzaldehyde, 57. Phenol, 30. o-nitro, 57. p-dimethyl-amino, 104. Benzaurine, 31. Benzoyl cocaine test, 108. Benzoyl-ecgonine, 106. Berberine, 116. Estimation, 282. Berthelot's alcohol test, 50. Bettendorff's reagent, 322. Test, 162. Biological test, Arsenic, 242. Blood, 3*4- Bile, Elimination of lead, 177. Bismuth, 165. Elimination, 180. Morphine, 135. Physiolog- ical action, 172. Toxicity, 1 80. Bitter almond water, 57. Blondlot-Dusart test, 8. Blood, Acetone, 55. Aniline, 44. Carbon disulphide, 46. Car- bon monoxide, 304. Chloral hydrate, 40. Chloroform, 35. Copper, 178. Defibrin- ated, 229. Heavy metals, 173. Hydrocyanic acid, 20. Lead, 177. Nitrobenzene, 42. Oxalates, 190. Phos- phorus, 17. Picric acid, 70. Potassium chlorate, 194. Saponins, 221. Stains, 307, 313. Sulphurous acid, 188. 334 INDEX Tests, 306, 310, 312, 314. Toxalbumins, 228. Boiling test for blood, 304. Bread, Ergot, 211. Bromine test, Aniline, 45. Digitonin, 208. Phenol, 28. Pyrami- done, 125. Salicylic acid, 78. Brucine, 86, 100, 143. Estimation, 296. Extract, 86. General reagents, 101. Nitric acid test, 186. Picrolonate, 296. Tests, 102. Bulb-tube test, Arsenic, 162. Cacao, Caffeine and theobromine, 298. Cadmium, 167. Caffeine, 66, 83, 87, 122, 142, 145, 147. Estimation, 271, 274, 298. Extract, 87. Extraction, 275. Fate, 84. Tests, 85. Thal- leioquin test, 121. Calabar bean, no. Canadine, 116. Cantharene, 204. Cantharic anhydride, acetyl-hydrato, 204. Cantharidin, 142, 203. Estimation, 263. Carbolic urine, 26. Carbon disulphide, 46. Air, 48. Tests, 47. Carbon monoxide blood, 304. Carbon tetrachloride, Caffeine, 274. Purification, 275. Separa- tion of theobromine, 299. Carboxyhaemoglobin, 304. Carmine, Absorption-bands, 311. Castor bean, 229. Cephaeline, 277. Test, 278. Cevadine, 93. Cevine, 94. Chavicine, 287. Chloral hydrate, 38. Blood, 313. Decomposition, 39. Estima- tion, 41. Fate, 40. Physio- logical action, 40. Powders, 40. Saponin test, 223. Sol- vent, 251. Tests, 39. Toxicological analysis, 40. Chlorine-ammonia test, Narceine, 139. Chlorine test, Hydrochloric acid, 183. Potassium chlorate, 195. Chlorine water, Preparation, 85. Thebaine, 228. Chloroform, 35. Estimation, 38. Tests, 36. Chocolate, Caffeine, 298. Cholesterine, Haemolysis, 222, 224. Melzer's reagent, 68. Sapo- nins, 221. Choline, Ergot, 211. Chrome yellow test, 170. Chromium, 169, 177. Cicutoxine, 66. Cinchona, Alkaloids, 264. Quinine, 268. Cinchonidine, 265. Cinchonine, 119. Claviceps purpurea, 209. Cocaine, 105, 144. Organism, 107. Physiological test, 109. Re- lation to atropine, 106. Tests, 107. Cocculus indicus, 66. Codeine, 87, in, 144. Estimation, 254. Extract, 87. Selen- ious acid, 215. Tests, in. Coffee, Caffeine, 271. Cola nuts; Caffeine, 273, 276. Colchicein, 68. Colchicin, 65, 68, 141. Estimation, 269. Hydrolysis, 68. Puri- fication, 70. Structure, 69. Tests, 69. Colchicum autumnale, 68. Colchicin, 269. Congo paper, 182. Conhydrine, 89. 7-Coniceine, 89. Coniine, 89, 142. Diazonium ' test, 137. Tests, 90. Conium maculatum, 89. Conjugated aceto-p-amino-phenol, 74. p-Amino-phenol, 44. Anti- INDEX 335 pyrine, 123. Benzoic acid, 58. Carbolic acid, 26. Chloral hydrate, 41. Mor- phine, 137. Salicylic acid, 79- Constitution, Acetanilide, 73. Anti- pyrine, 82. Apomorphine, 127. Atropine, 103. Caf- feine, 83. Cantharidin, 203. Cocaine, 105. Codeine, in. Coniine, 89. Homatropine. 105. Hydrastine, 117, Morphine, 131. Narceine. 138. Narcotine, 113. Nico, tine, 91. Papaverine, 215- Phenacetine, 75. Picric acid, 70. Piperine, 288. Pseudo- pelletierine, 2 70. Quinine, 119. Salicylic acid, 77. Santonin, 198. Thebaine, 227. Veratrine, 94. Vero- nal, 79. Copper, 155, 165, 167. Elimination, 178. Fusion, 155. Mercury test, 1 66. Nitric acid test, 1 86. Physiological action, 172, 178. Tests, 155, 167. Toxicity, 172. oxide, 156. sulphate, Blood test, 305. Phos- phorus, 14. sulphide, Solubility, 155. Corn smut, 211. Cornutine, 209. Corrosion, 173. Cotarnine, 114, 118. Couerbe's test, Narcotine, 115. Creatinine, 87. Cresols, 28. Crotin, 229. Croton Tiglium, 229. Crystallization test, Coniine, 90. Nic- otine, 92. Oxalic acid, 191. Cyanogen, 24. Cyano-haemoglobin, 20. Cystine, 17. Cytisine, 205. Tests, 206. Cytisus Laburnum, 205. Datura strammonium, Alkaloids, 300. Deniges test, Cocaine, 109. Destruction of organic matter, 148, 150-152. Dextrose, Digitalin, 208. Digitonin, 207. Hydrocyanic acid, 20. Phosphorus, 17. Dialysis, Potassium chlorate, 194. Diazonium test, Morphine, 136. Dichromate test, Strychnine, 98. Digitalis glucosides, 207. Dimethoxy-isoquinoline, 216. p-Dimethyl-amino-benzaldehyde, 104. Dionine, 136. Diphenylamine test, Nitric acid, 186. Reagent, 186, 235. Distillation test, Ammonia, 192. Hy- drochloric acid, 183. Nitric acid, 184. Distribution, Antimony, 175. Ar- senic, 173. Carbolic acid, 27. Chloroform, 35, 38. Ethyl alcohol, 49. Heavy metals, 173. Hydrocyanic acid, 20. Mercury, 178. Oxalic acid, 190. Tin, 181. Zinc, 1 80. Diuresis, 194. Dyeing test, Picric acid, 72. E Ecgonine,' 106. Electrolysis, Arsenic compounds, 233, 237- Elimination, Antimony, 175. Arsenic, 173. Bismuth, 180. Chro- mium, 178. Cocaine, 107. Copper, 178. Heavy metals, 173. Hydrocyanic acid, 19- Lead, 176. Mercury, 178. Picric acid, ?*> Potassium chlorate, 194- Strychnine, 97. Tin, 181. Veronal, 81. Emetine, 277. Diazonium test, 137. Epichlorohydrin, 92. 336 INDEX Erdmann's reagent, 320. Cocaine, 107. Colchicin, 69. Nar- ceine, 139. Narcotine, 115. Papaverine, 217. Strych- nine, 98. Thebaine, 228. Veratrine, 95. Ergot, 209. Ergotine, 209. Ergotinine, 209. Estimation, 211. Test, 212. Ergotoxine, 209. Erythrosine (see lodeosine). Eserine (see Physostigmine). Ether extracts, evaporation, 64. Ethyl acetate, Alcohol, 51. Caffeine, 275- Exhaustive methylation, 117. n- Methyl-granatic acid, 270. Extract belladonna, Alkaloids, 301, 302. cinchona, Alkaloids, 266, 302. Quinine, 269. hydrastis, Hydrastine, 281, 282. Berberine, 282. hyoscyamus, 301. nux vomica, Alkaloids, 295, 296, 303- opium, Morphine, 285. F s Faeces, lead in, 176. Fat, Phosphorus, 17. Fate, Acetanilide, 74. Benzaldehyde, 58. Caffeine, 84-. Chloral hydrate, 40. Cocaine, 107. Digitalis glucosides, 209. Ethyl alcohol, 49. Heavy metals, 173. Fehling's solution, 320. Chloroform, 37. Githagin, 224. Maltol, 251. Picrotoxin, 67. Sapo- nins, 224. Ferments, Hydrocyanic acid, 20. Ferric chloride, Antipyrine, 83. Apo- morphine, 129. Benzoic acid, 58. Carbolic acid, 29. Codeine, 112. Cytisine, 206. Ergotinine, 212. Maltol, 251. Meconic acid, 213. Morphine, 134. Pyrami- done, 125. Salicylic acid, 77, 250, 251. Santonin, 200. Ferric hydroxide, Arsenic, 235. Ferrous sulphate test, Nitric acid, 186. Fleury's test, Morphine, 135. Nitric acid, 185. Flour, Ergot, 211. Githagin, 223. Fluorescence test, Hydrastine, 118. Quinine, 120. Formaldehyde, Blood, 307. Phos- phorus, 4. Formaldehyde-sulphuric acid, 321. Codeine, 112. Morphine, 134. Pilocarpine, 219. Formamide test, Cocaine, 109. Fowler's solution, 247. Fresenius-v. Babo, Arsenic, 161. Or- ganic matter, 148. Froehde's reagent, 320. Apomor- phine, 128. Cephaeline, 278. Cocaine, 107. Codeine, 112. Emetine, 279. Hydrastine, 118. Morphine, 134. Nar- ceine, 139. Narcotine, 115. Papaverine, 217. Saponins, 222. Solanine, 227. Strych- nine, 98. Thebaine, 228. Veratrine, 95. Fuchsine, Absorption-band, 312. Furfural, Codeine, 112. Santonin, 200. Fusion of heavy metals, 155. Galactose, Digitonin, 207. Solanine, 225. General alkaloidal reagents (see Re- agents, general). German Pharmacopoeia (see Pharma- copeia, German). Githagin in flour, 223. Glucose (see Dextrose). Glycocoll (see Acid, amino-acetic). Glycosuria, 17. INDEX 337 Glycurone, 44. Glyoxaline, 218. Gold chloride, 317. Golden chain, 205. Grandeau's test, Veratrine, 96. Guaiac-copper paper, 2 1 . Guaiac resin, Blood, 312. Guarana, Caffeine, 276. Guglialmelli's test, Pyramidone, 125. Giinzburg's reagent, 321. Mineral acid, 182. Gutzeit's test, 163, 240. H Haematin, 310. Haematin, reduced (see Haemochro- mogen). Haematoporphyrin, Spectrum, 311. Urine, 202. Haematoxylin, 266, 303. Haemin, 310. Haemin crystals, 308. Haemochromogen, 310. Haemoglobin with metals, 173. Haemoglobinuria, 226. Haemolysis, 224. Cholesterin, 222. Saponins, 221. Herapathite test, Quinine, 120. Heroine, 136. Hirschsohn's test, Quinine, 122. Homatropine, 105. Hunef eld's solution, 321. Husemann's test, Morphine, 133. Hydrastal, 117. Hydrastine, 116, 144. Estimation, 281. Hydrastinine, 117. Hydrastis canadensis, 116. Hydrocotarnine, 114. Hydro-ergotinine, 209. Hydrogen sulphide, arsenic-free, 152. Phosphorus, 3. Hydrolysis, Aconitine, 262. Atropine, 103. Cocaine, 106. Colchi- cin,68. Digitalin, 208. Digi- tonin, 207. Digitoxin, 208. Narcotine, 114. Piperine, 22 287. Saponins, 221. Sola- nine, 225. Hydroquinol, 26. Hyoscyamine, 103. Hyoscyamus niger, Alkaloids, 300. Hyper-isotonic solutions, 224. Hyp-isotonic solutions, 224. Hypochlorite test, Acetanilide, 74. Aniline, 45. Carbolic acid, 29. 1 Ignatius beans, 96. Indigo test, Chloric acid, 195. Indigo tine test, Acetone, 57. Indophenol test, Acetanilide, 73. Phenacetine, 76. lodeosine, 322. Iodine test, Narceine, 139. Pyrami- done, 125. lodoform, 41. lodoform test, Acetone, 56. lodo-potassium iodide, 318. Alka- loids, 257. Ipecac, Alkaloids, 277, 279. Isomethyl-pelletierine, 270. Iso-pelletierine, 270. Iso-pilocarpine, 217. Isopurpuric acid test, Picric acid, 71. Iso-quinoline, 216. Isotonic solutions, 224. J Jaborandum, Alkaloids, 217. Pilo- carpine, 286. Jaborine, 217. Jequirity seeds, 229. Kiliani's test, Digitoxin, 208. Kjeldahl, Caffeine estimation, 274. de Konink's reagent, 193. Langley's test, Picrotoxin, 68. Lead, 167. Blood, 173- Elimination, 338 INDEX 176. Sheep, 177. Physio- logical action, 172. Lead acetate test, Blood, 304. Car- bon disulphide, 47. Lead paper, Phosphorus, 3. Lecithine-saponins, 221. Leucine, Phosphorus, 17. Leucocytosis, 46. Lichen's test, Alcohol, 50. Acetone, 56. Liver, Copper, 178. Lloyd's test, Morphine, 134. Lustgarten's test, lodoform, 42. Lytta vesicatoria, 203. Magnesia mixture, 7, 321. Mai's method, Organic matter, 152. Maltol, 251. Mandelin's reagent, 321. Hydrastine, 1 1 8. Pilocarpine, 219. Strychnine, 99. Marquis' reagent, 134. Marsh apparatus, 134. Marsh-Berzelius test, 156, 236. Mat6 (see Paraguay tea). Material to be examined, Arsenic, 173. Bismuth, 180. Carbolic acid, 26, 27. Chloroform, 38. Copper, 178. Digitalis glucosides, 209. Ethyl alco- 1 hoi, 49. Hydrocyanic acid, acid, 21. Mercury, 178. Potassium chlorate, 194. Veronal, 81. Zinc, 180. Mauch's solvent, 251. Brucine, 102. Meadow saffron, 68. Meat, Potassium chlorate, 197. Sali- cylic acid, 250. Sulphur dioxide, 189. Mecke's reagent, 322. Codeine, 113. Meconine, 213. Melting-point test, Salicylic acid, 78. Sulphonal, 201. Melzer's reagent, 67. Carbolic acid, 30. Picrotoxin, 67. Melzer's test. Nicotine, 92. Menispermum cocculus, 66. Mercuric chloride, 318. cyanide, 25, 65. iodide, Mercury, 166. Mercury, 165. Distribution, 178. Physiological action, 172. Tests, 166. Tin, 164. Toxicity, 172. Urine, 178. Meroquinene, 120. Metallic poisons (see Poisons, metallic). Metals, heavy, 172. Methaemoglobin, 308. Absorption- band, 310. n-Methyl-coniine, 89. n-Methyl-granatoline, 270. n-Methyl-granatonine (see Pseudo- pelletierine). Methyl orange, 182. Methyl-pelletierine, 270. Methyl violet, 182. Milk, Salicylic acid, 251. Toxalbu- mins, 228. Millon's reagent, 321. Carbolic acid, 28. Maltol, 251. Salicylic acid, 78. Mitscherlich's phosphorus test, 5, 15. Morphenol, 132. Morphine, 131, 146. Estimation, 254. General reagents, 137. Opium, 283. Organism, 137. Preliminary test, 130. Pu- trefaction, 137. Selenious- sulphuric acid, 215. Morphol, 132. Murexide reaction, 85. N Naphthol test, Chloroform, 36. Narceine, 138, 147. Selenious-sul- phuric acid, 215. Narcotine, 113, 145. Selenious-sul- phuric acid, 215. Nessler's reagent, 321. Ammonia, 192. Chloral hydrate, 39. Nicotine, 90, 143. Diazonium test, 137. Tobacco, 279. Nitrite test, Carbolic acid, 30. INDEX 339 Nitrobenzene, 42. Differentiation 58. Nitro-phenacetine, 76. Nitroprusside test, Acetone, 56. Hy- drocyanic acid, 22. Non- volatile poisons (see Poisons, non- volatile). Normal arsenic, 1 74. Nux vomica, 96. Alkaloids, 293-296. Odor test, Atropine, 104. Oil of bitter almonds, Hydrocyanic acid, 58. Oils, phosphorated, Phosphorus, 14, 231- Opium, 212. Morphine, 283. Mor- phine in tincture, 285. Mor- phine in wine, 285. Organic arsenic compounds, 245. Electrolysis, 234. Organic matter, Destruction, 148, 151, 152, 234. Oxalates in plants, 190. Oxidation test, Methyl alcohol, 53-55. Caffeine, 85. Codeine, in. Phenacetine, 76. Picro- toxin, 67. Oxy-dimorphine, 133. Oxy-haemoglobin, 304. Reduction, 37- Oxy-santonins, 199. Palet's test, Apomorphine, 129. Py- ramidone, 126. Palladous chloride test, Blood, 305. Papaveraldine, 216. Papaverine, 145, 215. Paracholesterine, 68. Paraguay tea, Caffeine, 276. Paraxanthine, 84, 300. Pellagri's test, Apomorphine, 128. Codeine, 112. Morphine, i34- Pelletierine, 270. Penzoldt's test, Acetone, 57. Pepper, 287. Pipeline, 288. Peptones, Phosphorus, 17. Animal matter, 87. Peptonuria, 17. Peronine, 136. Pharmacopoeia, German, Aconitine, 262. Alkaloids, 260. Bella- donna, 301. Cantharidin, 263. Cinchona, 264. Hy- drastine, 281. Hyoscyamus, 301. Ipecac, 279. Nux vom- ica, 294, 295. Opium, 283. Pomegranate, 271. Phenacetine, 66, 75, 141. Phenanthrene, 132. Phenol (see Acid, carbolic). Phenol, aceto-p-amino, 74. Phenolphthalein, 24. Phenolphthalin test, Hydrocyanic acid, 24. Phenylisocyanide test, Acetanilide, 73. Aniline, 45. Chloroform, 36. Phosphine, 8. Phosphorous acid (see Acid, phos- phorous). Phosphorus, yellow, 5. Antidote, 14. Blondlot-Dusart, 8. Frese- nius, Neubauer, 10. Hilger- Nattermann, n. Magnesium test, 7. Metabolism, 16. Mit- scherlich test, 5. Molybdate test, 7. Oils, 14, 231. Or- ganic compounds, 13. Scherer's test, 3. Spectrum, 12. Urine, 17. Physiological action, Acetanilide, 73. Acetone, 55. Alkalies, 192. Berberine, 282. Bismuth, 172. Cantharidin, 205. Carbolic acid, 26. Chloral hydrate, 4- Chlo- roform, 35. Chromium, 177. Copper, 172, 178- Ergot, 210. Heavy metals, 172- Homatropine, 105. Hydro- cyanic acid, 19. Lead, 172. Mercury, 172. Morphine, 340 INDEX 137. Nicotine, 91. Nitric acid, 184. Nitrobenzene, 42. Oxalic acid, 190. Phos- phorus, 16. Picric acid, 70. Pyramidone, 124. Santonin, 199. Saponins, 221. Silver, 172, 179. Strychnine, 97. Sulphuric acid, 186. Sul- phurous acid, 188. Tin, 181. Uranium, 172, 180. Vero- nal, 80. Physiological salt solution, 224. Physiological test, Alkaloids, 88. Atropine, 104. Cantharidin, 205. Cocaine, 109. Nico- tine, 93. Physostigmine, no. Strychnine, 99. Vera- trine, 94. Physostigma venenosum, no. Physostigmine, no, 145. Diazonium test, 137. Phytosterine, 68, 281. Picraconitine, 261. Picro-sclerotine, 209. Picrotin, 66. Picrotoxin, 65, 66, 140. Picrotoxinin, 66. Pilocarpidine, 217. 'Pilocarpine, 217. Estimation, 286. Pilocarpus pennatifolius (see Jaboran- dum). Piperidine, 287. Diazonium test, 137. Piperine, 287. Estimation, 288. Pisani's test, Cocaine, 109. Platinum chloride, 318. Poisons, metallic, 148. non- volatile, 61. volatile, 3. Distillation, 18. Pomegranate bark, Alkaloids, 270. Potassium arsenate, in. bismuthous iodide, 318. Alka- loids, 255. cadmium iodide, 318. chlorate, 194. Meat, 197. Or- ganic matter destroyed, 148. Putrefaction, 197. Urine, 197. ferrocyanide, 25. Blood, 305. Copper, 167. Potassium hydroxide, 192. Santonin, 200. mercuric iodide, 318. Alkaloids, 258. permanganate, Cocaine, 108. pyro-antimonate, Sodium, 193. zinc iodide, 319. Potatoes, Asparagine, 292. Sola- nine, 291. Precipitation test, Cocaine, 107. Copper, 167. Proteins, Phosphorus, 17. Chloro- form, 35. Heavy metals, 172. Prussian blue test, Hydrocyanic acid, 22. Morphine, 135. Tin, 164. Pseudo-conhydrine, 89. Pseudo-morphine (see Oxy-dimor- phine). Pseudo-narceine, 138. Pseudo-pelletierine, 270. Psychotrine, 277. Ptomaines, 219. Putrefaction, Carbolic acid, 27. Atro- pine, 103. Cantharidin, 205. Morphine, 137. Potassium chlorate, 197. Phosphorus, 16. Pyramidone, 87, 124, 145. Extract, 87. Pyridine, Chloroform, 37. Coffee, 275- Pyrocatechol, 26. Pyrone, 213. Quinidine, 264. Quinine, 87, 119, 145. Estimation, 258, 268. Extract, 87. Quinoline, 216. Radulescu's test, Morphine, 136. Ramverda's test, Cytisine, 206. Reagents, general alkaloidal, 317. INDEX 341 Resorcinol test, Chloroform, 36. Nar- ceine, 140. Reynold's test, Acetone, 57. Rhamnose, 225. Ricin, 229. Roussin's test, Nicotine, 92. Rubreserine test, Physostigmine, no. Santonin, 198. Estimation, 289. Sapogenins, 221. Saponins, 220. Beer, 223. Choles- te rides, 222. Schaer's test, Blood, 313, 314. Scherer's test, Phosphorus, 3. Schindelmeiser's test, Nicotine, 93. Schlererythrin, 209. Test, 211. Schmidt's test, Apomorphine, 129. Schonbein-Pagenstecher test, Hydro- cyanic acid, 21. Schonbein-Van Been test, Blood, 312. Scopolamine, 104. Secale cornutum, 209. Selenic-sulphuric acid test, Solanine, 226. Selenious-sulphuric acid, 214. Narco- tine, 116. Papaverine, 217. Selenium, Moulds, 243. Silver, 171. Estimation, 179. Or- ganism, 179. Physiological action, 172. Silver nitrate test, Hydrocyanic acid, 23. Potassium chlorate, 195. Silver phosphide, 3, 9. Sodium arsenate, 156. hydroxide, 192. Blood, 304. iodide, Haemin crystals, 308. nitroprusside, 56. perchlorate, Cocaine, 109. pyro-antimonate, 156. stannate, 156. thiosulphate, Chloral hydrate, 39. Solanidine, 225. Test, 226. Solanine, 225. Estimation, 291. Test, 226. Solanum tuberosum, 225. Solubility test, Coniine, 90. Sonnenschein-Jesserich, Destruction of organic matter, 151. Spanish flies, 263. Sparteine, 137. Spectroscopic test, Barium, 171. Blood, 306, 310. Hydrogen, 12. Phosphorus, 12. Spotted hemlock, 89. Stannic oxide, 156. Stannous chloride, 102, 322. Mer- cury, 1 66. Starch test, Sulphur dioxide, 189. Stas-Otto process, 63. Stibine, 160. Straub's test, Phosphorus, 14. Strychnine, 86, 96, 143. Estimation, 258, 298. Extract, 86. Pi- crolonate, 296. Stypticine, 254. Sugar test, Sulphuric acid, 187. Sulphocyanate test, Carbon disul- phide, 47. Hydrocyanic acid, 22. Mineral acid, 183. Sulphonal, 200. Extraction, 198. Sulphur dioxide test, Sulphuric acid, 187. Meat, 189. Synopsis of Group I, 59. Group II, 140. Group III, 171. Synthesis, Acetanilide, 73. Antipy- rine, 82. Nicotine, 91. Phenacetine, 75. Piperine, 287. Pyramidone, 1 24. Salicylic acid, 77. Sulpho- nal, 201. Veronal, 80. Tea, Caffeine, 272, 274, 276. Theo- phylline, 300. Teichmann's crystals (see Haemin crystals). Tellurium, Moulds, 243. Tetra-chloro-methane (see Carbon te- trachloride). Thalleioquin test, Quinine, 120. Thebaine, 146, 227. Selenious-sul- phuric acid, 215. 342 INDEX Thebaol, 227. Theine (see Caffeine). Theobromine, 84, 299, 300. Estima- tion, 277, 298. Theophylline, 84, 300. Thyroid gland, Arsenic, 174. Tin, 155. Distribution, 181. Tests, 164. Tincture of nux vomica, Alkaloids, 295, 297. opium, Morphine, 285. Tobacco, Nicotine, 278, 280. Toxalbumins, 228. Toxicity, Acetone, 55. Aniline, 44. Bismuth, 180. Carbolic acid, 26. Carbon disulphide, 46. Chloral hydrate, 40. Chromium, 177. Copper, 172. Cytisine, 206. Esti- mation by haemolysis, 258. Ethyl alcohol, 259. Homa- tropine, 105. Hydrocyanic acid, 19. mercury, 172. Metallic albummates, 172. Methyl alcohol, 52, 259. Ni- cotine, 91. Nitrobenzene, 42. Oxalic acid, 190. Pep- per, 287. Phosphorus, 16. Picrotoxin, 66. Potassium chlorate, 194. Saponins, 221. Solanine, 226. Sul- phurous acid, 188. Tin, 181. Toxalbumins, 229. Ura- nium, 1 80. Tribromophenol, 28. Trichlor-ethyl alcohol (see Alcohol, trichlor-ethyl). Trimethyl-amine, 211. Trional, 203. Triphenyl-methane, p-dihydroxy, 30. Troches, Santonin, 291. Tropidine, 106. Tropine, 103, 106. Turpentine, ozonized, Blood, 312. Tyrosine, 17, 28. U Ulex europaeus, 205. Ulexine (see Cytisine). Uranium, 172, 180. Urobilin, 17. Urotheobromine, 300. Vanadic-sulphuric acid test, Solanine, 226. Van der Moer's test, Cytisine, 206. Veratridine, 94. Veratrine, 87, 93, 143. Extract, 87. Veronal, 66, 79, 142. Vitali's test, Alcohol, 51. Alkalies, 193. Atropine, 104. Blood, 313. Carbon disulphide, 49. Veratrine, 96. Vortmann's test, Hydrocyanic acid, 2.3- W Wangerin's test, Apomorphine, 128. Narcotine, 115. Warren's test, Papaverine, 217. Weehuizen's test, Hydrocyanic acid, 24. Weppen's test, Veratrine, 95. White arsenic, 150. Wine of opium, Morphine, 285. Wine, Salicylic acid, 250. Saponins, 223. Wormseed, Santonin, 289. X Xanthine bases, Urine, 84. Xanthogenate test, Carbon disulphide, 47- o-Xylene, 204. Yellow phosphorus, 5. Z Zeisel's test, Colchicin, 69. Methoxyl, 101. Zinc, 1 68, 1 80. UNIVERSITY OF CALIFORNIA LIBRARY Los Angeles This book is DUE on the last date stamped below. Form L9-25m-9,'55(B4283s4)444