REESE LIBRARY DIVERSITY OF CALIFORNIA. APR 20 1893 : t of the pressure of the atmosphere. The spark is then passed. The gas becomes hot anal luminous, emitting lights of different colours, and giving, when examined with the spectroscope, a spec- trum different for each gas examined. Thudichum remarks, " It must be left to the future to decide how far these spectra can be practically utilised in the diagnosis of gases which occur in the animal economy. Such gases as occur in morbid emphysemata (in the cellular tissues of cows during cattle plague, in contused limbs with effused blood) in the blood in various conditions, in certain internal cavities or tissues as results of disease, have hitherto mostly escaped analysis, as their small quantity makes the ordinary methods inapplicable. The attention of inquirers must henceforth be directed upon their analysis by means of the electro-spectroscopical method." The Chemical Circulation. Secondly, the spectro- scope may be used to determine the time that certain salts take to reach any part of the body. Dr Bence Jones used it for this purpose. He imagined "that there are good grounds for believing that there exists within us, in addition to the mechanical or animal circulation of the blood, another and a greater, and a more strictly chemical circulation, closely resem- bling, if not identical with, that which obtains in the 40 CHEMICAL CIRCULATION. lower divisions of animals and in vegetables. A circu- lation in which substances continually pass from the outside of the body into the blood, and through the blood into the textures, and from the textures either into the ducts, by which they again pass back into the blood, or are thrown out of the body, or into the absorbents, by which they are again taken back into the blood, again to pass from it into the textures." He calls this " the chemical circulation," and his ex- periments were made in order to determine " where diffusing substances go to, how long they are in going out of the stomach into the textures, how long they stay in the textures, and how quickly they cease to appear in the excretions." With the assistance of Dr Dupre, he made use of the spectroscope to answer these questions. He first shows how the spectroscope can detect of Chlorate of soda . '. y^ millionth of a grain Carbonate of lithia i ,, ,, Chloride of strontium .1 ,, Chloride of barium . 1 ,, ,, Chlorate of potass. . . -^ thousandth of a grain Chloride of lithium . . Y- millionth of a grain Chloride of rubidium . yg- thousandth of a grain Chloride of caesium . . f^r They (Jones and Dupre) then searched for lithia in many vegetable and animal substances, and found it in the following : CHEMICAL CIRCULATION. 41 " In Potatoes seldom. In Tea slight traces. Apples sometimes. Coffee slight traces. Bread traces. Ale slight traces. Cabbage distinctly. Porterslight traces. Rhine wines always. Mutton none. French wines distinctly. Beef none. Sherry distinctly. Milk none. Port distinctly. " It had already been found " In Sea- water. Kelp. Spring-water sometimes. Ashes of wood grown in the Odenwald. Russian and other potashes. Tobacco. Vine leaves and grapes. Ashes of the produce of the fields in the Palatinate. Milk of animals eating the produce. Ash of human blood and muscle. Meteoric stones. All the drinking waters of London, &c. " The spectrum of lithium is very characteristic and very perceptible, and some approximation to a quanti- tative determination may be arrived at by observing the amount of substance that requires to be burnt to obtain the reaction ; and by the necessity, in some cases, for the removal of interfering substances previous to the combustion. Thus three degrees may readily be 42 CHEMICAL CIRCULATION. observed. The highest amount of lithia is present when each particle of the substance introduced into the flame gives the lithia reaction ; and a smaller amount of lithia is present when the whole of a lens or of an organ must be extracted with water, to remove the lithia previous to the combustion ; and the smallest trace is present when the substance has to be incine- rated, the ash treated with sulphuric acid, the excess of acid driven off, the dry residue extracted with absolute alcohol, the alcohol evaporated, and the dry residue tested. These three quantities may be designated as the slightest trace, a trace, and plenty. " As soon as experiments on man and animals showed that the infinitesimal quantities taken in with the food were rarely to be perceived in the textures, experiments were made to determine how quickly the lithium diffused from the stomach into the blood circulation, and from the circulation into the textures, and whether it was to be found in those distant parts of the textures where no circulation existed, and especially in the lens of the eye. " The following table gives the experiments made on the rate of the passage of chloride of lithium from the stomach, not only into the circulation, but out of the circulation into the textures of a guinea-pig : After 1^ grs. were taken. In 3 days plenty was found everywhere. 3 ,, 15 minutes everywhere except in the lens. o ,, oU CHEMICAL CIKCULATION. 43 After 3 grs. were taken. In 30 minutes everywhere, and traces in the lens. 3 30 ,, everywhere, and outer part of the lens. 3 * 60 3 60 everywhere ex- cept in the lens. 3 ,, 2| hours everywhere, and throughout the lens. " " 4 ,, 5J 8 * 8 ,, 3 24 3 M 26 J ,, 5J ,, everywhere except in the lens. " It follows from these experiments that three grains of chloride of lithium given on an empty stomach, may diffuse into the cartilage of the hip-joint, and into the aqueous humour of the eye in a quarter of an hour. In very young and very small pigs, the same quantity of lithium may in thirty or thirty-two minutes be found in the lens of the eye, but in an old pig in this time the lithium will have got no farther than the humours of the eye. If the stomach was empty when the chloride of lithium was taken, then in one hour the lithium may be very evident in the outer part of the lens, and very faintly in the inner part ; but if the stomach be full of fpod, the lithium does not in an hour reach the lens. Even in two hours and a half the lithium may be more marked in the outer than in the inner part of the lens. 44 CHEMICAL CIECULATION. In four hours the lithium will be in every part of the lens, but it will still be more evident in the humours than in the lens. Even in eight hours the centre of the lens may show less than the outer part. The lithium will be found in as great quantity in the centre of the lens as in the outside after twenty-six hours." When the lithium was injected " into the skin " instead of being put in by the stomach, " three grains of the chloride showed in twenty-four minutes lithium in the lens and every texture ; in ten minutes, slightly in the lens but plenty everywhere else ; in four minutes no lithium was in the lens, but plenty in the aqueous humour of the eye and in the bile ; one and a half grain in five minutes showed no lithium in the lens, but plenty in the aqueous humour and in the bile." It was thus shown " that lithium will pass everywhere into the textures in between four and fifteen minutes, when injected into the circulation, and between fifteen minutes and twenty-six hours when taken in by the stomach. Some experiments were afterwards made to determine after how many days the lithium ceased to be detected in the textures after it hadbeen taken. Usually three pigs were taken : to one no lithium was given, the second was killed in a few hours after a dose of lithium, and the third was given the same dose and killed after many days." "The following table shows the rate at which chloride of lithium passes out of the textures : 2 grs. in 6 hours gave plenty everywhere. In 6 days gave no trace in the alcoholic extract of the kid- neys, livers, or lenses. CHEMICAL CIRCULATION. 45 2 grs. in 6 hours gave plenty everywhere, and in 6 days result as before. 2 grs. in 6 hours gave plenty everywhere, and in 4 days gave none in the lens. 1 gr. in 5^ hours showed partly in the lens. In 3 days gave faint traces in the lens. c< It follows from these and other experiments that twice in six days, and once in four days, two grains of chloride of lithium, which in six hours gave lithium everywhere, in six days ceased to be detectable in the lens, and that even in three days the lithium is most probably diminishing in the lens." Dr Bence Jones then, by the help of Mr Bowman and Mr Oritchett, endeavoured to trace the passage of lithium " into that part of the body which is most distant from the blood circulation in man." Twenty grains of carbonate of lithium dissolved in water were given in a few minutes, or a few hours, or a few days before the extraction of a lens affected by cataract. (Some lenses affected with the same disease having been previously examined so as to determine whether lithia might not have been naturally present, and in only one instance was the faintest trace discovered.) These experiments taught " that in the human body twenty grains of carbonate of lithia taken into the stomach in two and a half hours will have partly passed into every particle of the textures and beyond the blood circulation even into the most distant parts, and in three and a half hours it will be distinctly present in each particle of the lens." 46 CHEMICAL CIRCULATION. " After four days it will still be distinctly present in each particle of the lens. " After five days it will have begun most clearly to pass out of the lens, and in seven days scarcely the smallest trace will be detectable there. "A long series of experiments on the passage of lithium out by the excretions, after it had been taken in by the mouth, showed nearly the same fact, namely, that after a dose of twenty grains, the lithium was not entirely thrown out of the body under six, seven, or eight days. " Thus, then, both in animals and man the same law obtains. A single dose of lithium in a few minutes passes through the circulation into all the ducts, and into every particle of the body, and even into the parts most distant from the blood circulation. There it remains for a much longer time than it took to get into the textures, probably for three or four days, varying with the amount taken ; then it diminishes, and finally, in six, seven, or eight days, the whole quantity is thrown out of the body." Chloride of rubidium and csesium are also proved to follow the same law as lithium, in short, these sub- stances were found to pass into the lens, being detected there ; but twenty grains of chloride of rubi- dium or chloride of csesium had to be given to guinea- pigs before the lenses of the animals gave the spec- trum reaction of these metals. I have given Dr Bence Jones's own words in most of the above extracts from his book, his experiments METALS IN MINERAL WATERS. 47 and deductions being in my opinion too interesting to allow of their being abridged. The spectroscope can also be used to detect the presence of various metals in mineral waters. I would refer the reader to Eoscoe's ' Lectures on Spectrum Analysis ' for an account of the discovery of caesium and rubidium in the waters of Diirkheim by Bunsen, &c. 48 PHYSIOLOGICAL ABSORPTION SPECTRA. CHAPTER IV. PHYSIOLOGICAL ABSORPTION SPECTRA. THE study of the absorption spectra of various physiological fluids has taught us more than their ordinary chemical analyses. More especially is this the case with blood ; for not only has the spectro- scope shown why arterial differs from venous blood in colour, but it also enables us to detect the minutest trace of blood; and also to understand how certain gases, &c., of a poisonous nature put an end to animal life. The study of the action of various re- agents upon blood out of the body has explained the action of reagents within the body. For just as acids and alkalies, respectively, split haemoglobin up into acid and alkaline haematin in the laboratory of the chemist, so also is haemoglobin split up by the same or similar agents in some pathological fluids within the body. As an instance, the haematin which occurs in ovarian and parovarian cysts may be men- tioned, which I shall describe for the first time further on. An examination of the bile of various animals has led me to believe that the so-called urobilin is present VOGEI/S EESE ARCHES. 49 in the bile of many of the lower animals,* and it may be said to be present in the bile of almost all animals that possess a receptacle for this fluid. This colouring matter is constantly present in human urine, and is abundantly present in the bile of the mouse, which gives its absorption band with great distinctness. It was known to be capable of production in the labora- tory by the action of acids on bile, and, as will be de- scribed further on, Maly has produced it from another bile pigment; but I can affirm that it is constantly present in all kinds of bile, and in most of them its spectrum can be seen without any treatment whatever. Before proceeding to the description of various physiological and pathological absorption spectra, it will be necessary to give an account of some recent researches on absorption spectra in general, which are of great importance, as they teach us the necessity of caution in drawing inferences as to identity in chemical composition between two bodies, from the fact that solutions of these bodies give bands in the same part of the spectrum. It would at first. sight appear that since identity of spectrum does not ensure chemical identity, the study of absorption spectra is entirely useless, because it is upon this principle that the whole of spectrum analysis, as applied to the study of the chemical elements, is based ; but a little consideration will show that the value of absorption spectra, as tests for the presence of pigmented substances, is not dimi- nished by these discoveries. Vogel's researches on absorption spectra. Experi- * i. e., without any treament whatever. 4 50 VOGEL'S RESEARCHES. ments undertaken by Herr Kundt had shown that the stronger the dispersion of the solution, the nearer to the red end will the absorption band given by the substance in solution be found. In Kundt's experi- ments he overlooked the fact that, when changing the solvent, the whole character of the spectrum was changed, so that comparison of one solution with another was rendered difficult. Herr Yogel has investigated the different effects produced upon the position of absorption bands in the spectrum by the nature of the medium (in which the body giving these absorption bands is dissolved) ; he was induced to undertake this research through having observed remarkable differences between the spectra of solids and of solutions of the same solids. In his experiments, instruments of small dispersion were used, because, as mentioned before, such instru- ments are more suitable for observing absorption spectra, owing to the bands being better defined, and since the whole spectrum can be observed at the same time. " The absorption spectra of solid salts and pigments were obtained from thin layers of these sub- stances, prepared upon glass plates, through evapora- tion of a few drops of solution." Vogel shows not only that differences occurred in the spectra of one and the same solid substance and its solution, but often a striking coincidence in the position of the absorption bands belonging to totally different substances (e. g. nitrate of uranium and potassium permanganate). The vapours of iodine, hyponitric acid, indigo, &c., had their spectra examined, and these were compared, VOGEI/S EESEAECHES. 51 in most cases, with the aqueous, alcoholic, and other solutions of the same substances. The results arrived at may be thus summed up : 1 . Considerable differences exist between the spectrum which a substance gives in the solid, liquid, or dissolved and gaseous state. Characteristic bands which are shown in the spectrum of one state, are either not repro- duced in that of the others (this is the case with chrome alum, chloride of cobalt, iodine, bromine, naphthaline red, fuchsine, indigo, cyanine, aniline blue, methyl violet, eosine, carmine, purp urine, alizarine, santaline), or they reappear in a different position, or of different in- tensity (e.g. nitrate of uranium, permanganate of pota*s- sium, hyponitric acid, alcanna red). Sulphate of copper and chlorophyll show the same absorption both in the dissolved and in the solid state. 2. The spectra given by the same substance when dissolved in different media are the same in some cases (purp urine in alcohol or sulphide of carbon, aldehyde green in water or alcohol, methyl violet and indigo- sulphuric acid in water or amylic alcohol) ; in other cases they differ only in the position of bands (chloride of cobalt, fuchsine, coralline, eosine, and iodine green, in aqueous or alcoholic solutions) ; and again in others, their character is totally different, so that no point of coincidence remains (iodine in sulphide of carbon or alcohol, naphthaline, aniline blue, purpurine, hsema- toxylin, brasiline, in water or alcohol). 3. The rule established by Kundt, viz. that the absorption bands of a body in solution lie the nearer towards the red end of the spectrum the greater the dis- 52 VOGEL'S RESEARCHES. persion of the dissolving medium is, has not been con- firmed in many cases; on the contrary, in some instances the absorption bands move towards the blue in a solu- tion of greater dispersion (nitrate of uranium and blue chloride of cobalt in water and alcohol) ; in other cases their position remains unaltered for various media (hyponitric acid in air and benzol, indigo -sulphuric acid and methyl violet in water and amylic alcohol, aldehyde green in water and alcohol, purpurine in sulphide of carbon and alcohol) . In some cases a great difference in the sense of Kundt's rule becomes ap- parent, while in others for the same spectral region but a 'very trifling one appears, according to the nature of the pigment (coralline and fuchsine) . Indeed, it happens sometimes that certain bands are in the same position with different dissolving media, while others which are simultaneously visible are displaced (nitrate of uranium in water and alcohol, oxide of cobalt in glass and in water, proto -nitrate of uranium in neutral solution and in a solution of oxalic acid, chlorophyll in alcohol and ether). 4. The position of absorption bands in the spectra of solid and dissolved bodies may be only exceptionally deemed characteristic for any certain body. Totally different bodies show absorption bands in exactly the same position (solid nitrate of uranium and potassium permanganate in the blue; naphthaline red, and coralline in the yellow ; indigo, aniline blue, and cyanine in the orange ; aldehyde green and malachite green in the orange). Closely related substances sometimes show remarkable differences in the position of their bands VOGEL'S KESEARCHES. 53 under perfectly equal conditions (solid uranium salts). 5. The rule set up for absorption spectra, " each body has its own spectrum," can be admitted only with great restrictions. The great number of polychromatic substances show different colours and different spectra in the solid state, according to the direction in which they are observed. Most other bodies show different spectra in the solid state from those of their solu- tions ; and in the latter case again, different ones according to the dissolving medium, and the question arises which of all these spectra is the body's own spectrum. These conclusions arrived at by Herr Vogel would appear to diminish the value of bands, but we have another method to fall back on besides observing the mere position of bands, that is, the changes in the spectra of the same body, caused by various solvents and reagents. (Thus cyanine and aniline blue dissolved in alcohol give a very similar spectrum, but in water a totally different one. The two bands of 0- haemoglobin are replaced by one when reducing agents are used ; those of carmine which resembles blood in its bands are not changed, and so on.) The recognition of a body becomes more certain if its spectrum consists of several absorption bands, but even the coincidence of these bands with those of another body, is not sufficient to enable us to infer chemical identity ; what enables us to do so with certainty is the fact : that the two solutions give bands of equal intensities in the same parts of the spectrum which 54 MIXED COLOURING MATTERS. undergo analogous changes on the addition of the same reagent.* Fortunately for medical spectroscopy we always use reagents in determining the nature of a substance present in solution; thus, in determining whether haemoglobin or hsematin be present, we always add to the solution supposed to contain them, either Stokes' fluid or ammonium sulphide. So that Vogel's re- searches do not interfere with the conclusions drawn in this book. But they teach us to accept with reserve conclusions hastily drawn from the mere position of bands in a spectrum, unsupported as they often are by more precise analysis. There is no doubt at the same time that Vogel's conclusions are not true for all pigments, the aqueous, the alcoholic, chlo- roformic, and etherial solution of some substances, do not, at least in the case of some physiological pig- ments, show such striking differences as the above would lead us to expect ; and, as a general rule, two bodies, giving exactly the same spectrum, will be found to have a close chemical relationship, if not an identical composition. f Nor does the colour of the solution always make such a difference to the position of bands, for often fluids of a different colour give the same spectrum, e.g., the bile of the ox and sheep, when fresh and when they are beginning to decompose. Mixed colouring matters. Mr Sorby has written an excellent paper on the " Examination of mixed Colour- ing Matters by the Microspectroscope," from which I * ' Nature/ vol. xix, p. 495. f See remarks on wave-lengths, Appendix I. SOBBY ON MIXED COLOURING MATTERS. 55 quote a few extracts, as his remarks will be found exceed- ingly useful by those who wish to understand the methods whichit is necessary to adopt before drawing conclusions from the position of bands in a spectrum. The paper will be found in the ' Monthly Microscopical Journal ' for 1871 (vol. vi, p. 124, et seq.). He says : " In study- ing the colouring matters, soluble in water, that may be obtained from various kinds of algse, for which special names have been proposed, as though they were single and simple substances, I have been led to conclude that they are in some cases mixtures of at least four, which are readily distinguished by their spectra. The facts which have thus presented themselves, have so impressed me with their importance in such inquiries, that I think it may be well to make the study of mixed colouring matters the subject of a special communica- tion." " The manner in which the mixed nature of some colouring matters may be ascertained from their spectra, has been already described by Professor Stokes and others, as well as in previous papers by myself; but in order to make this communication complete in itself, I must be allowed to again describe some of them. I shall not attempt to enter into the chemical part of the subject, or to treat of the separa- tion of different substances by purely chemical methods, such as the solubility or insolubility in various reagents, but confine myself almost entirely to those processes in which the examination of the spectra is of primary importance. I scarcely need say that the coloured material should be separated, as far as 56 SORBY ON MIXED COLOURING MATTERS. can be conveniently managed, into that which is soluble or insoluble in such simple solvents as water or alco- hol; but at the same time there are cases in which such a difference in solubility does not appear sufficient to prove that the colouring matter itself differs essen- tially. The spectra seem to show that occasionally the presence of some other substance, insoluble in water, which has a strong affinity for the colouring matter, is the true cause of their variations. I shall, therefore, presume that we have to deal with colouring matters, separated from any others that differ materially in their solubility. " There are few cases in which the mixed nature of a coloured aqueous solution can be more easily ascer- tained, than when the constituents differ so much in character, that the addition of some reagent will more or less completely destroy the spectrum of one, without having any effect on that of the other. For this pur- pose no substance is superior to sulphite of soda. Without producing any real decomposition, this almost entirely removes the detached absorption at the red end of the spectrum of certain colours, but has no effect whatever on that of the others. In the case of some colours it thus acts when the solution contains excess of ammonia, but in the case of others it has then little or no action, but removes the absorption when the solution contains excess of such a moderately weak acid as citric." Mr Sorby then goes on to show how this method is applied in the case of the colouring matter of certain plants. He then shows how we may separate two colouring matters from each other by SOEBY ON MIXED COLOURING MATTERS. 57 ether, when the etherial solution rises to the top, and the aqueous falls to the bottom. " It will be thus seen that, if a mixture were thus treated, a partial separa- tion might often be effected, and on evaporating to dryness, redissolving in water, and comparing the spectra, either in the natural state or after reagents had been added, the differences might clearly prove that two or more colouring matters were present." . . "When a solution contains more than two colouring matters the recognition of each becomes somewhat more difficult ; but still, by following out this system, and dividing the material into more than two portions, a very good opinion may be often formed of the general optical properties of each substance. When some of them give well-marked and characteristic absorption bands, and when the absorption of others may be removed by the addition of sulphite of soda, the study of a complex mixture is very greatly facilitated, and especially if the spectrum of one or more of the con- stituents is of such a marked character, that it can be at once recognised as that of some substance already known in a pure state. A tolerably good opinion may then be formed of the spectrum of the rest by, as it were, subtracting that of the known constituent. This leads me to the description of the spectra of certain -^louring matters, which are met with so far separated naturally, that their compound characters may be in- ferred without reasonable doubt, and confirmed by a more extended examination." " I have lately found that many interesting facts may be observed, by examining the spectra of sub- 58 SUMMAKY. stances in their natural state without extracting the colouring matter. Frequently they are so opaque that it is requisite to use a very bright light to penetrate through them." .... The consideration of various absorption spectra which are described in his paper led Mr Sorby to draw these conclusions : 1. "When a spectrum shows two absorption bands, they should not be considered due to one single sub- stance, until satisfactory evidence of the fact has been obtained. The solution should be allowed to undergo slow decomposition, * and be repeatedly examined, in order to ascertain whether both bands disappear in the same proportion, and also the reaction of various re- agents observed, in order to learn whether one band can be permanently removed without the other, making, of course, due allowance for any change that may depend merely on an acid or alkaline reaction." 2. " When more than two bands are seen in the spectrum, and they are not at nearly equal intervals, the compound nature of the substance may be con- sidered so probable that further examination should certainly not be neglected." 3. " When there is broad shading about a narrow absorption band it is important to ascertain whether or not it is due to the same substance. There are certainly many cases in which I have always concluded that both are due to the same," but examples show that there are exceptions to this rule. " The occurrence of so many associated colouring matters, as in algse, may be rare. It must not be MIXED COLOURING MATTERS. 59 supposed that I imagine whenever there are two or more absorption bands they are due to two or more independent substances. As an example of what I look upon as satisfactory proof of the contrary, I will describe some facts connected with the well-known spectrum of blood. If after exposure in a dry state to the air for some weeks, until the haemoglobin has been converted into methsemoglobin, a small quantity of the double tartrate of potash and soda be added to the aqueous solution, and afterwards a very minute portion of the double sulphate of protoxide of iron and ammonia, the methsemoglobin is deoxidised and re- converted into haemoglobin, as described in my late paper ' On Blood Stains' (vide Appendix). Here, then, we have a decomposition gradually effected by the atmosphere, and if two different substances had been present it is extremely probable that they would have varied in the rate of change, so that there would finally have been an alteration in their relative propor- tion, and thus, when deoxidised, there would not have been the same relation between the absorption bands as in fresh blood. I find, however, that the agree- ment is complete. Moreover, if the colouring matter had been a mixture of two substances, it is extremely probable that there would have been some such varia- tion in their relative amount in the blood of very different animals, as occurs in the colouring matters of various algae. In order to ascertain whether this is the case, I carefully compared side by side the spectra of human blood and that of the small annelids so common in stagnant pools, and found that the position and 60 METHODS OP OBSERVATION. relative intensity of the two bands were exactly the same." His conclusions as to relationship between pigments which give spectra like each other, and the light which the study of wave-lengths has thrown upon this sub- ject, will be noticed after the absorption spectra of the blood, bile, and urine, &c., have been described. Methods of observation. The general method of ob- serving the absorption spectra of fluids was described before (see Chapter II). For most purposes, when there is abundance of material, the chemical spectro- scope is the most satisfactory instrument for working at absorption spectra, because the solutions being placed in test-tubes we are the better able to study the action of reagents ; and when, as in examining the spectrum of Gmelin's test, there are strata of different densities, we can examine each separate stratum with great satisfac- tion. But when the fluid is small in amount, or when we want to get a spectrum with the light reflected from the surface of the body, or to examine blow-pipe beads, &c., the microspectroscope is to be preferred; moreover, the definition of the latter is much better than the former, for we can often see with the microspectroscope faint absorption bands which escape notice when examined with the chemical spectroscope. Different depths of fluids are also examined more easily with the micro- spectroscope, as we can increase or diminish the depth of fluid in a cell, in a much shorter space of time than it takes to bring a succession of different sized test- tubes before the slit of the chemical spectroscope. For detecting blood in urine, a small pocket spectro- SPECTEUM OF 0-HJ3MOGLOBIN. 61 scope such as that shown here, will be found very con- venient, and considering the size of the instrument, it is astonishing what an amount of work can be done with it. For those who can afford it, two spectroscopes will be found to give more satisfactory results than one, but if the additional cost is an object, the Sorby- Browning microspectroscope with the photographed scale is amply sufficient for all purposes. FIG. 13. The spectrum of oxidized haemoglobin. The blood bands. Blood, as is now known to every reader of physiology, owes its colour and its spectrum to haemoglobin ; that this is the case, is proved by the fact, that a solution of blood gives the same spectrum as haemoglobin, when the latter has been separated from the former either in the crystallised or amorphous condition. The defibrinated blood of the dog or pig, or any other vertebrate animal, is amply sufficient for the study of its optical characters, or a drop of blood from the finger can be made to show the spectra of oxidized and deoxidized haemoglobin, of alkaline and acid haematin, and of deoxidized haematin. Of course if we wish to study the characters of chemically pure solutions, we must separate the haemoglobin by one of the usual 62 O-H^HMOGLOBIN. methods.* Having obtained some blood, and having made a solution with water, we can see by putting it before the slit of the chemical spectroscope, or in a cell beneath the microspectroscope, the spectrum of haemoglobin in the oxygenated condition. If the solution be too strong all light will be stopped, but if it is diluted sufficiently, a little of the red and the orange appear ; diluted still more, a little green appears ; but between the orange and the green there is a broad, very dark band. If we compare its position with that of the Fraunhofer lines we find that it extends from beyond D on the red side, to near b on the violet side (Chart 1,2). On diluting still more, this band is found to be composed of two, one of which, next D, is darker and more strongly marked than the other, while the latter is broader and more washed out at the edges. By further dilution we narrow these bands, and we can go on diluting until the band next the violet end of the spectrum disappears, and that nearest D only is left. By diluting still more the latter band also dis- appears, and nothing but the continuous spectrum of the light-source is left.f By examining in the cell of a microspectroscope a solution sufficiently strong to allow only the red to come through, and then gradually diminishing the thickness of the layer, the same appearances as" those got by gradual dilution can be obtained. We can get * See Thudichum's ' Chemical Physiology,' or any good treatise on physiological chemistry. f These two bands are those of oxidized haemoglobin, the " scarlet cruorine " of Stokes, the "oxidized hematocrystalline " of Thudichum, and the appearances described above are shown in Chart I, 2 to 5. B C Cka s A..liicMimn a.a.nat.del. To face Paye 63. DISTRIBUTION OP HAEMOGLOBIN. 63 the same two bands by examining the frog's web with the microspectroscope, by reflecting light from the surface of human skin into the microspectroscope, or by holding the ear of a rabbit between the light and the slit of a spectroscope ; the red fluid of the earth- worm and of the house-fly yields the same spectrum. So delicate is the spectroscopic test for blood that we can detect by means of the microspectroscope as little as the -nroro'th of a grain of haemoglobin . Distribution of haemoglobin in the Animal Kingdom. Hemoglobin has been detected by means of the spectroscope: (1) In the blood of vertebrates located in the red corpuscles, with the exception of the Amphioxus, in which it is found in the plasma, not in the corpuscles. (2) In most of the striped muscles of mammals and birds ; but only in the cardiac muscles, and in certain very active muscles of other vertebrates. (3) In the unstriped muscle of the human rectum ; in other unstriped muscles it is usually absent. (4) Its presence is variable in the Annelida, in some of which it is accompanied by another dichroic substance very like haemoglobin in its spectroscopic relations. (5) It is present in the fluid from the perivisceral cavity of the leech. (6) It is distributed through the plasma of the so-called blood of the larva of Chironomus, but it has been sought for in vain in other insects, myria- pods, and arachnids. (7) In the blood-plasma of certain crustaceans, while it is absent in Others. (8) It is absent, for the most part, from the blood of molluscs, though it is present in the blood of a gasteropod (Planorbis). (9) It is met with in the 64 QUANTITATIVE ESTIMATION OF HEMOGLOBIN. muscular fibres from the pharynx of gasteropod mol- luscs, as Limnaeus and Paludina, although it is want- ing in their blood. Lankester has observed that among gasteropods it is those muscles which are most active and most powerful that are furnished with hemoglobin. This author also describes chloro- cruorin from the green blood fluid of Sabella ; it was found to give two absorption bands, not in the same position as those of oxidized haemoglobin, but which, on the addition of reducing agents, were changed into one. He also showed that chlorocruorin and haemo- globin have a common base in " cyano-sidphcem" * and perhaps in Stokes' reduced haematin, of which a description will be given afterwards. Preyer's method of calculating the percentage of haemoglobin by means of the spectroscope. Before describing the spectrum method of estimating the amount of haemoglobin in blood, it may be mentioned that Preyer had arrived in 1869 at the following conclusions in regard to the effect which solutions of haemoglobin of different strengths, when examined in a layer one centimetre deep, had upon the spectrum ; these numbers will be found very useful : (a) A solution containing from 0*003 to 0*009 parts per cent, showed very faintly one band. (b) A solution containing 0*01 per cent, gave two bands very feebly. (c) A solittion containing 0*09 per cent, showed a difference of intensity in the shading of the bands. (d) A solution of 0*8 per cent, gave only one broad * See Appendix for title of Prof. Lankester's paper. ESTIMATION OF HEMOGLOBIN. 65 band, both bands having coalesced ; in addition to the red from a, to near D, there was only a green stripe observable between b, and F, but near b. This green stripe is not seen if the solution contains 0*9 per cent., but is distinctly seen if the solution contains 0*7 per cent, of haemoglobin. Solutions containing more than 7 '3 per cent, of haemoglobin allow no light at all to pass. The solution (d) containing 0*8 per cent, of haemo- globin is taken as the normal solution for comparison in determining the percentage of haemoglobin in the blood. To estimate the amount of haemoglobin in a specimen of blood, we must make a solution of a measured or weighed quantity of blood in water, and then find, with the aid of the spectroscope, what degree of dilu- tion is necessary to bring it to such a strength that only the red rays are transmitted. The point of dilu- tion at which green is extinguished has been found by Preyer to be very constant (the solution containing 0*8 per cent., one centimetre in thickness, see above), and is therefore used as the standard fluid for com- parison. To make the standard solution, we introduce a concentrated solution of a known weight of pure haemoglobin crystals into a glass chamber (haema- tinometer) of which the parallel sides are one centi- metre apart from each other. The chamber is then placed in front of the spectroscope, the source of light being a paraffin lamp. Distilled water is then gra- dually added from a divided burette so long as all of the spectrum is extinguished except the red. The moment the green begins to appear the operation is ended. 5 66 ESTIMATION OF HEMOGLOBIN. The volume of the diluted solution is determined, and the exact conditions, viz. the distance of the lamp and the glass chamber, and the width of the slit are noted down. The percentage of haemoglobin in the solution is that at which, under the given conditions, complete absorption of the green takes place. It may be called Jc. In order to determine the percentage of haemoglobin in any given specimen of blood, all that is required is to repeat the operation just described with the blood ; thus, a small quantity of fresh blood, which has been well agitated with air and defibrinated, is introduced into a finely graduated small pipette, from which exactly one cubic centimetre is delivered into the glass chamber, and diluted before the slit of the spectroscope (the liquid being carefully stirred after each addition of water) until the green begins to appear. At the moment the green is seen the liquid contains a per- centage of haemoglobin equal to Jc. If the volume of distilled water, including the cubic centimetre origin- ally added, be designated c, and the original volume of blood b, the percentage of haemoglobin which the blood contains is readily calculated according to the formula : x b-{-c l~ ~T~ Therefore, as b = 1, we have x = Jc (1 + c) * According to M. Rajewski this method is less exact * 'Handbook for Physiological Laboratory.' 1873. By Klein, Sanderson, Foster, and Brunton. EEDUCED HEMOGLOBIN. 67 than that of Hoppe-Seyler. (M. Quincke made a useful modification in this method by making use of a prismatic vessel attached to a graduated scale, in which a 10 per cent, solution of blood was introduced.) Among the other methods of estimating hsemoglobin may be mentioned (1) By the estimation of iron. (2) By the estimation of oxygen. (3) By the estimation of hsematin. (4) By the method of comparison of Hoppe- Seyler. (5) By J. Worm Miiller's method. (6) By the fluid scale of Welcker. (7) By Welcker's scale of blood stains. (8) By the painted scale of M. Hayem. (9) By the globulimeter of Mantegazza. Or (10) By the hsemochromometer of Malassez.* It will be thus seen that there are a great many methods of estimating haemoglobin besides the spectroscopic one.f The absorption bands of blood were first described by F. HoppeJ in 1862, and his experiments being repeated by Stokes, the results he arrived at were confirmed. Reduced haemoglobin. Professor Stokes not only confirmed Hoppe's experiments, but he also found that, by adding certain reducing agents to blood, * See the 'London Medical Record' for July 15, 1879, p. 256; and title of Malassez's paper, Appendix II. f Probably we might get a method still more accurate by noting the readings (with the microspectroscope), and calculating the wave-lengths corresponding to, the edges of absorption bands in a solution containing a known amount of haemoglobin, and examined in a layer of the same depth, and comparing with this standard solution, solutions of blood, always diluted to the same amount with water, and examined at the same depth as the standard solution, and noting the wave-lengths of absorption bands given by the latter. I merely throw this out as a sug- gestion, not having had sufficient time to follow up the idea. J ' Virchow's Archiv, Bd. xxiii, p. 44-6. Appendix II. 68 KEDUCED HEMOGLOBIN. he could change the scarlet blood into purple (or, as he described it), c( scarlet cruorine" into t( purple cruorine." Supposing that the change in colour arose from reduction he added to blood the fluid which is now called after him, viz. an ammoniacal solution of ferrous sulphate, to which enough tartaric acid had been added to prevent precipitation by alkalies ; and he found not only that change of colour took place, but that the spectrum was changed, instead of two bands, only one was seen, having its darkest portion in the position formerly occupied by the space between the blood bands. The fluid he added had a greater affinity for oxygen than had the haemoglobin, so the latter was robbed of it. Dr M. Foster describes so accurately the spectrum of this solution, that I cannot resist quoting him. He says : " Examined by the spectroscope, the reduced solution, or solution of reduced hemoglobin, as we may now call it, offers a spectrum entirely different from that of the un- reduced solution. The two absorption bands have disappeared, and in their place is seen a single, much broader, but at the same time much fainter band (see Sp. 6, Chart I), whose middle occupies a position about midway between the two absorption bands of the un- reduced solution, though the redward edge of the band shades away rather farther towards the red than does the other edge towards the blue. At the same time the general absorption of the spectrum is dif- ferent from that of the unreduced solution, less of the blue end is absorbed. Even when the solutions become tolerably concentrated, the bluish-green rays EEDUCED HEMOGLOBIN. 69 to the blue side of the single band still pass through. Hence the difference in colour between haemoglobin which retains the loosely combined oxygen and hsemo- globin which has lost its oxygen and become reduced. In tolerably concentrated solutions, or tolerably thick layers, the former lets through the red and the orange- yellow rays, the latter the red and the bluish-green rays. Accordingly, the one appears scarlet, the other purple. " In dilute solutions, or in a thin layer, the reduced haemoglobin lets through so much of the green rays that they preponderate over the red, and the resulting impression is one of green. In the unreduced hsemo- globin or oxy-haemoglobin, the potent yellow which is blocked out in the reduced haemoglobin, makes itself felt, so that a very thin layer of haemoglobin, as in a single corpuscle seen under the microscope, appears yellow rather than red." It is exceedingly easy to cause the reduction of haemoglobin in solution, we can do it by means of indifferent gases, e. g. nitrogen, or we can remove the oxygen by means of the air-pump. But the easiest method is by the addition of ammonium sulphide to the solution of blood, or by the addition of Stokes' fluid ; the last is not as satisfactory as the ammonium sulphide, since it causes turbidity in fluids where the other does not, and it spoils by being kept ; moreover, in using it greater precautions 1 have to be adopted for the exclusion of the air, and it sometimes fails to reduce certain forms of haematin, which are at once reduced by ammonium sulphide. If, then, we add to a solution of 70 REDUCED HEMOGLOBIN. blood in a test-tube before the slit a few drops of ammonium sulphide, turn the test-tube upside down once or twice, holding the thumb against its mouth to prevent spilling of the contents, plug it with some cotton wool, pushing the wool down almost to the surface of the fluid, and then gently heat, we notice the red colour of the solution change to purple, and the spectrum presents the appearance so well described by Professor M. Foster, and shown in Chart I, 6. Or, we may add the reducing agent to the solution in a cell beneath the microspectro scope, having taken the precaution to cover the fluid in the cell with a micro- scopic cover-glass.* The importance of this discovery of Stokes cannot be over estimated, for not only does it explain the differ- ence in colour between arterial and venous blood, but it also shows us wherein the breathing-power of the red blood-corpuscle resides, and explains phenomena which, before its discovery, were inexplicable. If, after adding the reducing agent, and after the spectrum has changed as described above, we shake the fluid in the test-tube with air, or stir up the fluid in the cell, the single band again disappears to be replaced by the first observed bands of oxy-haemo- globin; left to itself for a few moments it again becomes reduced, but can be again changed as before by agitation. In fact, the same result can be made to take place as often as we wish. * This reduction test distinguishes blood from other pigments, the spectra of which somewhat resemble it, e.g. carmine, and turacine, a pigment discovered in the feathers of the Cape lory by Church. . SPECTEUM OF VENOUS BLOOD AFTEE DEATH. 71 It has been stated repeatedly, that arterial and venous blood owe the difference in their colour to this oxidized and deoxidized condition of the haemoglobin ; but it has been also asserted that venous blood always contains enough oxygen to make it give the spectrum of oxy-haemoglobin. I shall now show that the latter statement is not absolutely correct. In the case of venous blood after death, there is an exception to the rule. Spectrum of venous blood after death. In exam- ining the blood of a still-born foetus with the spectro- scope, I thought I had come across an additional test of the viability or rather the non-viability of the foetus, as the blood from the vena cava, right auricle, right ventricle, left auricle, and left ventricle gave the one band of reduced haemoglobin. The blood was examined on a microscopic glass slip, being quickly covered with a cover-glass so that it had not time to become oxyge- nated. I then examined the blood of adults who had died of various diseases, and in every instance I found that the blood in the right auricle and right ventricle gave the spectrum of reduced haemoglobin. At the time, I concluded that this reduction was an effect of decomposition, because blood out of the body becomes spontaneously reduced after the lapse of some hours ; but a series of experiments on the lower animals has taught me that hcemoglobin becomes reduced in the act of dying, provided death is not caused by starva- tion, cold, &c. (vide p. 74). In addition to the method of examination adopted above, I find Husband's capil- lary vaccine tubes are very useful for the same purpose. 72 SPECTRUM OF VENOUS BLOOD AFTER DEATH. One end of the tube is sealed in the flame, and the tube being then quickly drawn through the flame so as to expel the air, the other end is sealed ; in this way we have a partial vacuum in the tube. When blood has to be examined, an opening sufficiently large to allow of the introduction of the tube is made in a blood-vessel, and the former is pushed into it for the distance of half an inch or so ; the tube is then held with the forefinger and thumb of the left hand, while its apex within the vessel is broken off, or crushed off, with forceps. The blood rushes into the tube, which is then withdrawn and again sealed in the flame. When this is examined with the micro spectroscope, it is necessary to place it on a piece of black paper or platinum foil perforated with a pinhole, so as to stop all surplus light. The following experiments were made in order to determine within what space of time the reduced haemoglobin band appears after death. (1) A rabbit was killed by pithing, and its blood was examined twenty-seven minutes after death. The blood] in the right auricle, right ventricle, venae cavae, and left iliac vein gave the reduced haemoglobin band ; that from the left ventricle and left auricle showed a tendency to reduction ; that in the aorta gave the two- landed spectrum of oxidized hcemoglobin. (2) A rabbit was poisoned with strychnine ; ten minutes after death the blood in the right auricle, right ventricle, and venae cavae gave the land of reduced haemoglobin, that from the left auricle and ventricle and aorta gave the bands of 0-hcemoglobin. SPECTRUM OF VENOUS BLOOD AFTER DEATH. 73 (3) A rabbit was killed by pithing ; the blood from the right auricle and ventricle, and venae cavae gave the band of reduced haemoglobin three minutes after death. Within six minutes the blood in the left auricle and left ventricle showed a tendency to reduction. The blood in the aorta was not reduced, and the blood in the ear was not reduced. (4) A rabbit died from debility ; within half an hour the blood was examined ; that in the right auricle and right ventricle gave the spectrum of reduced hcemoglobin ; that in the left auricle, left ventricle, and aorta gave the oxidized haemoglobin spectrum. (5) A hedgehog died from chloroform narcosis. Its blood was examined eight minutes after death. It was found to give the spectrum of reduced haemoglobin in the right auricle, right ventricle, and venae cavae ; but that of oxidized hcemoglobin in the left auricle, left ventricle, and aorta. From these and other experiments we may conclude that the haemoglobin of the blood in the right side of the heart and in the veins is reduced as soon as the animal has ceased to breathe, but that the haemoglobin of the blood in the left side of the heart and aorta does not become reduced for some time after death, the time varying with the mode of death. At the time I made these experiments and had drawn these conclusions from them, I was not aware that Professor Hofmann, of Vienna, in the paper, the title of which is given in the catalogue at the end of this book, had come to the same conclusion, and that he had stated in addition the fact, that Koselanski had 74 SPECTRUM OF BLOOD IN DEATH FROM ASPHYXIA. proved the presence of reduced haemoglobin in the blood of every dead body, if certain precautions for excluding the air had been taken. Hofmann concludes that the tissues of the body take the oxygen from the blood a few minutes after the lungs have ceased to convey air to that liquid. Hoppe-Seyler has also con- firmed these observations, but Albert Schmidt has shown that there are exceptions to this condition in several kinds of death in warm-blooded animals. Thus, in death from breathing carbonic oxide (as will be referred to further on) ; in death from starvation or cold, in which the reducing power of the tissues is diminished ; or in death from passage of air into the veins, the haemoglobin is not reduced. But whether the blood retains the spectrum of oxidized haemoglobin permanently, or only for a time, in death from these latter causes, has not yet been proved. Hofmann considers, and his conclusion is probably correct, that the difference is only of a temporary nature, for the blood has in itself the power of consuming its own oxygen, " in the absence of any contact with organic tissues." * Spectrum of the blood in death from asphyxia. Stroganoff endeavoured to decide the question, whether the blood of an asphyxiated animal contained oxy-hsemo- globin. He placed between two glasses the completely isolated jugular vein, or carotid artery of a rabbit, and compressed them sufficiently to allow of their being examined with the spectroscope. " It was invariably * E.g. Blood corked up in a bottle soon becomes of a purplish colour, and gives the band of reduced haemoglobin. SPECTRUM OF BLOOD IN DEATH FBOM NITROUS OXIDE. 75 found on asphyxiating the animal that the blood, even at the last moment of the last cardiac contraction, always contained oxy-haemoglobin." But in death from as- phyxia the blood, arterial as well as venous, immediately after death, gives the spectrum of reduced haemoglobin ; the following experiment will prove the truth of this assertion. If a rabbit be asphyxiated by compression of the trachea, or by drowning, and if the blood be examined in the manner I have described, within two minutes after death, or as soon as the blood can be examined, reduced hcemoglobin will be indicated by the spectroscope in the left auricle, left ventricle, and in the aorta. This is an important fact, and the know- ledge of it will be useful to medical jurists. Spectrum of the blood in death from the prolonged inhalation of nitrous oxide. Knowing that nitrous oxide has the power of reducing haemoglobin in solution, in the same manner, it is said, as indifferent gases, such as nitrogen and hydrogen, I was anxious to determine whether the arterial blood of an animal poisoned by prolonged inhalation of this gas would give imme- diately after death the spectrum of reduced haemoglobin. Accordingly a guinea-pig had nitrous oxide adminis- tered to it until it ceased to live, the blood was examined within two minutes from the time of death, and the arterial blood all over the body, and also the muscles, gave the spectrum of reduced hcemoglobin. The fact of the muscles having yielded this spectrum is made more interesting, when the remark of Thudi- chum is remembered, viz. that in death during the collapse stage of cholera, the muscles were found by 76 CARBONIC OXIDE HEMOGLOBIN. him to yield the spectrum of reduced haemoglobin ; thus explaining the symptoms, which are due to " suspended oxidation." The spectroscope accordingly confirms the assertion made in ' Wood's Therapeutics/ 1878, p. 268, that nitrous oxide produces its ansesthetic effects partly by stopping the supply of oxygen to the blood.* And it also suggests wherein lies the danger of its administration, viz. too prolonged inhalation ; for the temporary thus becomes converted into a permanent deprivation of oxygen, Spectrum of the blood after death from the in- halation of carbonic oxide. In examining the blood reduced by nitrous oxide we find that shaking with air brings back the spectrum of oxidized hemoglobin, thus the gas did not enter into a combination with the hgemo- globin, it suspended its breathing power for the^time ; but in carbonic oxide we have an agent which is quite different in its action, for ib not only destroys the breathing power of the haemoglobin, but it actually enters into a combination with it, displacing the oxygen volume for volume ; and so firm is the com- bination, that reducing agents fail to rob the carbonic- oxide-hsemoglobin of its carbonic oxide ; so that the spectrum of the combination is unchanged when sulphide of ammonium or other reducing agent is added to it. The spectrum, got by passing carbonic oxide (or even coal gas since it contains 7 per cent. CO) through blood, or through a solution of it, or by poisoning an animal with the gas, is characterised * Of course this fact alone does not account for the anaesthesia pro- duced. THE BLOOD IN POISONING BY CHARCOAL FUMES. 77 by having two absorption bands resembling in their relative breadth, and their shading the bands of oxy- haemoglobin, but differing from the latter in being nearer the violet end of the spectrum. Sp. 7, Chart I, ' represents the spectrum, and was mapped from a solution of blood obtained from the body of a mouse poisoned in an atmosphere of carbonic oxide. The colour of blood, or of a solution of blood, is made more scarlet after carbonic oxide is passed through it. When sulphide of ammonium, or when Stokes 5 fluid was added to the blood of the mouse, no reduction had taken place at the end of forty-eight hours, but instead, the band of sulphsemoglobin appeared in the red, which will be described further on. In death from the inhalation of the fumes of smouldering char- coal it is carbonic oxide which exerts its deadly influence, and the blood of people poisoned in this manner exhibits the bands shown in the map, and cannot be made to yield the spectrum of reduced haemoglobin on the addition of reducing agents. The blood of mice and other small mammals poisoned by being placed in an atmosphere of coal gas, gives the same spectrum ; hence, if any doubt existed as to the cause of death in a given case where coal-gas poison- ing was suspected, the spectroscope would at once decide the question. Not only has this instrument enabled us to detect carbonic oxide poisoning after death, but the study of carbonic-oxide-hsemoglobin spectra has suggested a treatment in those cases where life is not extinct. A common form of suicide on the Continent, more especially in Paris, is performed by 78 CARBONIC OXIDE HAEMOGLOBIN. the intending victim shutting himself or herself up in a room from which all air is excluded, and then having lighted some charcoal, inhaling the fumes ; hence it is as well to be aware of the treatment which is likely to be of use where life has not become wholly extinct. " Bonders * states that carbon-monoxide may be expelled from blood saturated with it by oxygen, carbon dioxide, and hydrogen, even at ; oxygen does not convert the monoxide into dioxide, but simply drives it out. If this be the case, it should be possible to pump the carbon-monoxide out of blood saturated with it, although it may not be removed quite so easily as oxygen." The experiments of Zuntz t show that this is possible. " When blood saturated with carbon-mon- oxide was placed in a receiver connected with an exhausting pump, and warmed to 37 42, an active escape of gas took place, ceasing apparently at the end of half an hour. When, however, the pumping was continued at various intervals, fresh quantities of gas were given off, and a further quantity was obtained by heating the receiver to 60. The blood so exhausted was found to give the spectrum of reduced haemoglobin, which was replaced by the spectrum of oxy-hajmoglobin after standing in the air." These results did not coincide with those of other observers, because the latter were not able to extract the carbon monoxide from the blood by exhaustion, " inasmuch as the process was supposed to be complete when no more gas was evolved after the first pumping." So that in cases of poison- ing by carbonic oxide artificial respiration should be * Pfliiger's Archiv,' iv, 28. f Ibid., v, 584. SPECTEOSCOPIC SUGGESTIONS FOE, TREATMENT. 79 kept up vigorously for some time. It has also been proposed to treat such cases by the inhalation of oxygen, and it has actually been carried out, it appears with some success in Berlin. Professor Baeblich de- monstrated the interesting fact that if oxygen be passed through carbonic oxide blood, it becomes reduced upon the subsequent addition of ammonium sulphide,* hence the reason of its adoption as an antidote. Podolinki t has also shown that blood saturated with carbon-monoxide is completely deprived of that gas by agitation for half an hour with hydrogen, and more rapidly with oxygen. Nitrogen dioxide is also expelled by hydrogen, but less rapidly than car- bon monoxide. Hence it appears that the compounds of haemoglobin with carbon monoxide and nitrogen dioxide are similar in character to oxy-hsemoglobin ; the order of stability being, oxy-hsemoglobin, carboxy- haemoglobin, nitroxy-hsemoglobin. Each of the three gases, 0, CO, NO, can be expelled by the one im- mediately following, and each also more easily by the one immediately preceding it, than by any other indifferent gas. Carboxy-hsemoglobin can be obtained in the crys- tallized condition by passing a stream of the gas through an aqueous solution of haemoglobin crys- tals, blood-corpuscles, or even defibrinated blood, cooling to 0, mixing with -J- vol. cold alcohol, and allowing the mixture to remain for some time at 0. Bluish-red crystals form, less soluble and less easily * ' Nature/ vol. xv, p. 362. f ' Pfliiger's Archiv,' vi, 553. 80 BLOOD TREATED WITH NITRIC OXIDE. decomposable than those of oxy-haemoglobin, but similar in form. " According to Koschlakoff and Bogomoloff,* solu- tions of oxy-haemoglobin and carboxy-haemoglobin, through which ammonia is passed gradually, turn brownish-green and no longer exhibit any absorption bands. Arsine colours solutions of oxy-hsemoglobin first yellow-brown, then green-brown, the absorption bands gradually disappearing and being replaced by the band of reduced haemoglobin, whereupon the solu- tion becomes somewhat red ; on the next day, however, the last-mentioned band disappears. On the other hand, carboxy-hgeinoglobin is coloured dingy green by arsenic, and its absorption bands are destroyed." f Spectrum of blood treated with nitrogen dioxide The oxygen of haemoglobin is displaced by nitrogen dioxide, and the latter forms a new combination with the haemoglobin. Arterial blood, however, takes up but a small quantity of the gas. " Thus the blood from the crural artery of the dog took up after addition of baryta water from 25*4 to 27'6 vols. per cent, of NO ; defibrinated dog's blood 23 vols. per cent. ; defibrinated ox blood 31*8 vols. per cent, (reduced to and at 1 metre pressure) ." J Nitrogen dioxide (= nitric oxide) and hyponitric acid, give spectra very like that of oxy-haemoglobin, but, as in the case of carbonic oxide, reducing agents fail to displace them from their com- binations with haemoglobin. Spectrum of blood treated with sulphuretted hydro- * ' Zeitschr. Anal. Chem.,' viii, 228. f Watts, ' Diet. Chem.,' 2nd Supp., 1875. J Watts, ' Diet. Chem./ 1st Supp., 1872. SULPHJBMOGLOBIN. 81 gen. If sulphuretted hydrogen be passed through a solution of blood, the haemoglobin first becomes reduced and then an additional band appears in the red. On shaking the solution with air, the broad band disappears and is replaced by those of oxy-hsemoglobin, but the band in red still remains ; these appearances are shown in Chart I, Sp. 8 and 9.* And Chart III, Sp. 5, shows Dr Thudichum's map of blood treated with sulphu- retted hydrogen ; the latter map is different from that of mine in some respects, which will be observed when they are compared. To the body giving this spectrum which was first described by Nawrocki, the name sulphsemoglobin has been given by Lankester ; it can be produced by adding excess of sulphide of ammonium to blood, especially if the latter be rather old, when the band in red appears quickly, but if it. be fresh it only appears after some time, or with a large excess of the sulphide, or on being heated after the addition of the sulphide. Any alkaline sulphide according to Preyer produces sulphaemoglobin. If a strong solution of blood be corked up for three days with ^yth its volume of the sulphide the spectrum will be obtained with great distinctness (Lankester). " I have carefully fixed its position, and find it to be quite distinct from that of any other haemoglobin band," says Lankester in the ' Journal of Anatomy,' 1869, p. 119. He also observes that blood treated sufficiently * I find that when ammonium sulphide is added to blood previously treated with sulphuretted hydrogen that we sometimes get the first band of reduced hsematin within that of reduced haemoglobin, and then those of reduced hsematin (see methsemoglobin, cyanhaematin, and nitrite blood), but the band in red still persists. 6 82 SULPHJ1MOGLOBIN. long with a sulphide gives eventually the bands of reduced hsematin. I may here remark that this is an important observation, because it teaches the necessity of caution in using sulphide of ammonium as a reducing agent, for an excess of sulphide will not only develop the band of sulpheemoglobin, but after a little time it may, if added to a product of hsemoglobin, such as methsemoglobin, intermediate between the former body and hsematin, bring out the bands of reduced haematin, and thus lead to an erroneous inference. " The previous addition to the blood of gallic acid and an alkaline carbonate has the same effect as putrefaction in making the sulphaemoglobin band occur at once when the sulphide of ammonium is added. This was pointed out by Professor Stokes, of Cambridge " (Purser). Preyer has noticed that sulphas moglobin is not formed if the sulphuretted hydrogen is made to act on reduced hsemoglobin. Hence " water contain- ing sulphuretted hydrogen can be drunk or injected into the veins without danger to life, while the danger of breathing the gas or of injecting its solution into the arteries is very great."* Solutions of haemoglobin, free from oxygen, even in presence of ammonia, are not affected by sul- phuretted hydrogen, but oxidized hsemoglobin, as stated before, is reduced, the first effect of this gas being the separation of the loosely combined oxygen from the hasmoglobin, this action being hastened by heat. In an ammoniacal solution of oxidized hsemo- globin abstraction of oxygen is the only action that * Purser, after Preyer. SULPH2EMOGLOB1N. 83 takes place, but in neutral solutions a band appears in the red. The colouring matter which gives this band differs from hsematin and from methaemoglobin (of which a description is given further on) in this, that the solutions of the latter substances when treated with ammonia and ammonium sulphide exhibit certain bands in green, whereas the sulphaemoglobin, got by passing sulphuretted hydrogen through a solution of oxyhsemoglobin, remains unaltered when thus treated.* Hoppe-Seyler regards sulphsemoglobin as a sulphur compound of haematin and haemoglobin. By the long-continued action of sulphuretted hydrogen this compound is decomposed, sulphur and an albu- minous substance being separated, and another body is formed, which is olive-green in thin layers, and brown-red in thicker layers; this dries up into a brittle hygroscopic mass having a pitchy lustre. This sub- stance is coagulated by heating its aqueous solution, as well as by acids and by alcohol. " It contains all the iron (0*44 p. c.) of the hemoglobin, and about four times as much sulphur (1*57 p. c. instead of 0'415) " (Hoppe-Seyler). A solution of iron sulphide (as ob- tained with very dilute ferrous sulphate, tartaric acid, and ammonium sulphide) gives a band in red like that of the solution of haemoglobin treated with oxygen and with sulphuretted hydrogen. But no formation of iron sulphide takes place in the above reaction on account of the presence of oxygen ; besides, the product contains all the iron of the haemoglobin. Nawrockif states that ammonium sulphide exerts on * Not always, see note, p. 81. f ' Zeitsch. Anal. Chem.,' vi, 285, and ' Jahresb.,' 1867, p. 802. 84 SULPH^EMOGLOBIN. haemoglobin at first a reducing, and afterwards a decomposing, action. A solution of haemoglobin mixed with ^ vol. of ordinary ammonium sulphide gives a dark band in the red at Fraunhofer's line C ; the broad reduction band between D and E becomes narrower and more sharply defined, and afterwards a second broader band appears, covering E and extend- ing beyond b. After the appearance of these bands, which disappear in about twenty-four hours, those of oxyhsemoglobin are no longer produced by agitation with air. According to Preyer* potassium persulphide also causes the bands of oxyhsemoglobin to disappear, at first bringing out the band of reduced haemoglobin, but afterwards, especially on heating gently, a sharply- defined black band, beginning at -^ of the distance from D to E, and ending at ^ f the same distance, and another band beginning at ^ spectrum being also obscured. See Sp. 21, Chart I. ' In stating this fact, which can be easily verified, I am not in accordance with some observers, who state that hydrocyanic acid has no effect .upon blood, and that it is only with cyanide of potassium this spectrum is got ; but as the map was drawn from an actual speci- men, and as the result arrived at was constant after many experiments, the only essential being the appli- cation of a gentle heat, I prefer again reiterating the statement that hydrocyanic acid does affect the spec- trum in this manner. With Scheele's prussic acid we get if we add it in sufficient quantity not this spectrum, but that of acid hsematin ; and I found that with the ordinary 2 per cent, acid of the 6 British Pharmacopseia ' a different result could be obtained according to the amount added. By deoxidizing a solution of blood, in which the spectrum just described is well marked, we get two bands like the carbonic oxide bands ; this spectrum is shown in Chart I, Sp. 22. But this result is not always constant, for in using the 2 per cent, acid, and if after the broad band has 86 ACTION OF CYANIDES. appeared we add a reducing agent we sometimes get a narrow dark band, in the position of the first one of reduced hsematin, superimposed upon a broad band, in the position of that of reduced hemoglobin. The reason of this is that too much hydrocyanic acid has been added, and the spectrum described above is entirely missed. In such cases it is probable that cyanha3moglobin and cyanhsematin are formed simul- taneously, as compounds behaving like this are pro- duced by other reagents as well as by hydrocyanic acid. And, further, if to blood which has been treated with hydrocyanic acid, and afterwards ammonium sul- phide, and which presents this abnormal behaviour, we add before the addition of the ammonium sulphide, a little ammonic hydrate we get the bands of reduced haematin. Hence the above solution of the problem is the correct one, for the ammonia converts all that part of the hemoglobin which remains, into haematin, and so we only get the bands of reduced haematin when the sulphide of ammonium is added. A concentrated solution of guinea pig's or dog's blood mixed with hydrocyanic acid, when Jth its volume of alcohol is added to it, and when it is cooled to 0, deposits crystals which are exactly like those of oxyhaemoglobin, but they retain hydrocyanic acid even after repeated crystallisation and drying in a vacuum ; the hydrocyanic acid can, however, be separated from them by distillation with water and a few drops of sulphuric acid (dilute). The compound of hydro- cyanic acid with haemoglobin crystallizes easily, but that of potassium cyanide does not. Neither com- ACTION OF CYANIDES. 87 pound can be reconverted into oxyhsemoglobin ; and neither can ozonise atmospheric oxygen. When blood has been treated with cyanide of potassium and after the band described above has appeared, a stream of oxygen may be passed into the solution, without affecting the spectrum. According to Preyer, as stated before, ammonium sulphide developes two bands, the first extending from - 2 ^ to -| of the distance from D to E, the other from -|-, between D and E as far as f from E to b. So that this spectrum re- sembles that got by acting on blood with carbonic oxide. On acting upon the solution reduced by ammonium sulphide, with oxygen, the broad band reappears, and on adding ammonium sulphide repeatedly the first two bands are reproduced. Moreover, the solution can now be coagulated by heat, whereas the solution of blood acted on by hydrocyanic acid is not coagulated by heat. It would appear from the behaviour of cyanide of potassium and of hydrogen cyanide with blood, and from the behaviour of these compounds with ammo- nium sulphide, that the latter contain oxygen which is removed by sulphide of ammonium, but which is more intimately combined with the hemoglobin than in oxyhaemoglobin. According to Nawrocki,* the broad band, got on adding hydrocyanic acid to blood, belongs to hasmatin, not to haemoglobin ; he shews that it is obtained at once and without the aid of heat,^f the blood- solution is first mixed with caustic potash. * ' Jahresb.,' 1867, p. 204. 88 ACTION OF CYANIDES. Lankester found that cyanhaematin was formed from cyanhaemoglobin if the latter solution had stood for two hours or more, "the pinkish-red colour being changed to an orange brown ;" the cyanhaemoglobin solution was obtained by passing cyanogen gas into a solution of blood, and Lankester thinks that we may infer the existence of a compound between cyanogen and haemoglobin, like CO -haemoglobin, NO- haemoglobin, &c. This view is opposed to that of Laschkewitsch,* as the latter states that cyanogen merely reduces haemoglobin. " He is probably led to this conclusion by the observation of the cyanhaematin of Hoppe-Seyler (having missed altogether the spec- trum seen by me), which has a single broad band resembling, but quite distinct from, that of reduced haemoglobin." t Professor Gamgee, in his report on physiology,! remarks : " Is it not likely that in this case a compound of the oxygenised blood colouring matter is formed with cyanogen, similar to the com- pounds with cyanide of potassium and with the nitrites, and that the spectrum described as that of reduced haemoglobin is really the spectrum of the new sub- stance ? " When potassium cyanide is added to an aqueous solution of blood which has been treated with carbonic oxide, the characteristic absorption bands do not dis- appear until the mixture has been heated to 40, when the broad band of cyanhaematin appears, and now on the addition of sulrphide of ammonium the reduced * Reichert's u. Reymond's ' Archiv,' 1868, p. 649. f Lankester, in ' Journ. Anat. and Phy.,' November, 1869. % ' Journ. of Anat. and Phy.,' May, 1869, p. 469. ACTION OF CYANIDES. 89 cyanhgematin bands appear ; by agitation with air the broad band is reproduced, and afterwards the original spectrum of the carbonic oxide compound. Hydrocyanic acid and ammonium cyanide act in the same manner with the aid of heat ; but the filtered solution after agitation with air finally exhibits the bands of oxidized haemoglobin.* The blood of animals poisoned with hydrocyanic acid gives no characteristic spectrum, nothing beyond that of oxyhasmoglobin. * Preyer, ' Zeitschr. Anal. Chem., vi, 289; ' Jahresb.,' 1867, p. 803. 90 ACTION OF NITELTES, CHAPTER Y. ABSORPTION SPECTEA OF BLOOD (continued). Action of nitrites on blood. The blood of ani- mals, which have been made to inhale nitrite of amyl until the cessation of life, exhibits a choco- late colour, and when this blood is examined with the spectroscope, the change of colour is seen to be accompanied by a change of spectrum. There are three bands visible in the aqueous solution of such blood, the spectrum is shown in Chart I, Sp. 11.* This fact was first discovered by Professor Gamgee, of the Owens College, Manchester, and his paper bearing upon the subject will be found in the ' Philosophical Transactions ' of the Royal Society of London for 1868, vol. 158, part II, p. [589] ; he had previously published a paper on the action of the nitrites in the e Transac- tions of the Royal Society of Edinburgh ' (v. Appendix). After laying down a few propositions, Professor Gamgee shows that no one had hitherto investigated the action of nitrites on blood, his own attention having been first called to the matter by noticing the chocolate colour, which the blood of mice assumed when poisoned with the nitrite of amyl vapour. In his paper read before the Royal Society of Edinburgh * A fourth band is also shown. The reason of this is explained in p. 97. ACTION OF NITRITES. 91 he had discussed the optical characters only, but in this he discusses the changes and influences which the nitrites exert on the relation of the blood to various gases. - But here we shall only refer to the optical characters of the blood : (1.) As to colour. When defibrinated and well- arterialised blood is mixed with a solution of nitrite of potassium or sodium, its colour becomes almost imme- diately altered, changing to a chocolate brown. But the nitrites differ in the rapidity with which they act upon blood, for the blood of the dog is almost instantaneously affected, while the blood of the ox and sheep may take twenty minutes or even longer. The blood-cells of the dog are recommended for experi- mental purposes because they burst more readily. The nitrite should be dissolved in alcohol, and a few drops of the alcoholic solution added to the blood. Ammonia in solution turns the chocolate-coloured blood of a red colour, which change was proved by experiment to have been independent of any alteration in the shape of the corpuscles. (2.) As to the spectrum. If after blood is diluted enough to show the bands of oxy haemoglobin, we add a solution of the nitrites, when the solution begins to change colour to a brownish tint, the blood bands undergo remarkable changes. The two bands become fainter and fainter, and are only visible when a com- paratively thick layer of the fluid is examined. At the same time if the layer be sufficiently thick, an addi- tional though faint band appears in red. This band appears absolutely to coincide with that of acid hsematin. 92 ACTION OF NITEITES. It is seen to best advantage where so thick a layer of solution is examined as to cut off all but the red rays. The complete change in spectrum is always coincident with the change in colour. By the addition of ammonia to alkalinity the colour changes from chocolate-brown to blood-red again. Simultaneously the band in red disappears and the bands between D and B become again more distinct. In addition, that part of the spectrum which is between yellow and orange becomes shaded by a well-defined absorption band. According to Prof. Gamgee, the spectrum of the nitrite blood consists of three bands, which may be called, according to their amount of shading, S, a, |3. is between C and D, near C. a is shown covering D. )3 occurs between D and E, near E. The spectrum of the compound of nitrites with the haDmoglobin after the addition of ammonia showed a faint, and a darker band, touching each other, the dark one covering D, the light one on the red side of D, and a band between D and E. (In these investigations a single-prism spectroscope was used.) On adding sul- phide of ammonium, or Stokes 5 fluid, to the nitrite blood treated with ammonia, an extraordinary change ensued. First of all, the spectrum of the nitrite blood treated with the ammonia appeared, which was then replaced by the spectrum of reduced haemoglobin, and when the last was shaken with air the spectrum of oxidized haemoglobin appeared. ACTION OF NITEITES. 93 Potassium nitrite also gave a band in red, and after the blood had been treated with this reagent, ammonia was added to the mixture. The band in red disap- peared at once, and the two bands became more in- tense. The orange was again shaded, a faint band appearing to overshadow it, and on adding a re- ducing agent the oxyhasmoglobin bands appeared darker than ever, and after some time they gave way to the band of reduced hasmoglobin. These experiments so far shewed (1) That nitrites exert a marked influence both on the colour and on the spectrum of blood, due obviously to a chemical change exerted on the hemoglobin. (2) This chemical change does not alter the com- position of the colouring matter, as proved by the action of reducing agents. (3) Nitrites neither expel nor remove the loose oxygen of the blood, because reducing agents develop the spectrum of oxyhsemoglobin before that of reduced haemoglobin without the intervention of atmospheric oxygen. The author then shews how nitrites modify the respiratory function of the blood by several interest- ing experiments, for which the reader may consult the original paper. His conclusions were as follows : (1) When a solution of any nitrite acts upon blood, peculiar changes occur in the colour, and simul- taneously in the absorption spectrum. (2) These changes in the optical properties of blood are due to the formation of compounds presenting the same crystalline form, colour, and spectrum, whatever 94 ACTION OF NITRITES. the nitrite which has been employed in their prepa- ration. (3) These bodies appear to be compounds of the nitrite used, with oxidized haemoglobin. (4) The substances formed by this process of chemi- cal addition, although isomorphous with haemoglobin, differ from it in many of their most remarkable pro- perties upon which its functions in the economy of the body depend. By this process of addition the blood- colouring matter appears to have lost its power of absorbing oxygen. (5) The addition of nitrites to haemoglobin appears to result in the locking up of the loosely-combined oxygen, so as to make it irremovable by carbonic oxide, or by a vacuum. The author makes a few observations at the end of his paper which are worth quoting. "We have hitherto been acquainted with haemoglobin itself, as well as with its 0-, CO-, and N 2 2 -compounds. These compounds are all isomorphous, possess almost the same physical characters ; in all the oxygen-free haemoglobin has apparently linked itself to a molecule of 0, CO, and N 2 2 respectively, the stability of the compound being least in the case of the 0-compound, and greatest in the case of the N 2 2 compound. "All these bodies, and pre-eminently the 0-com- pound, appear to be examples of a class of bodies which stand, as it were, on the boundary line which separates chemical from physical combinations to be, in fact, examples of the class of so-called molecular com- pounds. Like other molecular compounds, their com- ACTION OF NITEITES. 95 position varies greatly within certain limits, and is influenced by circumstances and conditions which have no action on chemical compounds proper. " That a body possessing such a very complicated molecular structure as haemoglobin should present numerous points of attachment, as it were, for the linking on of such active, condensed bodies as the nitrites, is more than probable, and it is not remark- able that, as in the case of other combinations of a molecular kind, such as the union of salts with their water of crystallisation, of bases with sugar, of albu- men with metallic oxides, of iodine with the com- pound ammonias, the amount of t^e simple body added to the more complex should vary within wide limits." " The experiments of Hoppe-Seyler and of Preyer show that hydrocyanic acid possesses the property of linking itself to haemoglobin, forming a body isomorphous with it, but which, physiologically, is an inert body, having lost the power which, normally, haemoglobin seems to possess, of ozonising atmo- spheric oxygen." Professor E. Ray Lankester, on repeating these experiments, found, when sulphide of ammonium was added to the nitrite blood to which a little ammonia had been previously added, that the darker band of reduced haematin became visible in the midst of the broad band of reduced haemoglobin ; and on adding ammonia to the nitrite blood a clouding occurred in the position of the alkaline haematin band. "The band in the extreme red of the nitrite blood agrees 96 ACTION OF NITRITES. exactly with that of acid haematin, as Dr Gamgee observes. It therefore seems not improbable that, on addition of ammonia, a small quantity of ha3matin is really formed, which was partially developed on the first addition of the nitrite to the blood, as indicated by the band in red but which does not separate clinging, like the nitrite itself, in definite quantity, to the crystals of the blood thus treated " (Lankester). This appearance of Stokes' reduced haematin band within that of reduced haemoglobin would indicate probably the fact, that the spectrum observed was that of a mixture of haemoglobin and hasmatin. I have ob- served exactly the same appearances in three in- stances : (1) in a solution of blood treated with 2 per cent, hydrocyanic acid when reduced ; (2) in the fluid vomited, in a case of haematemesis, when reduced ; and (3) in blood treated with sulphuretted hydrogen and then with ammonium sulphide. At the same time the spectrum of the nitrite blood is exceedingly like that of methaemoglobin, both as regards the position of the bands and in its behaviour with reducing agents. I found, on repeating Gamgee's experiments, but in a somewhat modified manner four absorption bands, the blood (that of the cat) had a few drops of amyl nitrite added to it, the characteristic change in colour very soon occurred, and on exa- mining with the spectroscope three bands appeared, one near C, between C and D, one close to D, on the violet side of it, and another between D and E, slightly covering E, the violet end of the spectrum being shaded up to near b. On adding to this fluid, ammo- ACTION OF NITRITES. 97 nium sulphide I observed a band in the position of that of reduced haemoglobin . But if, after adding nitrite of amyl to blood, we shake the mixture with alcohol, a four-bonded spec- trum is obtained, three of the bands of which are evidently those of the aqueous solution, and the fourth band is placed between b and F.* The etherial solu- tion is also characterised by having four bands, which differ slightly in their position and in their relative shading from those of the alcoholic solution. But that the bands of the etherial and of the alcoholic solution belong to the same chemical compound is proved by adding a reducing agent, as the latter soon develops a band in the position of that of reduced haemoglobin. One curious fact which is observable about the nitrite blood is, that ammonia in solution develops a band in the same position as ammonium sulphide, but no further change beyond darkening took place in this band on adding a reducing agent to it. It appears to me from what I have myself observed that the body formed by the action of nitrites on blood is very like, if not identical, with the body which will be next described, i.e., methaemoglobin, because the spectrum of the latter not only closely resembles the nitrite spectrum, but also gives a band in the same place, that of reduced haemoglobin, when reducing agents are added. It would seem that agents not sufficiently strong to split haemoglobin up into haematin develop from it, or link themselves on to it * It is best seen by illuminating the slit with direct sunlight. 7 98 METHAEMOGLOBIN. to form compounds which are characterised by giving, upon the addition of reducing agents, the spectrum of reduced haemoglobin. The spectrum got by treating fresh cat blood with nitrite of amyl and shaking with alcohol is shown in Chart I, Sp. 11.* Methaemoglobin. If a solution of haemoglobin be left exposed to the air for some time it undergoes a change in colour, losing its brightness, and at the same time a change occurs in its spectrum, as a new band has appeared in the red ; the same change occurs if solutions of haemoglobin be evaporated at tempera- tures above 100 0. ; and if the edges of a filtering paper, through which a solution of blood has been filtered, be examined with the microspectroscope the same band will sometimes be found. This band owes its presence to a brown colouring matter, which was called by Hoppe-Seyler methaemoglobin. It is said by some to resemble haematin in its optical characters, but this statement is incorrect, as solutions of methaemo- globin do not give the bands of reduced hsematin, but that of reduced haemoglobin when reducing agents are added to them. It differs from haematin also in the fact that it is soluble in water and in very dilute acids. A certain band in red, which is said by the authors of some books on the microspectroscope (and, indeed, by some physiological chemists) to be that of acid haematin, I find is really due to methaemoglobin, as the band of methaemoglobin can be produced * The blood of an animal made to inhale the nitrite until it dies, gives the three bands described by Professor Gamgee, which differ slightly in position from those in the map. METH2EMOGLOBIN. 99 by adding weak acids to solutions of blood. The same band can be made to appear by passing carbonic acid gas through dilute solutions of hasmoglobin, or by adding a very small quantity of glacial acetic acid to solutions of blood. Permanganate of potassium causes a like transformation in haemoglobin. If a crystal of the permanganate be dissolved in water and added to a very dilute solution of blood before the slit of the spectroscope, and kept at a temperature of 25 C., the hemoglobin bands gradually disappear, and a new spectrum appears instead, which has not only the band in red, but also two* others, which occur nearly in the position of the haemoglobin bands. The fact that methaemoglobin is often present in pathological fluids makes it especially interesting to the student of medicine. I have found it in the urine of acute desquamative nephritis, in that of post-scarla- tinal nephritis, and in the blackish-brown fluid vomited in some cases of haematemesis. The spectrum of a solution obtained by the action of potassium perman- ganate on blood is shown in Chart I, Sp. 10, and the spectrum of the same body was noticed in the fluid vomited in a case of hsematernesis. In the last case, in addition to methaemoglobin, hsematin was also probably present, as sulphide of ammonium developed the first band of Stokes' acid ha3matin within that of reduced haemoglobin. There is a danger of con- fusing methasmoglobin with sulphsemoglobin, but the * A fourth band is sometimes seen in some pathological fluids ; it occurs to the violet side of the third band mentioned above. The fourth band, which is described under the nitrite spectrum will repre- sent its position. See Chart I, Sp. 11. 100 ACTION OF AMMONIA GAS, ETC. latter is distinguished from the former by not losing the band in red on adding ammonium sulphide to its solutions. The chemical nature of methaemoglobin is not accu- rately determined, but some have considered it a hyperoxide of haemoglobin ; Hoppe-Seyler* shows that this view is incorrect, since in some cases where metha3moglobin is found, the fact of any oxidation having taken place is out of the question. It is more correct to assume that metha3moglobin is a mixture of haBmatin with a soluble albumen, and from what has been already said about it, this appears to be the most rational view. Action of ammonia gas, arseniuretted and anti- moniuretted hydrogen on haemoglobin. According to Koschlakoff: and Bogomoloff when ammonia gas is passed into solutions of oxy-ha3moglobin or CO-haemo- globin, the solution assumes a yellow colour, the bands disappear, and are not replaced by that of reduced haemoglobin or those of haematin. Arseniuretted hydrogen, according to the same authorities, and also according to Thudichum, causes the appearance of the reduced haemoglobin spectrum when passed into solutions of 0-haemoglobin. When passed into a solution of CD-haemoglobin, this gas exerts a similar action to NH 3 and PH 3 , i.e. the entire dis- appearance of the absorption-bands. When it is passed into alkaline solutions of haematin the bands of reduced haematin appear. * ' Zeitschrift fiir Physiol. Chemie,' ii, p. 148, and Centralblatt f. Med. Wiss./ January 25th, 1879. HJ1MOCHEOMOGEN. 101 Antimoniuretted hydrogen exerts a similar action on 0-hgemoglobin and on CO-hgemoglobin.* HaBinatin and haemochromogen. Before discussing the spectra of hsematin it will be necessary to refer to a fact which was discovered by Hoppe-Seyler, and published in 1871. It was considered up to that time that by the action of acids and alkalies the hemoglobin of the blood was split up into a colouring matter hcematin, and an albuminous body globin, but it appears from Hoppe-Seyler's discovery that hsematin is not a direct product of the splitting up of haemoglobin, but results from such a decomposition accompanied by oxidation. " This oxidation takes place so rapidly, that it is only by special precautions that the non- oxidised products can be obtained. When, however, a solution of haemoglobin is reduced by hydrogen and decomposed by alcohol containing sulphuric acid or caustic potash, in an apparatus from which oxygen is completely excluded, a colouring-matter is produced, which is acid, has a purple-red colour in alkaline solu- tions, and is characterised by certain definite absorp- tion bands." This is hcemochromogen, which yields hsematin by oxidation. " It has not yet been isolated or regenerated by reduction of hsematin, but its spectrum agrees generally with that of reduced hsema- tin. Hoppe-Seyler supposes it to have the composition C 34 H 36 N 4 Fe0 5 , and represents the formation of haematin from it by the equation : 2C 34 H 36 N 4 Fe 5 + 3 == C 68 H 68 N 8 ]0 2H 2 0.t * Gamgee, ' Journ. of Anat. and Physiol.,' 1869. f Watts' ' Diet.,' 2nd supp., 1875. This equation does not show what becomes of Fe. 102 THUDICHUM' s KESEAECHES. Haematin. Although haematin lias been already re- ferred to, the consideration of its spectrum and of the methods of procuring the different kinds of hoamatin has been purposely deferred till now. The most com- plete account that I know of the various kinds of hsematin is given by Thudichum in the ' Tenth Report of the Medical Officer of the Privy Council,' which is referred to in the Appendix. I have drawn in Chart III, all the most important spectra given by that author in the plates accompanying his paper, and I have myself repeated his experiments, and thus can vouch for their accuracy. After an account of his methods and those of others, a brief summary of easy methods, by which all the important spectra of hsematin and cruentin can be procured, will be given, but it was not until after I had studied Dr Thudichum' s methods, that I was able to procure the various decomposition- products of haemoglobin, by simple and rapid processes. Judging, therefore, from my own experience, I believe his methods ought to be more generally known, and I must also take this opportunity of observing that he has not been sufficiently thanked by the members of his own profession for the great and valuable additions to our knowledge of physiological and pathological chemistry which he has made. To German chemists and to others who have made absorption spectra their study, his methods are well known, and his results have but too frequently been made to appear by some of them as if they belonged to them- selves. " Blood treated with Alcohol and Ammonia." Sp. 6 A a B C D a:- A . MacMunn. "fe cit . Hanliart litL. To fact Pay* J02 VARIETIES OF HJEMATIN. 103 Chart III, represents the spectrum of a solution of blood in alcohol and ammonia which was thus prepared. 66 Blood was mixed with twice its volume of alcohol, and the coagulum filtered from the liquid, the latter showed no spectrum bands. The coagulum dissolved in ammonia showed three bands which had a general resemblance to the sulphuretted hydrogen blood spectrum (Sp. 5, Chart III), but showed different measurements ; when to this Stokes 5 fluid was added the spectrum of reduced hematine appeared." I have also given a drawing of the spectrum, which I obtained by pouring alcohol and ammonia on to some defi- brinated cat's blood, a method somewhat different from Dr Thudichum's ; this spectrum is shown in Chart I, Sp. 14, and gave the reduced hsematin bands when treated with ammonium sulphide. " Spectral Phenomena of Hematine." "When he- matocrystalline is treated with acids, or alkalies and alcohol, it is split up into albuminous substances, and a coloured matter, which retains all the iron, but none of the sulphur of the original compound. This is hematine. It appears from my researches that there are at least three different kinds of hematine recognis- able by the spectroscope, besides a number of combi- nations which may be formed by one or perhaps all of them." " Five-banded Hematine" " Blood-corpuscles iso- lated from serum by sulphate of soda, are treated at the ordinary temperature with alcohol to which a little sulphuric acid has been added. This solution was found by me to possess five absorption bands, three of 104 VAEIETIES OF H^MATIN. them being those of acid hematine, first described by Stokes, two others being situated in red and orange, and narrow, one very fine, like a narrow bundle of sun-lines " (Chart III, Sp. 3). " Four-banded Hematine by a modified Process" " When the corpuscles are boiled in water after treat- ment with sodium sulphate, so that all sulphate is removed, and are then treated with acidified alcohol, a solution is obtained which gives a spectrum similar to the " last in three of its bands, but having one fine line in the orange instead of two. " This is identical with the spectrum of hematine first described by Stokes. It was also obtained by dissolving pure crys- tallised hematine in alcohol and a little sulphuric acid by the aid of a gentle heat " (Chart III, Sp. 2). "Blood treated with Acid" " The simple addition of acid to blood changes its spectrum. When an organic acid, such as acetic or tartaric, is taken, one band in red appears, and great obscuration of the rest of the spectrum ensues. The one band in red belongs to acid hematine, and the darkness in green is due to two other bands. These, first correctly described by Stokes, it is not easy to define without the aid of sun- light or Drummond's light. They have, therefore, not been noticed by later authors. Upon the purest specimens of acid hematine they can, however, be observed and measured with tolerable accuracy. The acid solution of hematine was hitherto believed to contain a particular body, which was termed hemine. It is, however, quite easy to show that it is hematine, for its spectrum can, by alternate acidification and ALKALINE HfflMATIN. 105 alkalification, be made to yield the bands either of acid or alkaline hematine." " Alkaline Hematine"-" In order to fully study the phenomena of hematine, I produced a quantity of it in a neutral state. A large quantity of amorphous hemato-crystalline was made from ten gallons of blood and a hundredweight of potassium carbonate. The isolated material was dried at 40 C., and extracted with cold absolute alcohol. The splendid ruby red solution was treated with a solution of tartaric acid in absolute alcohol as long as a precipitate fell down. The filtered solution was then slowly evaporated at 40 0. until it deposited all colouring matter as a fine powder of black, somewhat violet colour. This was filtered off, the powder washed with alcohol, lastly with water. It was then redissolved in absolute alcohol and potassium carbonate. Tartaric acid was again added and crystallisation completed as before. Ulti- mately a black violet powder remained, consisting, under high powers of the microscope, of little rhombic scales, mostly crossed, and imitating well the shape of the hemine crystals. I believe this to be pure crystal- lised hematine. It certainly contains no hydrochloric acid, and negatived the assumption hitherto made by some animal chemists, that all crystallised hematine was identical with hemine, and like hemine, was a hydrochlorate of hematine. " The neutral hematine is insoluble in water, alcohol, and ether, but dissolves in caustic alkaline water and in alkaline or acid alcohol. The spectrum of the acid solution is that just described." The pure hematine, 106 CRUENTIN. prepared as above, gave, wlien dissolved in alkaline alcoholic solution, one broad band covering D (see Chart III, Sp. 4). Treated with a little sulphuric acid, the spectrum became that of acid " hematine." " Reduced Hematine" " When an alkaline watery solution of hematine is mixed with a deoxydising agent, such as the alkaline tartrate solution of ammonio- sulphate of iron suboxyde," the spectrum gave the bands of Stokes' reduced hasmatin (see Chart I, Sp. 15). " Cruentine, a new derivative of Hemato-e/rystalline and of Hematine'' " When human or animal hemato- crystalline is boiled with sulphuric acid it becomes chemolysed, the albumen dissolves and yields its par- ticular products, a portion of the hematine also dis- solves and colours the fluid ruby red, while a brownish red, grumous matter remains suspended in the fluid in an insoluble state. This is a mixture of neutral cruentine with its sulphate. By washing with water, this matter loses sulphuric acid, and becomes ultimately free from it. Treated with sulphuric acid it dissolves completely, and is now sulphate of cruentine." " Cruentine Sulphate." A concentrated solution of this body gives one black band in red to orange, the blue end of the spectrum being shaded. On dilution this band splits up into two, " and a third very feeble band in green becomes visible just to disappear." Chart III, Sp. 7, shows this spectrum. " Neutral fluorescent Cruentine" " The insoluble residue from the sulphuric acid treatment is washed to neutrality and dried, a portion of it is soluble in ether CRUENTIN. 107 and chloroform. The ether solution has four bands which are a little less shaded, but nearly identical with the bands of the chloroform solution." (My own draw- ing of the spectrum of the latter is shown in Chart I, Sp. 18.) This solution of cruentine fluoresced " with a splendid blood-red colour in the sun cone. This is the first body which is known to fluoresce with homo- geneous light, that is to say, the same kind of light or colours which it transmits." " Alkaline four-banded Cruentine" The solution of cruentine in alcohol is made alkaline by ammonia, and gives the spectrum which I have figured in Chart I, Sp. 17,* from a solution which I prepared by the method I shall describe. " Neutral five-banded Cruentine in Alcohol. 99 "When the preparation from which chloroform extracts the neutral fluorescent cruentine is treated with alcohol it dissolves easily and almost entirely. The concen- trated solution allows a little red to pass. On further dilution three bands appear, ultimately five, one in red feeble, one in yellow, also feeble, both narrow, and three dense and dark bands in green." Sp. 8, Chart III, represents this spectrum. "Reduced Cruentine" This spectrum was got by adding Stokes' solution to an ammoniacal solution of cruentine in alcohol. It is shown in Sp. 9, Chart III. On acidifying this solution with sulphuric acid, a preci- pitate fell, and after filtration, the filtrate gave the bands of cruentine sulphate. " Cruentine, therefore, exhibits this peculiar property, that it can be deoxydised (in * I found five bands. 108 REACTIONS OF HJ1MATIN. alkaline) and reoxydised (in acid) solutions. During this process, however, much colouring matter is lost by changes not yet scrutinised. In its alkaline solu- tion it is deoxydised and reoxydised as easily as hematocrystalline. It is a most remarkable fact in science that a decomposition product of hemato- crystalline of the second order retains what I will term the breathing power of the blood-corpuscle." "Cruentine and Hydrochloric Acid." " The chloro- form solution treated with HOI and water becomes turbid. Warmed, it clears up and appears more rose coloured. On cooling it becomes, however, again turbid. Its spectrum shows three bands," which are figured in Chart I, Sp. 19. These are the principal blood spectra described by Thudichum. Other observers call by different names the products which he describes. General account of haematin and its reactions. The foregoing account of the methods of Thudichum, has shown, how the substances which have been called by English observers alkaline and acid hsematin, and that called by the author just quoted cruentine, can be prepared, but what he has said is not sufficient to enable any one who has not read the subject before to understand it thoroughly, so that I shall give a short account of what authorities say upon this matter. A short repetition of what has been said before may be pardoned, as it is unavoidable.* * Thudichum holds opinions exclusively his own on hsematin and cruentin, and he does not agree with Preyer or Hoppe-Seyler in their views as to the composition of heematin. REACTIONS OF HJ1MATIN. 109 Hgematin was at one time supposed to be the colouring matter of the blood, but Hoppe-Seyler showed that it does not exist preformed in that fluid, but is produced, together with globin, an albuminous body, by the action of acids and alkalies on the hemo- globin of the blood.* It may, according to this authority, be obtained pure by dissolving its hydro- chloride (ha3min) in ammonia, evaporating to dryness, heating the residue to 130, dissolving out the ammo- nium chloride by water, and again heating to 130. It may also be obtained by mixing defibrinated blood with a strong solution of potassium carbonate until the liquid adhering to the separated coagulum becomes colourless, drying the coagulum at a temperature not above 50 0., and digesting it for some days with absolute alcohol in a close vessel at a moderate tem- perature (below 50). The red liquid, when filtered, is an alcoholic solution of hgematin. In acid liquids the spectrum is a four-banded one, if an alcoholic or etherial solution be used, and differs in no essential respect from that which is got by merely acting upon blood with acetic acid and shaking up with ether, which I shall describe. The brown solution of haematin in potash or potas- sium cyanide exhibits least absorption of light near C ; on diluting this solution there remains a band between D and E, but nearer to D, which, however, disappears while the solution still exhibits a strong colour. According to Nawrocki an alkaline solution of * Hsemochromogen, an intermediate product being first formed. See p. 101. 110 IRON-FREE HSEMATIN. hasmatin (haamin crystals) dissolved in ammonia gives a spectrum with a broad band between C and D, but after treatment with a ferrous salt, or with stannous chloride, it shows two other bands, which do not disappear on agitation with air, and are likewise visible for some time in the red etherial solutions obtained by mixing the ammoniacal liquid with ether and glacial acetic acid, but in this they merge into the three bands of the normal has matin solution. If, on the other hand, the alkaline solution of hsematin be mixed with ammonium sulphide, the liquid exhibits the same bands as haemoglobin when similarly treated, and no longer yields up anything to ether on addition of acetic acid (?). " Hsematin or hsemin heated for some time with ammonia, or a fixed alkali, is converted into a body, the solution of which in acidulated alcohol, or in an alkali, has a dingy olive-green colour, dark red in thick layers, and after treatment with reducing agents does not exhibit the spectrum of reduced hasmatin, neither can haemin crystals be obtained from it."* Iron-free hsematin. " By dissolving haamin in strong sulphuric acid, and adding water to the solution, a substance is precipitated resembling hasmatin, but not containing iron ; it is soluble in alkalies. The solution of this non-ferruginous haematin in strong sulphuric acid absorbs blue and violet light strongly; on diluting it with sulphuric acid a very dark, well-defined band appears about midway between D and E, and a narrow band between C and D (near D), the spectrum being * Hoppe-Seyler, quoted in Watts' ' Dictionary.' HJSMATOPOBPHTBIK. Ill very darkly shaded between D and the dark absorp- tion-band. The solution of non-ferruginous hsematin in dilute ammonia exhibits the smallest absorption for red light. On diluting with water a band appears between and D, and on further dilution three others. Reducing agents alter this fluid in the same manner as ordinary hsematin."* It is quite evident that the body whose spectrum is thus described is the same as the substance called by Thudichum " cruentine," which the latter author has shown to contain 1*51 per cent, of iron. On the other hand, Foster states that "by the action of sulphuric acid hsematin may be robbed of all its iron." Preyer maintain sf that the body which goes into solution when blood is treated with acetic acid and shaken with ether, which Stokes called acid hsematin, is really free from iron, and he calls it heematoin. But, if acid haematin is free from iron, so also ought alkaline haematin to be, as I have found that there are some acids which when added to blood give a four-banded spectrum, that can be made to give the same spectrum as that yielded by alkaline hse- matin on the addition of reducing agents. { The long names hcematoporpkyrin and hcematolin have been proposed for bodies, the former of which appears to be practically identical with cruentin, from the descrip- tion of its spectrum by Hoppe-Seyler. It is got by filtering a solution of haematin in oil of vitriol through * Hoppe-Seyler, 1865. f ' Die Blutkrystalle,' p. 181. J This, however, is merely a spectroscopic reason. ' Med. Chem. Unter.,' 523, 1871. 112 asbestos, when a fine purple-red solution is obtained, which gives a small, dark absorption-band just before the line D, and another sharply-defined band between D and E. When this solution is mixed with water the greater part is precipitated, the precipitation being increased by the addition of alkalies to neutralisation. The alkaline aqueous solutions give a faint band between C and D, another faint baud between D and E, nearer D, a dark band in the same interval near E, and a dark band between b and F. This substance, hcematoporpkyrin, is free from iron, and gives by analysis 68'42 p. c. C., 9*58 N., 6'07 H., and 15*93 0. Its formation is represented by the equation : C 68 H 70 N 8 Fe 2 10 +0 3 +2H 2 S0 4 =C 68 H 74 N 8 13 +2FeS0 4 . "When, on the other hand, haematin is acted on by sulphuric acid in closed vessels, hsematolin, C 68 H 78 N" 8 7 , is formed, which is but very slightly soluble in sul- phuric acid, and very slightly soluble in caustic potash. Hsematin hydrochloride. In describing the process by which Hoppe-Seyler recommends hgematin to be procured, hsemin was incidentally mentioned. This sub- stance has been known under the name of Teichmann's crystals, and has assigned to it the formula C 96 H 142 Fe 3 O 18 . It is obtained in regular crystals by treating haemoglobin or methsemoglobin with common salt and glacial acetic acid. The crystals are rhombic or six- sided plates, dark blue by reflected, dirty brown by transmitted, light; insoluble in water, alcohol, and ether ; soluble in acids and alkalies, but decomposed by all acids except acetic and hydrochloric. 113 It may be prepared as follows : Defibrinated blood dried at the ordinary temperature, or blood clot cut up and dried, is powdered in a mortar with one fifth part pure carbonate of potassium, and the dried mass digested with alcohol of 94 per cent, at 40 45 until the resulting dark, garnet-coloured solution no longer becomes darker in colour. The solution is filtered; the residue again treated with alcohol; the united extracts mixed with rather more than an equal volume of water, and then with enough acetic acid to produce a slightly acid reaction. The brown flocculent precipitate which is produced is col- lected on a filter and dried slowly, the heat being finally raised to 100; it is triturated with one fifth part sodium chloride and from twenty to thirty parts glacial acetic acid, and the mixture digested for some time at 60 until a crystalline mass separates. The whole is heated to 100 and left to cool ; the crystals are then washed on a filter with warm glacial acetic acid, pressed, dried, and again boiled with water (J. Gwosden). Hoppe-Seyler has modified this process; he causes a coagulum to separate from the blood by pouring it into alcohol or boiling water ; the clot, separated by filtration, and still moist, is warmed with alcohol to which a few drops of strong sulphuric acid have been added; the filtered brown solution is mixed with a warm, saturated solution of sodium acetate, then 'im- mediately neutralised with sodium carbonate, and, in order to separate the hsemin, if this has not already separated, it is mixed with water, or freed from alcohol 8 114 ACTION OF ACIDS ON HAEMOGLOBIN. by distillation. The precipitate, after being washed on a filter and dried in the air, is then ready for treat- ment with common salt and acetic acid, as in the former method. Although this substance is of no interest directly to the spectroscopist, it is indirectly, as it has been used to prepare pure haematin, accord- ingly I considered that its mode of preparation ought to be mentioned. The exact amount of sodium carbonate required to convert haemoglobin into haematin.-^When a small quantity of sodium carbonate is added to a solution of haemoglobin, no coagulation takes place, even after the fluid has been heated to 100 0. At 54 C. the sub- stance is obviously decomposed, as this solution be- comes of a dark, brown-red colour, and examined with the spectroscope it exhibits the spectrum of haematin in alkaline solution, instead of that of oxyhsemoglobin. The fluid is alkaline and remains clear after boiling. At the temperature at which the decomposition takes place, it is probable that haemoglobin splits up into haematin and albumen, and Preyer calculated the amount of sodium carbonate which had to be added in order to prevent the coagulation by heat ; the mean of two observations showed that one gramme of haemo- globin in distilled water required 0'0238 gramme of the carbonate. Accordingly, he concludes that one mole- cule reacts on three molecules of haemoglobin in order to produce the non-coagulating compound (Gamgee). The action of various acids on haemoglobin in pro- ducing haematin. Preyer has studied the action of the following acids on haemoglobin : CARBONIC OXIDE AND HJIMATIN, 115 Phosphoric. Phosphorous. Sulphurous. Oxalic. Monochloracetic. Phosphomolybdic. Gallic. Nitric. Pyrogallic. Acetic. Formic. Butyric. Propionic. Metaphosphoric. Benzoic. Hydrochloric. Hippuric. Carbonic. Lactic. Citric. Tartaric. Malic. Succinic. Carbolic. Uric. Sulphuric. Chromic. All act somewhat similarly, but some of them preci- pitate the haemoglobin, and upon the occurrence of a precipitate and its character Preyer bases a classifica- tion of their action. The action of alkalies and alka- line solutions is more uniformly similar than that of the acids. Action of carbonic oxide on hsematin. Dr L. Popoff has studied the action of carbonic oxide on solutions of haematin, and has arrived at the following conclu- sions : (1) Carbonic oxide causes no change in acid or alka- line solutions of haematin when passed through them. (2) If, however, a reducing agent had been added at the same time that the carbonic oxide was being passed through the solution, a new compound was formed, which, in the case of an ammoniacal solution of haematin, was deposited in the form of a floccu- lent red precipitate. (3) The spectrum of this consists of two bands similar to, and identical with, those of reduced haematin. 116 H^EMATIN AND PHOSPHOROUS CHLORIDE. Action of tin and hydrochloric acid on hsematin. Tin and hydrochloric acid act on haematin in the pre- sence of alcohol differently, according as the hsematin is fully dissolved or not. When a concentrated solu- tion, or one containing excess of hgematin, is heated with tin, copper, or zinc, on the water bath, a purple- red colour is produced, and after a time a resinous, dark violet precipitate forms*. The solution gives two dark bands between D and E. Haematin, or haemin crystals, dissolved in alcohol containing sulphuric acid, when decomposed by hydrochloric acid and tin and heated, give a purple solution, which has a character- istic spectrum. One band between D and E, another before D, but close to it, and a broad band between b and F, covering the latter line. HaBxnatin treated with phosphorous chloride con- taining free phosphorus. When haemin crystals are heated with this substance to a temperature of 104 in closed tubes for from six to eight hours, a purple- brown fluid is obtained, which gives three absorption bands, one between and D, close to C, another between D and E, near E, and a third between b and F. No gas escapes on opening the tube, but a crust forms on the sides of the tube, which is easily sepa- rated. Part of this is soluble in water, and the aqueous solution gives the same spectrum as haemato- porphyrin. From that part which is insoluble in water a substance can be got resembling haematin, but containing phosphorus. The agreement of the spec- trum of this compound with that of haematoporphyrin seems to show that the latter consists of AMMONIA GAS, ETC., AND HyEMATIN. 117 C 68 H 70 N 8 10 .2H a O, or that it is a hydrate of the same molecule as that contained in the phosphorus compound (Hoppe- Seyler). Action of ammonia, arsine, and stibine on haematin. Ammonia (gas) colours alkaline solutions of haematin orange, and the absorption bands become paler ; a broad but diffuse shadow appears in the green part of the spectrum, and after a little time an amorphous precipitate forms. This precipitate dissolves in acetic acid, and the solution gives the spectrum of acid hsematin. When arsine is passed into alkaline solu- tions of haematin, the colour of the liquid gradually becomes red, and the bands of reduced hsematin appear. Shaking with air restores the colour of the alkaline hsematin, but after a few seconds the solution begins quickly to redden, and this alternation -may be repeated about ten times. If the arsine be passed for a longer time, the alkaline solution turns brown, and may deposit next day crystals of arsenic of a steel-grey colour. After this time no more bands are seen; nevertheless, the presence of hsematin may be demon- strated by means of reducing agents. The bands of reduced haematin may be recognised at a much greater degree of dilution than those of the alkaline or of the acid solution, a fact which seems to show that, in spite of the action of the arsine, part of the haematin has remained undecomposed. The action of stibine is the same as that of arsine (Koschlakoff). 118 ALKALINE H^E MATIN. CHAPTER VI. EASY METHODS OF PEEPAEING THE MOST IMPOETANT OF THE SPECTEA WHICH HAVE "BEEN HITHEETO DESCEIBED ; AND APPLICATION OF THESE METHODS. ANY one reading the last chapter might be led to suppose that the study of the blood-spectra is an exceedingly difficult one, but such is not the case ; and I shall now describe how all the spectra of import- ance can be prepared by very rapid and very simple processes ; and, in addition, other spectra will be mentioned which have not been hitherto described. Oxidized and deoxidized haemoglobin have already been mentioned, so that their consideration need not be repeated. Alkaline haematin. Make a saturated solution of carbonate of potash in alcohol, and pour a few drops of blood into the solution, the colour of the blood imme- diately changes, and when examined the spectrum shown in Chart I, Sp. 13 is seen, abroad, lightly-shaded band covering D. The action of caustic alkalies on de- fibrinated fresh cat blood was found to give different spectra from those which are often described, thus : (1) Caustic potash and caustic soda gave, in alcoholic solution, when added to blood, each the same spec- trum, but different, in some respects, from that got from ammonic hydrate and from carbonate of potash. REDUCED H2EMATIN. 119 It consisted of three bands, two of which were like the blood bands, but that the product giving this spec- trum was hsematin was proved on adding a reducing agent, as the spectrum of reduced hasmatin now appeared.* (2) An ammoniacal solution of alcohol was now added to a few drops of blood (the same blood as before) when a faint band in red, and a dark band nearer violet appeared. On further dilution the latter was seen to be composed of two, and on adding a reducing agent the bands of reduced haematin appeared (see Sp. 14, Chart I). The feeble band in red has not been generally noticed, and I believe the reason of this is, that observers generally work at old blood in which the haemoglobin has been reduced.! I often found a great discrepancy arise when the same experiment was performed respectively on old pig blood, or old ox blood, and fresh blood removed from the body imme- diately after death. Reduced hsematin. This can be got by adding Stokes' fluid, or ammonium sulphide, to the solution of blood treated by alcohol and carbonate of potash, or by the alcoholic solution of caustic soda, of caustic potash, or of ammonia; but if an alkali alone be added to blood previously, the reducing agent will not develop this spectrum. The reduced hsematin spectrum can be also got from the haBmatin formed by the action of some acids on haemoglobin, thus, from that got by acting on blood * This spectrum is so easily procured that it is unnecessary to give a map of it. f The band in red is only seen with the microspectroscope. 120 ACTION OF BROMINE. with salicylic acid and dissolving the hgematin formed in alcohol, we can, by adding ammonium sulphide, get the spectrum of reduced hasmatin ; in the same way the alcoholic solution of blood treated by lactic acid can be made to yield the spectrum of reduced hsematin. Sp. 15, Chart I, is that of reduced hsematin. . Acid hsematin. Aftd a few drops of acetic acid to blood and shake with ether ; the brown-red etherial solution gives four well-marked bands. This spec- trum is shown in Chart I, Sp. 12. The feeble band near D is with difficulty seen at that degree of dilution, which shows the bands in green to best advantage. In examining with the microspectroscope, it is neces- sary to narrow the slit sufficiently and focus carefully, and in working with the chemical spectroscope it is a good plan to move the telescope from side to side, when a faint shadow, in the position of this feeble band, will be seen to move across the field of view. By looking obliquely, too, feeble bands are sometimes seen with the latter instrument which would otherwise be missed. Action of bromine on blood. This spectrum was first described by me in the 'Dublin Journal,' June, 1877. (1) Ox blood treated with bromine and shaken with alcohol gives four absorption bands, which are practi- cally identical with those of acid haematin. (2) Ammonium sulphide added to this gives, not only the bands of reduced hasmatin, but a third feeble band in orange, close upon D (see Chart I, Sp. 24). (3) If ammonia be added to the first solution it develops a band at D, like that of alkaline hgematin. CBUENTIN SULPHATE. 121 (4) Ammonium sulphide added to this develops the same bands as in (2). (5) A solution of blood treated with an aqueous solution of bromine gives a band just like alkaline ha3matin, or that of (3). (Chart I, Sp. 23.) (6) When ammonium sulphide is added to this it gives exactly the same spectrum as that got in (2) and (4). Thus, bromine teaches an important fact, viz. that a different kind of haematin is produced according to the amount of chemical action on hasmoglobin. It would appear from its action that alkaline hsernatin is a body which is produced from haemoglobin by a less complete " splitting up " than in the case of acid hasmatin. This form of haematin is, however, dif- ferent from that of ordinary haematin, as it gives three bands, instead of two, on the addition of re- ducing agents. The compound is insoluble in chloro- form, sparingly, or not at all, in ether, slightly in absolute alcohol, but more freely in rectified spirit. Action of iodine. Added to blood an aqueous solu- tion (with iodide of potassium) produced a precipitate, and the supernatant fluid gave no spectrum, although there was evidently a little haematm present in solu- tion, since the addition of ammonium sulphide gave the bands of reduced haBmatin. The precipitate, when dissolved in alcohol, gave no spectrum. Chlorine seems to act much in the same manner, by forming an insoluble form of haematin. Sulphate of cruentin. Boil defibrinated blood with strong sulphuric acid, add water and filter, wash the mass on the filter until the washings are neutral to 122 METH^MOGLOBIN. test paper, and dry. This can be used for the prepa- ration of all those kinds of cruentin which Dr Thudi- chum describes. Dissolve some in sulphuric acid, it gives a fine ruby-red colour, giving two absorption bands; this is the spectrum of sulphate of cruentin (Sp. 16, Chart I). Alkaline cruentin. Dissolve some of the neutral dried precipitate of the last experiment in alcohol and ammonia when a red fluid will be obtained, giving five (or four) bands (Sp. 17, Chart I). Neutral cruentin. Dissolve some of the neutral dried precipitate in chloroform, this gives four bands, but differing in their positions from those of alkaline cruentin (Sp. 18, Chart I). Hydrochloric product of neutral cruentin. Add hydrochloric acid to the last fluid it becomes turbid, heat, the turbidity disappears ; it is now of a purplish colour, and gives three bands (Sp. 19, Chart I). Reduced cruentin. Add ammonium sulphide to the alkaline solution of cruentin ; this solution gives three bands, which are shown in Chart I, Sp. 20. It is noticeable that the darkest band of this spectrum is nearly in the same place as the first reduced ha3inatin band. Methaemoglobin. Add a solution of permanganate of potassium to a solution of blood ; notice the posi- tion of the band in red. This is the only band of importance; it is shown in Sp. 10, Chart I, in which the other bands are also seen. Dilute and add ammo- nium sulphide; notice the band of reduced hemo- globin. PATHOLOGICAL APPLICATIONS. 123 Sulphaemoglobin. Pass sulphuretted hydrogen for some time through a solution of blood; in this case also a band in red is developed, but there is also a broad dark band in the position of that of reduced hemoglobin. (See Chart I, Sp. 8.)* Add ammo- nium sulphide, and notice that the band in red per- sists, thus presenting a contrast to methaemoglobin, in which the band in red disappeared. Cyanhaematin. -Add a solution of cyanide of potas- sium to a solution of blood and heat gently for some time ; the blood bands disappear and a band is deve- loped which has been already described, p. 85. (See Chart I, Sp. 21.) Add a reducing agent, and notice the appearance of two other bands nearly in the position of the blood bands, but nearer the violet (Chart I, Sp. 22).t CO Haemoglobin. Pass coal gas through a solution of blood for some time, the solution becomes red and looks clearer than before; examine with the spectro- scope, two bands are seen nearer the violet than the blood bands. Add ammonium sulphide, no effect is produced (Chart I, Sp. 7). Pathological application of the study of the spectra of haemoglobin and of haematin. Having learned the appearances of the various decomposition products of hemoglobin, the reader will be now in a position to * On shaking with air this band is replaced by two. (Sp. 9.) f Having repeated these experiments several times I can promise that, if any one will take the trouble to repeat them for himself, he will have no difficulty in obtaining the spectra which are shown in Chart I ; and, indeed, without actually performing them for himself, he will not be in a position to draw any inferences from the spectra of pathological fluids. 124 BLOOD IN URINE. examine pathological fluids, and by studying the action of reducing agents, to draw correct inferences by means of the spectroscope. There are certain spectra which ought to be most familiar to the medical spectro- scopist, viz. that of haemoglobin, oxidized and reduced, that of methaemoglobin, that of acid, and of alkaline and reduced hsematin. Blood which has not been kept in contact with an acid secretion for too long a time, as in haemorrhage from the bladder or urethra, gives the spectrum of oxidized haemoglobin in the majority of cases.* In haemorrhage from the kidney, when the haemor- rhage is gradual, and a result of inflammation, the spectrum of methaemoglobin will generally be seen. In haemoglobinuria, intermittent cruenturesis, or paroxysmal haematuria, or, as it has been incorrectly called, haematinuria, the spectrum is generally that of methaemoglobin. When the blood has been acted on by the acids of the gastric juice, as in various forms of haematemesis, the spectrum of methaemoglobin, with a mixture of acid haematin, will be found ; for on adding sulphide of ammonium to such fluids I have sometimes found the band of reduced haematin within that of reduced haemoglobin. If the blood has been shut up within a cyst and kept at the temperature of the body for a considerable time, it may, as in the case of ovarian and par- ovarian cysts, have been converted into a form of * Provided the urine be examined soon after removal from the bladder. BLOOD IN URINE. 125 hgematin, this can be proved by the addition of sulphide of ammonium. Blood in the urine. Blood may be present either in a soluble or insoluble state. It may readily be detected when present in the soluble condition by holding the vessel containing it between the eye and the source of light, and examining with an ordinary pocket spec- troscope ; if it contains blood two bands are visible as shown in Chart I, Sp. 4; if in the form of methsemo- globin, a band will be seen in red in addition to the other two. (If no blood be present a shadowy band may be noticed between green and blue, that of urobilin, which will be referred to again). To prove that the band in red is not due to acid hsematin, some of the urine is placed in a test-tube and a reducing agent (sulphide of ammonium) added, when if the band be due to methsenaoglobin, the spectrum of reduced haemo- globin will appear, if to acid hsematin, that of reduced hsematin. (Or the reducing agent may be added in a small cell beneath the microspectroscope.) But the blood may be present in an insoluble state ; in which case the urine (both before and after nitration) may show no absorption bands, In this case, filter the urine, digest the filtering paper with the deposit upon it in alcohol and ammonia, and examine the fluid with the spectroscope ; there may be the faintest possible shadow in the orange if there is but little blood present, but on adding ammonium sulphide, the first dark band, and occasionally the second band, of reduced hsematin at once show themselves ; shake up with air, the bands disappear, again to reappear on standing. The same 126 HJEMATIN IN OVARIAN CYSTS. direction will apply in the case of other fluids supposed to contain blood. A small blood-clot, the nature of which is doubtful, may be treated in the same way, or it may be digested in alcohol acidulated with sulphuric acid, and the spectrum of the solution examined, which will be found to be that of acid hsematin. Haematin in parovarian and ovarian cysts. Thudi- chum states that the contents of certain kinds of ovarian cysts give the spectrum of lutein, but he does not say what kind of cyst gives the spectrum, and lie does not notice the very important fact that a kind of hsematin is sometimes present in parovarian and ovarian cysts, which if it should be peculiar to these cysts, will be a great help in diagnosis. I discovered this fact, thanks to the kindness of Mr Lawson Tait, of Birmingham, who has taken the greatest trouble in sending me specimens. Three specimens of parovarian fluid, and one of ovarian out of five, gave the same spectrum ; the fifth was almost colourless and gave no spectrum whatever. The first specimen of parovarian fluid was olive brown in very thin layers, in deeper, blackish brown, and in very deep almost black, with a greenish shade. Before the slit of the chemical spectroscope, the fluid itself gave a band in red, and two in green, one of which latter was sufficiently distinct to allow of its being mapped. One band was placed between and D, nearer C, another between D and E. (See Sp. 20, Chart II.) On adding ammonium sulphide to this fluid the bands of reduced hcematin appeared at once. (The sulphide of ammonium was quite OVARIAN CYSTS. 127 pure* and its action on haemoglobin previously tested.) Acetic acid caused the band in red to become less distinct, and ammonia intensified it. ' There might have been a feeble band close to F, but this was not quite certain. f Microscopic examination, with a r^th immersion, showed crystals of cholesterin in great abundance, leucocytes, granular round bodies, very large round granular bodies which kept rolling about, no crystals of any kind beyond the cholesterin ; very few blood- corpuscles. The reaction was alkaline (due to fixed alkali). Specific gravity determined by bottle 1024'6. The fluid gave no precipitate with acetic acid, but a copious one with nitric acid, and with nitrate of silver soluble in ammonia (chlorides). It was coagulable by heat, the precipitate when burned smelling of burnt feathers. A quantitative analysis gave the following result : Water . . 90'346 Solid organic matter . 8*736 Inorganic salts . . '918 100-000 The inorganic salts consisted of chlorides, sulphates, phosphates, and carbonates of sodium, potassium, and calcium. Iron was also present, probably from the haematin. * This is a matter of the greatest importance, as disappointment will be experienced with changed sulphide. f In some specimens a fourth band close upon D appeared. Com- pare Sp. 12, Chart I. 128 OVARIAN CYSTS. The next specimen of parovarian fluid was of a brown colour with a tinge of yellow round the edges in thin layers, while it was dark brown in deeper layers. It gave exactly the same spectrum as the first specimen, both in the original condition, and on the addition of ammonium sulphide. Its reaction was faintly alkaline and its specific gravity was 1012*6. Under the microscope there was an absence of cholesterin, but the same large round granular bodies as before. No blood-corpuscles, but refracting granules were visible. It evidently contained paralbumin, since in using the test of Koeberle this was proved to be present ; i.e. a precipitate with nitric acid partially soluble in acetic acid. It gave no preci- pitate with a solution of tungstate of sodium acidulated with acetic acid. The third specimen was ovarian fluid, it was turbid, and of a dirty -brown colour ; its reaction was feebly alkaline, and specific gravity 1013. Its spectrum was identical as regards the position of the absorption bands with those of the other two specimens, and on the addition of reducing agents the spectrum of reduced haematin again appeared. A remarkable change in the spectrum, and even in the colour of this speci- men was found to have taken place after the lapse of twenty-four hours, for it then showed a band between C and E, slightly covering D, and another at F, the latter, however, being feebly marked ; it is shown in Sp. 22, Chart II. On adding to the solution giving this last spectrum ammonium sulphide, the bands of reduced haematin became visible. OVARIAN FLUIDS. 129 The microscopic and chemical characters of this speci- men were almost the same as those of the last one.* Here, then, we have a fluid which, although alkaline in reaction, yet gave the spectrum of acid hsematin, and which in the last case gave a band very like that of alkaline haematin after standing some time ; it is not impossible that while in the body the re- action was acid, but that after exposure to the air decomposition had set in and an alkaline reaction was developed, which went on increasing until the fluid was sufficiently alkaline to convert its con- tained acid hsernatin into the alkaline variety, as shewn by its changed spectrum. The examination of this fluid teaches the importance of a knowledge of the action of reagents upon haemoglobin, for no one could have imagined that acid haematin, which was supposed to be only capable of being produced out of the body by the action of acids on the blood, could have been produced actually in the body by the action of reagents furnished by the human body itself. I believe that the spectroscope will enable the contents of ovarian and parovarian cysts to be diagnosed, from other fluids resembling them, in many cases, but even if it should not of itself be able to do so, we have other corroborative tests to fall back on, such as that of Koeberle a precipitate with nitric acid which is soluble in acetic acid. I would refer the reader to the ' London Medical Eecord ' for 1876, p. 269, for an account of his researches. * Hence the spectroscope will not enable parovarian to be distin- guished from ovarian cysts. 9 130 DETECTION OF BLOOD-STAINS. The presence or absence of the band of lutein will not help the diagnosis, as serous fluids yield the spectrum of this body (see last Chapter).* The application of the study of the absorption bands of haemoglobin and haematin to Medical Jurisprudence. The detection of blood-stains. As Mr Sorby is the great authority on this subject, I here give an abstract of a paper of his which appeared in the 'Monthly Microscopical Journal' (1871, vol. vi, pp. 9 17), entitled " On some Improvements in the Spectrum Method of Detecting Blood." " There does not appear to be any probability of our being able to decide, by this means, whether blood is, or is not, human."t The spectrum microscope used in these inquiries should have a compound prism, with enough, but not too great, dispersive power, or else the bands become, as it were, diluted, and made less distinct. Cells, fyc. The cells should be made from barometer tubing, and be about one eighth of an inch in internal diameter, and half an inch long, one end being fastened to a piece of plate glass with purified gutta percha, like an ordinary cell for mounting objects in liquids. It is, however, of great advantage to insert between the plate and the cell a diaphragm of platinum foil, having a circular hole about two thirds of the internal * There is also another test for paralbumin, which, with the micro- scopic character of the deposit from these fluids, will be found described in Thomas's ' Diseases of Women/ j" Krauss (' Jahresb.,' 1861) states that hspmin crystals from human blood are different from those got from the blood of oxen, sheep, pigs, and poultry. BLOOD-STAINS. 131 diameter of the tube, fixed so that its centre corre- sponds with that of the cell. This prevents any light passing upwards that has not penetrated through the whole length of the solution, which is very important when using direct concentrated sunlight to penetrate through turbid or very opaque liquids. A small spatula made of stout platinum wire, flattened at the end, is very convenient for adding small quantities of the reagent ; and they should be stirred up in the cells with a platinum wire flattened and turned up at the end, like a small hoe. Reagents required. A diluted solution of ammonia, citric acid, double tartrate of potash and soda, the last being used to prevent the precipitation of oxide of iron, and the double sulphate of the protoxide of iron and ammonia employed to deoxidise. In some special cases dilute hydrochloric acid, purified boric acid, and sulphite of soda are required. Character of stains and action of reagents. The character of a stain varies with its age, and with the nature of the substance on which it occurs. If quite recent, and if the substance has no immediate influence on blood, the stain contains little or no colouring matter but haemoglobin. This is easily dissolved in water, and when sufficiently diluted it gives the spec- trum of oxy-haemoglobin, which, on the addition of ammonia, and of a small quantity of the double tar- trate, and then a small piece of ferrous salt, about 4^th of an inch in diameter, and stirring carefully, avoiding access of air as much as possible, changes to the spectrum of reduced haemoglobin. When stirred 132 SOEBT ON THE so as to expose the solution as much as possible to the air, the two bands again appear. On gradually adding citric acid in small quantities until the colour begins to change, these bands slowly fade away, and if there had been much blood present a band appears in the red. Wl^en previously deoxidised, this solution may be turbid, but not so as to interfere with the result. The addition of excess of ammonia makes all clear again, but does not restore the original bands, or only to a slight degree, for the haemoglobin has been changed into haematin. This reaction alone distin- guishes blood from most coloured substances, which latter, after being changed by acids, are restored by alkalies to the original state. On adding the ferrous salt to the ammoniacal solution we get the spectrum of reduced haematin (Chart I, Sp. 15), though, if the quantity of blood be small, only the first of the two bands may be seen. If too much citric acid or double tartrate had been added this solution might be turbid, but if all had been properly managed it would be quite clear. As the deoxidisation takes place slowly, espe- cially in cold weather, it is well to slightly stir up the ferrous salt at the bottom, completely fill up the cell, cover it with a piece of thin glass, remove the excess of liquid with blotting paper, and mix the solution by turning the tube upside down over and over again. On reoxidising the solution by stirring, the bands of deoxidised haematin disappear, and the two bands of haemoglobin will probably be recognised, owing to citric acid not changing the original merely into haematin, but also giving rise to some methaemoglobin. DETECTION OF BLOOD-STAINS. 133 The whole of these facts may be seen with a single cell containing about ro^th of a grain of blood. Very faint bands are best seen by lamplight. Exposed to the air in a damp place, a blood-stain may be com- pletely decomposed by the growth of mould, but when not thus destroyed, it is partly changed into haematin. If it had been kept dry the hemoglobin has become changed into a variable mixture of methaemoglobin, haematin, and a brown substance not yet much studied. This change takes place more rapidly in the acid atmosphere of towns and houses, especially when gas is burned, than in the open country ; but it does occur even in the purest air, and in glass tubes herme- tically sealed. The presence of a weak acid in perspi- ration may also cause a stain on a worn garment to be completely changed in a very short time, and the pre- sence of a stronger acid on dirty clothes may at once alter the haemoglobin into haematin. On digesting a stain in which all the hsemoglobin has changed into methaemoglobin from being kept a long time, the methaemoglobin dissolves. When the solution is sufficiently strong, this shows a band in red, and two fainter bands in green. The addition of ammonia removes that in red, but makes those in green much darker, and develops a special, very narrow band in orange. Deoxidised, this gives the spectrum of reduced haemoglobin. Since methaemo- globin is formed at once from haemoglobin by the action of a great number of different oxidising agents, and since it can be reconverted into oxidised haemo- globin by slight deoxidation, Mr Sorby is inclined to 134 SOEBY ON THE look upon it as a peculiar oxidised modification. On adding a little of the double tartrate and of the ferrous salt to even a dilute solution from an old stain, the niethgemoglobin is deoxidised, and the well-marked spectrum of fresh blood can be seen. If left too long, the spectrum of deoxidised haemoglobin is developed, but on irell stirring, that of the oxidised reappears, and the various other spectra may be obtained as described above. That part of the stain insoluble in water, which is chiefly haematin, may be dissolved in dilute citric acid or ammonia, and when deoxidised the spectrum seen to even greater advantage than when fresh blood is employed, because there is no general shading in the green due to there having been met- hsemoglobin mixed with the haematin. We may thus obtain an excellent spectrum from a blood-stain nearly fifty years old. In very old stains all the methaemo- globin has disappeared, and sometimes even a con- siderable part of the haematin has been altered into another brown colouring matter, which does not give any well-marked spectrum. When a blood- stain has been made sufficiently hot to coagulate the albumen, neither water, citric acid, nor cold ammonia will dissolve it, but by heating in dilute ammonia the haematin is easily dissolved, and may be detected either before or after concentrating the solution by evaporation. The spectrum of deoxi- dised hsematin can in no way be better seen than by deoxidising a solution of fresh blood that has been boiled with dilute ammonia, which gives rise to a very pure haematin. DETECTION OF BLOOD-STAINS. 135 Examination of the stain. In applying these prin- ciples to the detection of suspected stains, it is desirable, in the first place, to examine a portion of the unstained fabric, to ascertain whether any colour is dissolved from the fabric by dilute citric acid or dilute ammonia, and if so, to determine whether this would in any way interfere with the recognition of blood by the processes described above. In the case of scarlet cloth, and of some other red fabrics, much colour is dissolved out by ammonia, but not by citric acid, which ought, therefore, to be used ; whereas, in other cases, am- monia is the best solvent. Unless the stain be faint, a portion should be soaked in a few drops of water in a watch-glass, the liquid squeezed out, allowed to stand a short time in the glass, so as to deposit any small portion of the fabric, and poured into one of the experiment cells. If the stain had been recently made, and had not been changed by any special action, a solution of haemoglobin would be obtained, and the various spectra could be seen one after the other, as already described. If, however, the stain were a few days, or a few weeks old, we should obtain a mixture of haemoglobin and methaBmoglobin, or the latter alone. The various spectra could then be developed, and compared, side by side, with those from fresh blood, to be sure that there is complete correspondence in the position and relative intensity of the bands. The residue insoluble in water should then be dissolved in dilute citric acid or ammonia, according to the nature of the fabric, and the spec- trum of deoxidised hsematin developed. If insoluble 136 SORBY ON THE in cold citric acid or ammonia, hot ammonia should be tried, since the stain might have been so heated as to coagulate the albumen. If it be desirable to keep the specimen of deoxidised haematin for subsequent refer- ence, the cell may be covered with a piece of thin glass, and after removing the excess of liquid, the edge of 1^he cover painted round with gold size. When properly managed, such an object will show a per- fectly good spectrum, even after many weeks. The most important absorption spectra in detecting blood stains. If, therefore, we have a sufficient amount of a moderately old stain, we may easily see, in suc- cession, the seven very different spectra of the follow- ing solutions : (1) Neutral methgemoglobin ; (2) alka- line methsemoglobin ; (3) deoxidised methsemoglobin ; (4) oxidised haemoglobin ; (5) acid hasmatin ; (6) alka- line hasmatin ; (7) deoxidised hsematin. If the amount was very small, only (4) and (7) would show distinct bands, and the rest would be characterised rather by their comparative absence ; and it must always be borne in mind that (1) and (2) may be modified by the presence of unaltered haemoglobin, (3) by that of dis- solved haematin, and (5), (6), and (7) by that of unde- eornposed haemoglobin or methaemoglobin. Mr Sorby considers that these spectra afford as satisfactory a test for blood as could be desired. Examination of faint blood-stains. The foregoing directions apply to simple cases, when there is enough material at command, and when the fabric on which the stain is found does not contain anything which makes the blood insoluble, or interferes with the various DETECTION OF BLOOD-STAINS. 137 tests. If the stain be very faint, from the presence of but little blood, or from partial removal by washing, it might be necessary to examine the whole at once. In this case the stained portion should be digested in a few drops of dilute citric acid or ammonia and the presence of hsematin determined, as already described. If faint and spread over a considerable surface, it might be well to digest in citric acid or ammonia diluted with much more water than would fill the experiment cell, and the solution afterwards concen- trated by gentle evaporation. By this means blood could be detected, even when considerable effort had been made to remove it, and only a faint brown tinge left, just visible on white linen. There would generally be no difficulty in the case of a stain on cloth which had been sponged, for enough blood solution would be left in the fabric. The effect of mordants in the detection of blood-stains. The presence of mordants in cloth or prints may require a modification of these proceedings, especially if the stains had been wetted, and to a great extent removed, so that we have only the dried-up solution of blood, thoroughly incorporated with the mordant. In the case of a piece of brown cloth, portions of which with a wetted stain were sent by Mr Sorby to a number of the highest authorities who pronounced it impos- sible to recognise blood on it, he found that after the lapse of six years, the presence of blood could be detected, by digesting a portion of the cloth in dilute ammonia and squeezing it over and over again with a pair of forceps, and finally with the finger and thumb, 138 SORBY ON THE so as to obtain as much of the solution as possible. This solution was very turbid, but when deoxidised in the usual manner, and illuminated by concentrated light direct from the sun itself, the band of deoxidised hsematin was quite distinct. When the cell was kept for some time, so that the insoluble part settled to the side, no T^and was visible, and therefore the hsematin was evidently combined with the mordant. He there- fore recommends the solution in such cases to be examined with a sufficiently strong light, and the sediment not to be allowed time to settle. If the sun could not be made use of, the lime or electric light would, no doubt, be the best substitute. Effect of vegetable soil on blood- colouring matter. When fresh blood solution is agitated in a test-tube with vegetable soil, and left until quite clear, the colouring matter is completely carried down with the earth. Dilute ammonia, however, dissolves out haematin, and therefore, in testing portions of soil, they should be digested in considerably more of that solvent than will fill an experiment cell, and after the solution has become quite clear it should be concentrated by eva- poration. The spectrum of deoxidised hsematin may then be seen by following the ordinary method. The same process should be adopted in examining stains on clothes impregnated with earth or earthy dust, and marks on iron contaminated with much rust, if water will not dissolve out the unaltered blood or methsemo- globin. Detection of blood-stains on leather. The importance of being able to detect blood-stains on leather was pro- DETECTION OF BLOOD-STAINS. * 139 minently brought before Mr Sorby by a case in which the trial of a suspected person depended on the nature of certain dark marks on his gaiters. The presence of tannic acid so completely mordants the blood, that neither water nor citric acid will dissolve it, and ammonia gives rise to a most inconveniently dark solution. If the stain is on the surface, and has never been wetted, a thin shaving should be cut off, so as to have as much blood, and as little leather as possible, and the blood should be dissolved off without exposing the solution to the action of the leather itself. This may be accomplished by taking one of the experiment cells, nearly filled with water, bending the shaving, and inserting it into the upper part of the water, so as to touch the water, being careful to arrange it so that the stain may be on the convex side of the leather, and in contact with the water. When a drop of blood falls on leather, many red globules are filtered out from the serum and left on the surface, and when thus treated, they dissolve, and the coloured solution sinks at once to the bottom of the cell, without coming into contact with the leather. The various spectra may then be observed in the usual manner. This method would be of little or no use if the stains had been wetted, and for a long time Mr Sorby concluded that after such treatment it would be impossible to recognise blood. However, after many experiments, and after having again and again almost given up the inquiry in despair, he found that the difficulty could be over- come in a very simple manner. The best solvent for the insoluble compound of the colouring matter of the 140 SORBY ON THE blood with tannic acid, is hydrochloric acid diluted with about fifty times its bulk of water. If stronger or weaker the result is not so good. When a portion of unstained common brown leather is digested in this dilute acid, the solution is scarcely tinged yellow. On adding excess of ammonia, the colour becomes pale purple, or neutral tint, made deeper when the double tartrate and the ferrous salt are added, but remaining nearly clear. This gives a spectrum very dull all over, but without any trace of definite bands in any part. The depth of colour varies much with different speci- mens of leather. A portion of similar material soaked with wetted blood gives a yellow solution, made brown- purple and turbid by the double tartrate and ammonia, and remains so when deoxidised. The. band of de- oxidised haematin can, however, be distinctly seen with a light sufficiently strong to penetrate the turbid and dark solution. Before examining the suspected stain, it would be well to make out how much of the unstained leather could be used without giving too dark a solution, and to use no more of the stain. If the deoxidised solution be too turbid, the cell may be kept for a while horizontal, until the deposit has subsided sufficiently to allow the principal absorption-band to be seen ; but it is not so distinct, when all has subsided, as though the greater part of the haematin still existed as a com- pound insoluble in dilute ammonia. The presence of tannic acid in wood and other sub- stances might make it necessary to employ a similar process, if the relative amount of blood be so small DETECTION OF BLOOD-STAINS. 141 that none could be dissolved out by water, or dilute citric acid. Precautions necessary when other coloured matters besides blood are present. Cases might occur when it would be necessary to decide whether blood were present, along with some other coloured substance soluble in water. The method to be employed would depend much on the nature of this impurity. If it were a colouring matter belonging to those pigments in which the absorption is removed by sulphite of soda, in an alkaline solution, there would be no difficulty in seeing all the spectra. Thus, for example, it is easy to add so much magenta to the solution of a little blood that its absorption bands are entirely hidden ; but a small quantity of sulphite of soda so completely removes the colour of the magenta, that the various spectra of the blood may be seen almost as well as if it had been pure. If the colouring matters are those of fruits, the presence of free acid would be almost certain to have changed the haemoglobin into haematin. The best plan would then be to add excess of ammonia, and, if the solution was made too dark, to dilute it with so much water that the strongest light at our command would show the green part of the spectrum sufficiently bright to prove that no absorption band occurred there. On deoxidising in the usual manner the solution may be made somewhat darker by the presence of tannic acid, but the dark band of deoxidised hgematin could be recognised without material diffi- culty. Cochineal is a colouring matter thajLre^uires special ELIB Of THE UNIVERSITY 142 SORBY ON THE attention. The addition of ammonia to its solution in water gives rise to two bands in the green, which though differing materially from those of blood, are yet so nearly in the same situation, that they com- pletely disguise the presence of a small amount of blood. However, on adding a small excess of boric acid, the bands of the cochineal are made more faint, and very considerably raised towards the blue end, so as to leave the red end of the green clear, whilst those of oxy -haemoglobin are not changed, and by that means the red end, if not both, can be seen perfectly well. By proceeding in the usual manner there is no great difficulty in recognising the darker band of deoxidised hsematin. Other difficulties which may occur. We need never despair of detecting blood so long as any haematin remains undecomposed. Fortunately it resists decom- position so well, that this would rarely happen in ordinary circumstances; but yet there are cases in which it does occur, as, for example, when acted on by strong ozone, or other powerful oxidising reagents. It is quite possible that stained garments might have been washed, and some of the water employed might be obtained. If no soap had been used, this water could be examined in a long tube of thick glass, ten inches or more in length, and a quarter of an inch in internal diameter, permanently closed at one end with a circular piece of plate glass, and, when filled, covered over at the other with another glass. A pocket spec- troscope is the best instrument for using in this examination, such as that made by Mr Browning. If DETECTION OF BLOOD-STAINS. 143 only two or three days old, tlie bands of oxidised haemoglobin might be seen ; but if the solution had been kept longer, and these bands could not be detected, it should be concentrated by evaporation at a gentle heat, and tested for ha3matin. If during evaporation any deposit be formed, insoluble in cold dilute am- monia, it should be dissolved by the aid of heat. When soap has been used to wash off a stain, the alkali of the soap has converted the haemoglobin into hsematin, and the soap has made the solution inconveniently turbid and opaque. It is best in such a case to agitate the suspected soap and water with ether, remove it with a pipette, after the two liquids have completely separated, and repeat the process over and over again with fresh ether, until the aqueous solution at the bottom has become quite clear and free from soap. It should then be concentrated by evaporation, and exa- mined for haematin, as usual. Of course, in such cases it would be desirable to test the solution as soon as possible, lest decomposition should occur; but by these means a very small quantity of blood, that would show no colour, might be recognised within a week or two, but probably not after.* Richardson's method of detecting blood-stains. In the c Monthly Microscopical Journal' for 1876, vol. xv, pp. 30 32, Dr Joseph Gr. Richardson, of Pennsylvania, describes his method of detecting blood * In the foregoing pages I have given almost the exact words of Mr Sorby, changing the sentences slightly here and there for the sake of brevity ; and I have also divided the paper under separate headings, so as to facilitate reference. 144 RICHARDSON'S METHOD. in medico-legal cases. He says : " The value to medi- cal jurisprudence of spectrum analysis, as employed for the detection of dried blood, is so fully established by the researches of H. G. Sorby, Dr W. B. Herepath, Professor A. S. Taylor, W. Preyer, and others, that it seems unnecessary for me to do more than state that the demonstration of the two dark bands in the green, caused by scarlet cruorine (haemoglobin), such as that contained in a recent blood stain, enables experts to discriminate positively blood from other red colouring matters soluble in water, whether mineral, vegetable, or animal, except an extract of the red feathers from the Turacus Albocristatus, a bird found in the East Indies, and quite unknown on our continent of America. Valuable as this test is thus seen to be, there are, unfortunately, several circumstances which limit its general application, as, for example, the changes in the constitution of haemoglobin which occur from prolonged, and frequently from compara- tively brief, exposure to the air, the modification of the absorption bands caused by the presence of other substances, and last, but not least in many instances, the difficulty of procuring sufficient material for ex- periment. The insuperable nature of this latter obstacle will be at once appreciated when I mention that whilst the smallest amount which Sorby, Here- path, and Taylor furnish directions for is a spot ' one tenth of an inch in diameter, or a quantity of the red colouring matter amounting to no more than one thousandth part of a grain/ the important stain upon an axe handle, supposed to have been used in a murder, EICHARDSON'S METHOD. 145 I am now investigating, probably weighed less than one three thousandth of a grain when entire and un- injured." Dr Richardson then goes on to describe the method he adopted which enabled him to reveal the presence of blood in a quantity of matter " one hundredth the amount directed by Mr Sorby." " Procure," he says, " a glass slide with a circular excavation in the middle, called by dealers ' a concave centre/ and moisten it around the edges of the cavity with a small drop of diluted glycerine. Thoroughly clean a thin glass cover, about one-eighth of an inch larger than the excavation, lay it on white paper, and upon it place the tiniest visible fragment of a freshly dried blood clot (this fragment will weigh from one twenty-five-thousandth to one fifty-thousandth of a grain). Then, with a cataract needle, deposit on the centre of the cover, near your blood spot, a drop of glycerine about the size of this period (.), and with a dry needle gently push the blood to the brink of your microscopic pond, so that it may be just moistened by the fluid. Finally, invert your slide upon the thin glass cover in such a manner that the glycerined edges of the cavity in the former may adhere to the margins of the latter, and, turning the slide face upwards, transfer it to the stage of the microscope. " By this method it is obvious we obtain an ex- tremely minute quantity of strong solution of haemo- globin, whose point of greatest density (generally in the centre of the clot) is readily found under a j-inch objective, and tested by the adjustment of the spectro- scopic eyepiece. After a little practice it will be 10 146 RICHAEDSON'S METHOD. found quite possible to modify the bands by the addi- tion of sulphuret of sodium solution, as advised by Preyer. " In order to compare the delicacy of my plan with that of Mr Sorby, a spot of blood one-tenth of an inch square may be made on a piece of white muslin, the threads of which average one hundred to an inch. When the stain is dry, ravel out one of the coloured threads and cut off and test a fragment as long as the diameter of the filament, which will, of course, be a particle of stained fabric measuring one one-hundredth of the minimum-sized piece directed by Mr Sorby. When the drop of blood is old, a larger amount of material becomes requisite, and you may be obliged to moisten it with aqua ammonia?, or with solution of tartrate of ammonium and protosulphate of iron ; but in the criminal case referred to, five months after the murder, I am able, from a scrap of stained muslin, one- fiftieth of an inch square, to obtain well-marked ab- sorption bands, easily discriminated from those pro- duced by a solution of alkanet root with alum, and those caused by infusion of cochineal with the same salt. " In cases of this kind, where the greatest possible economy, or even parsimony, of material is needful, I would advise the following mode of procedure for proving and corroborating your proof of the existence of blood, so that its presence in a stain may be affirmed with absolute certainty : " From a suspected blood-spot upon metal, wood, leather, paper, muslin, or cloth, scrape with a fine RICHARDSON'S METHOD. 147 sharp knife two or three, or more, minute particles of the reddish substance, causing them to fall near the middle of a large thin glass cover. Apply in close proximity to them a very small drop of three fourths per cent, salt solution, bring the particles of supposed blood clot to its edge, and proceed as I have already directed. " After thus examining the spectrum of the sub- stance, you may generally, by rotating the stage, cause the coloured fluid to partly drain away from the solid portion, wherein, under favorable circumstances, should the specimen be blood, the granular white blood- globules become plainly visible, as do also cell- walls of the red disks. Among the latter, if your mental and physical vision is keen enough, you can, by the aid of a -^gth-immersion lens and an eyepiece micrometer, measure a series of corpuscles accurately enough to discriminate human blood from that of an ox, pig, horse, or sheep. " Lastly, to make assurance triply sure, lift up the thin glass cover, wipe off the tiny drop of blood solu- tion and clot you have been examining on the folded edge of a thin piece of moistened blotting paper, let fall upon it a little fresh tincture of guaiacum, and then a drop of ozonised ether, which will at once strike the deep blue colour of the guaiacum test for blood. In this way I have actually obtained these three kinds of evidence, to wit, that of spectrum analysis, that of the micro- scope, and that of chemical reaction, from one single particle of blood, which, judged by a definite standard, certainly weighed less than one fifteen-thousandth, and 148 RICHARDSON'S METHOD. probably less than one twenty-five-thousandth of a grain." Dr Richardson then goes on to criticise other methods, but the reader will not fail to notice that he goes wide of the mark, as he has to bring in the aid of the microscope while professing to have found an improvement over Mr Sorby's method of detecting blood by means of the spectroscope. I would advise those who are engaged in such research to follow Mr Sorby's advice, as his methods were arrived at after prolonged and careful study; indeed, he never lays down rules without good reason, and he may well be considered the greatest living authority in this special branch of inquiry.* * In most cases I believe the blood-stained cloth could be made to yield haematin, either acid, or alkaline in a very simple way ; thus, by digesting it in alcohol containing sulphuric acid, acid hsematin may be obtained, or in alcohol containing ammonia, that variety of alkaline hsematin before described would be obtained. By adding sulphide of ammonium to the latter fluid the bands of reduced heomatin would appear, although there might be but the slightest trace of ha3matin in solution. HUMAN BILE. 149 CHAPTER VII. ABSORPTION SPECTRA OF BILE, URINE, ETC. Brief sketch of the chemistry of the bile. Before describing the spectra yielded by human bile when treated with reagents, or those of the bile of other animals, a few words on the chemistry of this fluid may not be uninteresting ; moreover, a knowledge of its chemistry is absolutely necessary before commencing the study of its spectra. The bile of man, of carnivorous and omnivorous animals, is "a bright golden red ;" of graminivo- rous animals " a golden green, or a bright green, or dirty green," but its colour in various animals will be referred to again. The reaction is alkaline. According to Frerichs the following is the average composition of human bile in 1000 parts : Water 859*2 Bile salts . . . . 91/4T Fats, &c 9-2 Cholesterin . . . . 2'6 - 140'8 Mucus and pigment . . .29' Inorganic salts . * . .7 1000- Of these constituents, we are here concerned only with the pigments and the bile salts. 150 BILIFUSCIN. The colour of human bile is said to be due to Bilirubin* a pigment which is also found in gall- stones, in jaundiced urine, and which can be pre- pared by the following method : " Extract some powdered ox gall-stone successively with water, alcohol, dilute hydrochloric acid, boiling alcohol, and ether; then boil the dry powder with chloroform, and exharust with this agent. Distil the chloroform from the red solutions, but not quite to dryness. To the residue add several volumes of absolute alcohol, and let stand twenty-four hours. There will be depo- sited a brilliant red powder mixed with steel-blue or brown crystals. Both the powder and the crystals are pure bilirubin, and can be separated by levigation with much absolute alcohol.' J f Human gall-stones, by this treatment, will, after the extraction of the bilifuscin, also yield bilirubin, but in very small quantity. By exposing an alkaline solution of bilirubin to the air it turns green, and is converted into JBiliverdin, the green pigment found in herbivorous bile (?). Bilifuscin, another colouring matter, can be ob- tained from brown human gall-stones, by powdering the stone and extracting with ether, which removes the cholesterin. The powder is then treated with water and a little hydrochloric acid, and washed to neutrality. It is again extracted with boiling ether to remove fatty acids, and the powder boiled with absolute alcohol. Bilifuscin, will form a brown solution, and remains after evaporation of the alcohol as a black, shining, brittle mass, or as a dark brown powder. J * Thudicfcum, ' Chemical Physiology,' 1872. f Ibid. J Ibid. SPECTEUM OF HUMAN BILE. 151 The salts consist of sodium glycocholate and tauro- cholate. In ox-gall sodium glycocholate is abundant, and taurocholate scanty, while human bile contains chiefly sodium taurocholate, with a small quantity of the other salt. The bile of the cat, .dog, bear, and other carnivora, contains only sodium taurocholate.* This brief statement will recall to the reader's mind all that will be necessary to enable him to understand what follows. Of the spectrum of bile itself. Human, or dog, or cat bile gives no spectrum whatever! when it is fresh, but when it begins to decompose, or when extracted with alcohol it gives a spectrum, which will be described again. The bile of the lower animals with a few exceptions besides those mentioned above gives a spectrum which in some cases is a rather complicated one, and in most is more or less independent of the colour of this fluid. Although none of the bile-pigments mentioned above give any spectrum, there are other colouring-matters got from bile by treatment with stronger reagents than those required in the separation of bilirubin, biliverdin and bilifuscin, which, as the study of their spectra throws considerable light on what is to follow, will now be described. The pure chemical substances obtained by different observers from the oxidation of the bile-pigments must be mentioned nYst, * We also find in the bile of the pig two peculiar acids united with sodium, viz. taurohyocholic and glycohyocholic. Again, goose-bile con- tains taurochenocholic acid. f Although no spectrum is seen when the bile itself is examined, yet by careful dilution we can generally bring out a shading at F. t Nor do Bilifuscin, Bilirubin, or Biliverdin. 152 BILICYANIN. and afterwards an account of the spectra which can be obtained by acting on bile itself with reagents, and of various kinds of bile, will follow. Bilicyanin Maly, Heynsius, and Campbell have shown that bilirubin and other pigments treated with oxidizing agents yield a blue pigment, which, on account of its colour, has been named bilicyanin. This pigment is produced by mild oxidation, while by stronger oxidation choletelin is produced. Bilicyanin was obtained by adding an alcoholic solution of bromine to bilirubin suspended in chloro- form. As soon as the liquid assumes a blue colour it is left to evaporate, when there remains a substance which appears dark green when spread in a thin layer on porcelain, and which when dissolved in alcohol exhibits a fine blue colour. It is partially soluble in ether, and more so in a mixture of ether and alcohol. The alcoholic solution, on gentle heating with nitric acid, changes from blue to violet, then to purple red, and lastly to light brown ; caustic potash causes it to assume a dingy sap-green colour, which turns to blue on the addition of hydrochloric acid ; ammonia changes the blue solution to indigo-blue, which becomes a bright blue on adding hydrochloric acid. Sulphuretted hydrogen forms with bilicyanin in solution at first a bright green solution, and then flocks of biliverdin are precipitated, after the separation of which the liquid becomes colourless. As two or three different kinds of spectra are yielded by bilicyanin characterised by the appearance of bands * Or cholophaeine. CHOLETELIN, ETC. 153 in the yellow and green, it cannot be considered a perfectly definite product. It is not found in bile- pigment without exposure to the air, or until the pigment has been oxidized in some other manner, but it exists in gall-stones and probably in urine, since hydrochloric acid acts upon it in the same manner as it acts upon indican, for which substance it has pro- bably been mistaken in the urine. Choletelin. By passing nitrous vapours into alcohol in which bilirubin is suspended, this end-product of the oxidation process is obtained : by pouring the alcoholic solution in water after being thus treated, nearly all the colouring matter separates in the form of flakes, which dry up to a brown powder. This is soluble in alcohol, ether, and chloroform. It does not give any play of colour with nitric acid, but it has a more con- stant spectrum than that of bilicyanin ; thus, when in acid solution it gives one broad band extending from b to a little beyond F ; in an alkaline solution the band is less refrangible. It resembles thus both in its spectrum, and in the changes by acids and alkalies which are caused by the latter in the spectrum, some- what the urobilin of Jaffe, which will be referred to further on. Another blue colouring matter, which is probably a constituent of the bile itself, is described by E. Hitter.* It is not, like the last, produced by oxidation. It is found in the bile of man, the ox, sheep, pig, dog, and cat. He prepared it by shaking bile with chloro- form until a yellow solution formed, and this was * ' N. Rep. Pharm.,' xx, 569. 154 CHOLOCYANINE, ETC. treated with sodium carbonate until colourless. It was then neutralised with hydrochloric acid, when two strata formed, one containing the yellow chloroform solution, the other the blue colouring matter in a state of suspension. The latter is insoluble in acids and chloroform, soluble in alkalis, forming a colourless or yellow solution, which, when exposed to the air, forms a brown precipitate, which after some time again becomes blue. In this respect it differs from reduced indigo, which turns blue when dissolved in alkalis and when exposed to the air. Some bile-pigments described by Thudichum which give a spectrum. In addition to choletelin and bili- cyanin Dr Thudichum describes, in the report quoted before, some other colouring matters produced by the action of various acids on bile which gave well-marked spectra : among these may be men- tioned, first, one which was obtained by the action of concentrated nitric acid on an ammoniacal solution of bilirubin, when a blue precipitate was formed, which, after being quickly isolated by filtration and washing with water, was dissolved in alcohol. It showed an absorption band in yellow, and was called by the discoverer cholocyanine. Its spectrum is represented in Chart III, Sp. 18. By the action of fuming sul- phuric acid on bilirubin he also obtained a sulphate of cholocyanine having much the same spectrum. Two other bodies, sulpho-cholocyanine and cholo- thalline, were obtained by the action of sulphuric acid on bilirubin, but their consideration is of such little importance that they need not be further mentioned ; BILE SPECTEA. 155 their description will be found in p. 252 of Thudichum's Report. Cholonematine, another substance, was ob- tained by the same observer from the alcoholic extract of the colouring matter of human gall-stones, which, on account of its remarkable spectrum, is figured in Chart III, Sp. 19.* This is the spectrum of an ethereal solution, which appeared green in reflected light, and " brown in transparent dilute solution." Boviprasine, a green colouring matter, giving the spectrum shown in Chart III, Sp. 20, was obtained from the alcoholic extract of the gall-stones of the ox ; the spectrum was that of an ethereal solution of the resinous residue left after evaporation of the alcoholic solution. Hyocceruline is a blue matter obtained by the same observer from the gall-stones of a pig, the spectrum of which, dissolved in alcohol, is shown in Chart III, Sp. 21. Another colouring matter from the same source is called hyoflavine, which, when dissolved in alcohol and potash, boiled, and nitric acid added, gave a blue solution, showing two bands and a third broad and dark band. This spectrum is shown in Chart III, Sp. 22. My object in figuring these spectra is for the sake of comparison, e.g. compare the last spectrum with Chart II, Sp. 5, which latter was got by acting on human-bile solution with nitric acid, or with pig-bile solution acted on by the same acid, when almost the same spectrum is obtained. There are various other bile spectra described and figured by Thudichum, but they are not of sufficient medical interest to call for their being mentioned here. * Copied from Plate IY of ' Report/ 156 BILE SPECTRA Spectra obtained from the bile of some of the lower animals. I have been engaged for a considerable time in investigating the spectrum of the bile in various animals, and as this has not been done by others, except Dr Dalton, of New York, at least, so far as I have been able to ascertain, I shall now give the results at which I have arrived. The animals whose bile has been examined are : 1. Man. 7. Mouse. 13. Chicken. 2. Pig. 8. Sheep. 14. Goose. 3. Dog. 9. Hedgehog. 15. Wild duck. 4. Cat. 10. Ox. 16. Duck. 5. Guinea-pig. 11. Crow. 17. Frog. 6. Rabbit. 12. Blackbird. Among these animals the bile of the following gave a characteristic spectrum : Guinea-pig. Mouse. Ox. Rabbit. Sheep. Crow. It is a curious fact that the darkest green or golden-red bile gave the least characteristic spectrum. The carnivorous and omnivorous bile gave a negative result with slight exceptions; that of the herbivora and graminivora gave a characteristic one, while the insectivorous specimen was negative among the mam- mals ; and the bile of the frog, which was the only reptilian one examined, showed a resemblance to the mammalian insectivorous specimen. Among the birds the opposite fact was noticed. While those birds, such as the blackbird, chicken, goose, wild duck, and OF LOWER ANIMALS. 157 duck, gave nothing very remarkable, yet the bile of the crow, which may well be considered an omni- vorous bird, gave a most characteristic spectrum. There is one feature in common, viz. that by care- ful dilution a band can, in almost every specimen of bile, be made to appear at F, which is intensified by acids, and made almost, or altogether, to disappear by alkalies, besides the play of colours with nitric acid which all specimens of bile give. Human bile, as was referred to before, gives no spectrum,* except that by very careful dilution we can generally bring out a shading at F, which shading is intensified by hydrochloric acid and diminished by caustic alkalies. Pig-bile, when fresh, is said to give no spectrum ; four hours after it had been removed from the gall- bladder the fluid itself appeared brownish red in deep, and yellow in thin, layers, and gave a feeble absorption band, which is shown in Chart II, Sp. 6. Dog-bile, obtained fresh from the gall-bladder, was a golden-brown colour, and gave the feeblest possible shadow between D and B, but no well-marked band. On examining this bile in thinner and thinner layers it gave no other bands. Gat-bile, obtained perfectly fresh, was of a golden- yellow colour with a greenish tinge ; in deep layers (5 mm.) no bands were visible, but in a thin layer a band was seen, which read on my scale from 46 to 55, so that it was placed at F, which it covered. The * When old, or when extracted by alcohol, it gives at band at D. Vide antea, p. 151. 158 BILE SPECTRA band of urobilin, Chart II, Sp. 16, will represent its position. This band was intensified by treating solu- tions of the bile with hydrochloric acid, and dimi- nished by caustic potash, &c. Bile of the guinea-pig, obtained fresh ; its colour was golden yellow, and it was free from blood. It gave the spectrum shown in Chart II, Sp. 10. By dilution, or by thinning the layer, a feeble shadow could be seen at F. Bile of the rabbit. Freshly examined its colour was sap-green in thin layers. It showed at a suitable depth three bands, which are seen in Chart II, Sp. 11. Bile of _ the mouse. Fresh; yellow in colour. It gave, when examined in a thin layer or in solution, a splendidly marked band at F, which was made still darker by adding mineral acids, and diminished by caustic alkalis, so that it would appear the colouring matter of the bile of the mouse is identical with the bile-pigment called urobilin,* constantly present in human urine. Bile of the sheep and ox. When obtained fresh it is green, but soon changes to reddish brown, and pre- sents exactly the same spectrum when obtained from the ox that it does when it is got from the sheep. This spectrum is a very fine one, and presents in a deep layer three bands, in a thinner one four bands, and in a still thinner a fifth band at F is visible. Chart II, Sp. 7, shows the spectrum of the four bands ; the fifth may be represented by that seen in Sp. 8, which is the spectrum of this fluid treated by nitric * See the account of its spectrum, infra. OF LOWER ANIMALS. 159 acid. Sp. 9 is that of a solution treated with hydro- chloric acid, the precipitate of cholic acid, &c., being in both cases redissolved in boiling alcohol. Bile of the hedgehog. This bile, obtained fresh from the gall-bladder, was a fine blue-green colour. It gave no absorption band, but a feeble shading was noticed at F. Bile of the crow. Of a yellowish-green colour ; it gave a very remarkable spectrum, which is shown in Chart II, Sp. 13. The band in the extreme red is best seen by lamp -light. Bile of the blackbird. Of a yellowish colour ; gave only a shadow at F. Bile of the chicken. The colour was dark sap-green ; at first no band was visible, but after having been exposed to the air for a few minutes the usual band at F appeared. Bile of the goose. Colour dirty green ; there was no band except a shadow at F. Bile of the wild duck. Sap-green in colour ; this bile gave only the band at F. Bile of the domestic duck. It was of a dark sap- green colour; but gave no characteristic spectrum except a band at F. Bile of the frog. From the colour of the bile of this animal, which was a fine bright green, one would be led to expect a characteristic spectrum, but, with the exception of the shading in the red and violet ends of the spectrum, and an uncertain shading at F, there was nothing remarkable. Remarks on the above spectra. The absorption band 160 GMELIN'S KEACTION. at F thus appears to be the link which binds all these specimens of bile together, and which is seen with the same distinctness in the bile of the mouse as it appears in febrile human urine. Thus, the examination of the bile of an insignificant little mammal has thrown light upon an obscure fact in physiological chemistry, a statement with which the reader will agree when he has read what follows. The spectrum of Gmelin's reaction When to a solu- tion of bile in a test tube we add strong nitric acid the solution at once undergoes a change of colour, becoming green, blue, violet, red, and lastly yellow or brownish- yellow, and if the experiment has been performed by pouring the nitric acid into a test tube before the slit of the chemical spectroscope we notice a broad shading (probably composed of two distinct bands) in orange and yellow, and a black band extending from near b to beyond F; this is shown in Chart II, Sp. 5. In a very short time the shading in orange begins to fade, and at the time the oxidation-process is completed and the colour of the solution has become yellow, nothing but the band at F is left. Or the experiment may be varied in this way : A little of the bile itself may be put into a glass cell, nitric acid added, and boiling alcohol then poured into the cell, which has the effect, not only of dis- solving the precipitate, but of .arresting the oxidation process for a short time, and thus enables any particular colour to be observed. On examining such a solution we get, if we have acted on human bile, the spectrum shown in Chart II, Sp. 5, and we may by GMELIN'S REACTION. 161 adding more and more alcohol cause the disappear- ance of the band in orange until, as before mentioned, that at F only is left. Ox bile treated in the same manner gives the spec- trum shown in the same chart, Sp. 8, or treated with hydrochloric acid, Sp. 9.* This spectrum teaches that many colouring matters derived from bile were actually formed by reagents upon that fluid, and to me it appears that bilirubin, and perhaps bilifuscin, in carnivora, biliverdin (when it will have been properly obtained) in herbivora, &c., and the so-called urobilin, are the only bile-pigments which are normally present. The reaction of nitric acid upon bile-pigment has been studied by Jaffe,t who states that the different colours produced corre- spond to characteristic alterations of the spectrum ; in fact, his description | corresponds with what has been already said. He, however, succeeded in isolating the pigment present in the blue-violet solution, by treating an alcoholic solution of biliverdin or an ammoniacal solution of bilirubin mixed with alcohol with a mix- ture of ordinary concentrated nitric acid, until a sample showed the bands on each side D, the solution being then mixed with chloroform and shaken with water. The water was then removed, the chloroform layer containing the blue pigment was filtered from the * If pig -bile is treated in this way, we get a band on each side D, separated by an interval of yellow colour, which may be compared with Thudichum's oxidation product from hyoflavine, Chart III, Sp. 22. f ' Zeitsch. f. Chem.,' v, 666. J When I first described this spectrum in 1877, I was quite ignorant of Jaffe's researches on * Gmelin's Reaction.' 11 162 TJROBTLIN OF JAFF biliverdin and evaporated to dryness, and the residue purified by repeated solution in chloroform. The pigment thus obtained is of a deep violet colour, insoluble in water, soluble in alcohol, ether, and chloroform, forming solutions of a violet colour. Alkalis dissolve it, the solutions being brown-violet ; acids also form solutions with it of a blue colour. The neutral and alkaline solutions exhibit no absorp- tion bands, but a small trace of an acid causes the appearance of the original spectrum. Urobilin. Jaffe also isolated the pigment which gives the band at F, having obtained it by the same process as in the last case. The substance which was got was brown red, soluble in alcohol, ether, and chloroform, the solution being a fine red colour, unaltered by acids or alkalis ; but the acid solution only exhibited the original absorption band. By the action of hydrochloric acid on the bile of the dog, the same observer obtained a red solution which gave exactly the same band. This red solution becomes yellow on adding alkalies to it, and then gives another band between C and F, but nearer to C.* Chloroform extracts the colour from the solution, and on evaporating the chloroform a red residue is left, soluble in water, in alcohol, and in chloroform. It can be precipitated from the aqueous solution by lead acetate or calcium chloride. The great pathological importance of this discovery of Jaffe's cannot be over-estimated, for the pigment which he succeeded in isolating from the bile of the * Watts' ' Diet.,' 1st Supp. Is not C a misprint for E ? EESEARCHES OF STOKVIS. 163 dog, is identical with the urobilin which I have found in every specimen of human urine. A substance giving the same spectrum may be obtained from human bile by the simple addition of hydrochloric acid to a solu- tion of the former. Detection of bile-pigment in urine by Gmelin's test and its spectrum, By means of the spectrum described above we can be certain that the colour reaction developed on the addition of nitric acid is due to bile-pigment ; the appearance of a shadow on each side D, which soon disappears, being absolute proof that bile-pigment is present. This shows the importance of using the spectroscope in clinical work, since the colour reaction alone is attended with difficulties, which are considerably diminished by using this instrument. Reducible product of the oxidation of bile-pigment. According to Stokvis,* " this substance is formed as a secondary product in most cases of the oxidation of biliary colouring matter whereby Gmelin's reaction is produced. It is colourless, or of a light yellow tint, and is soluble in water, alcohol, and dilute acids. It becomes of a beautiful rose-red colour when boiled with reducing agents in alkaline solutions. The red solution gives in the spectrum a broad band in green. In thick strata or concentrated solutions the band begins close to D and extends to b. In dilute solu- tions (thin strata) it occupies only two thirds of the distance between D and E, ending short of E. Shak- ing with air causes both the rose colour and the band * " N. Rep. Pharm.," xxi, 123, Watts' ' Diet,' 2nd Supp., 1875. 164 RESEARCHES OF STOKVIS. to disappear. This by-product differs from the bile- colouring matter and other oxidation products of the same, in being insoluble in chloroform and ether, and in not forming insoluble compounds with neutral or basic lead acetate. It is precipitated, however, by ammonia and basic lead acetate. " This substance exists as such in the gall-stones of man and of the ox, and it can be obtained from them by boiling with distilled water and extracting with dilute acids. It does not exist in fresh bile. It occurs in the urine of animals which has been standing for some days previously, in jaundiced urine, and in the urine of febrile diseases, e.g. smallpox, typhus, &c. It is not found in healthy urine. It seems to be present in the alimentary canal, although in direct experiments with different kinds of food little or none could be found. In alkaline solutions it soon loses its charac- teristic properties. Its occurrence in any liquid of neutral or acid reaction affords an indication of the previous existence of bile-pigments therein. In apply- ing the test, the liquid is to be precipitated with lead acetate, excess of lead removed by oxalic acid, and the filtrate concentrated and boiled with alkalies and a reducing agent. If no reduction takes place, and if the other tests for biliary colouring matter have given a negative result, their absence may be safely in- ferred." This is probably the colouring matter which I have found several times in various morbid urines on adding to them strong nitric acid, and accordingly its descrip- tion has been fully given ; this fact will be referred PETTENKOFER'S TEST. 165 to when the spectra of these specimens of urine are described. The spectrum of Pettenkofer's test for bile acids. I have in a former paper* described Pettenkofer's test as consisting of two bands, but I now find that the band at F must have been due to the action of sul- phuric acid on the bile-pigment. In Chart II, Sp. 14, is a representation of the spectrum, which was obtained as follows : Human bile was treated with absolute alcohol and filtered; the filtrate was thoroughly de- colorised with animal charcoal, and Pettenkofer's test was applied with every possible precaution to a small portion of the alcoholic solution ; it gave two bands nearly in the same position as those represented in Sp. 14, which is the spectrum of the aqueous solution of the bile salts obtained by evaporating off the alcohol and dissolving the residue in water. I then examined the spectrum of Pettenkofer's test as applied to the bile salts of the pig, the solution being prepared as before, and the result was the spectrum shown in Chart II, Sp. 15. The band near D in Sp. 14 is hardly, if at all, visible in the chemical spectroscope, and is probably of little consequence. This spectrum enables us to distinguish the purple reaction given by the action of sulphuric acid and sugar on bile salts from the reaction developed by the same reagents with many other bodies, but I fear its use in examining for * ' Dub. Journal/ 1877. According to Heynsius and Campbell, the colour developed by the action of sulphuric acid and sugar with sodium taurocholate gives a spectrum of three bands, one between C and D, the next between D and B, and the third between b and F. Pfluger's ' Archiv. f. Physiol.,' iv, 497. 166 UEOBILTN IN URINE. them in urine is exceedingly limited for obvious reasons. Thudichum's map of Pettenkofer's reaction is shown in Chart III, Sp. 16. According to Thudichum the salts are absent from bile in certain "diseased conditions" of this fluid, and in fatal cases of cholera.* Urobilin in human urine, In the concluding part of this chapter the importance of a knowledge of the researches of Jaffe on bile-pigments will be shown, in enabling us to understand the nature of the colouring matter present in urine in health ; and the discovery of the other oxidation product of bile-pigment by Stokvis is important, since it throws light upon a spectrum which is seen in urine in certain pathological conditions when this fluid is treated with nitric acid. The accusation of being a " test-tube chemist " may be brought against any one who accepts Jaffa's views, but the test-tube has done a great deal for medi- cine, and in this instance, assisted by the spectro- scope, it has enabled us to understand a subject which will be of the greatest benefit to clinical medicine, and which, when it has been sufficiently worked out, will be of immense value both in diagnosis and in treatment. It would be beyond the scope of this little volume were I to give an account of all the pigments which have been separated from urine ; it will suffice to say that there is only one pigment which gives a well- marked absorption band, and there are two proposi- tions which may be laid down with regard to this pigment : * ' Chemical Physiology,' 1872, p. 74. UBOBILIN IN URINE. 167 I. That normal human urine always contains a pig- ment, the spectrum of which in acid solution is charac- terised by an absorption band at Fraunhofer's line F. II. This absorption band behaves on treatment with reagents in the same manner as that of the substance obtained by the action of hydrochloric acid on bile by Jaffe, and like that obtained by Maly from bilirubin 9 'which he called hydrobilirubin, so that we may conclude that the substance in solution which gives the band at F is urobilin. I do not maintain that urine owes all its colour to urobilin, as there are no doubt other pigments present as occasional ingredients, but there is no doubt what- ever that urobilin is constantly present in every speci- men of human urine in health, that when this fluid is high coloured the absorption band at F is dark, that when it is not high coloured then the band is feeble, and it may not be visible in very alkaline urine. That it is present in the last case can be demonstrated, however, by the addition of nitric, or of hydrochloric, or even acetic acid, when it appears at once. What- ever the pathological significance of the other pig- ments that have been described may be, urobilin is the pigment which ought to have the greatest atten- tion paid to it by clinical observers, for although con- stantly present in healthy human urine it is sometimes absent in disease. I am not at present in a position to say in what diseases it is absent, but that it is so is a fact beyond dispute. The spectrum of normal human urine of acid reaction is shown in Chart II, Sp. 16. 168 ANOTHER BILE-PIGMENT This band is especially well marked in some febrile urines, but it is not quite correct to say that caustic potash and caustic soda cause it to be replaced by another one, for although this does sometimes happen in the urine of disease, it is not the case in healthy urine. The fact that acids intensify the band and that alkalis, more especially caustic potash and ammonia, cause it to disappear, constitutes the test which distinguishes urobilin from other pigments which give an absorption band in the same part of the spectrum. Urobilin in pink urates. A hot alcoholic solution of pink urates gave the band of urobilin with remark- able distinctness, intensified by acids, and disappearing on the addition of alkalies . Another bile-pigment in the urine of disease. Be- sides urobilin, the spectroscope teaches that another bile-pigment appears in certain morbid conditions in the urine upon the addition of nitric acid ; it is indicated by an absorption-band which occurs on the red side of the band of urobilin ; it is probably that discovered by Stokvis, which was mentioned in page 163. It was found to occur in the following specimens of urine :* (1) Urine of rheumatic fever ; patient being treated by salicylate of soda (10 grains ter die). The colour of the fluid was a light yellow ; reaction acid ; perchloride of iron in solution developed the usual salicylate reaction ; no albumen. On examining the urine itself a band in the position of that of urobilin appeared ; * In none of the specimens could the colour- reaction known as Gmelin's test be developed. IN THE UKINE OF DISEASE. 169 tf on adding nitric acid the colour of the fluid became slightly pink or lavender-pink, and it now gave another band, extending from about half way between D and E to near E ; the urobilin band was still visible (Chart II, Sp. 17). The urine from the same patient, and taking the same drug, failed to give this reaction three days after the last examination. The salicylic acid had nothing to do with the appearance of this absorption band, as another specimen of urine from a case of phthisis treated with the salicylate failed to give the band ; nor is it constantly present in rheu- matic fever urine, as several specimens from other cases were examined with a negative result. (2) The urine of a case of pregnancy (6th month) when treated with nitric acid developed a band nearly in the same position, in addition to that of urobilin, but it is not constantly present in the urine of pregnant women^ (3) The urine of a case of thoracic aneurism ac- companied by albuminuria ; dark orange yellow in colour. It gave the spectrum of urobilin, but when treated with nitric acid a dark absorption band made its appearance, in a different position from that of the first specimens. This will be observed if Sp. 18, that of this specimen, is compared with Sp. 17 (Chart II). Hydrochloric acid only intensified the urobilin band, but caustic potash when added to the original urine at first caused the disappearance of the urobilin band, which was soon replaced by another nearer the red (see Chart II, Sp. 19). Another speci- men of urine obtained some days after this examination 170 CONNECTION BETWEEN THE I from the same patient behaved in an exactly similar manner. (4) The urine of a case of cirrhosis of the liver, of a dark straw colour, gave a spectrum almost identical with that shown in Sp. 17. (5) From a case of cancer of the pylorus, the urine, which was a straw colour, gave a spectrum closely resembling the last in the presence of two bands, but that near the red was a little nearer b than in the last case. Out of some hundreds of specimens of urine, from patients suffering from trifling ailments, which I have examined with the spectroscope I have never got a second absorption band. That which was seen was always due to the presence of urobilin only. Pro- bably the appearance of this second band indicates grave disturbance in the system, as it appears only in those cases where there is undoubted disease of a severe character, with the exception of preg- nancy.* Connection between the colouring matters of blood, bile, and urine. Though such a connection is denied by some chemists, who assert that it is impossible that bile-pigment can be derived from haemoglobin, there is undoubtedly a time approaching when con- vincing proof of this fact will be forthcoming, and also of the no less important one that the colouring matter of urine is derived from bile-pigment ; rnean- * The presence of urobilin is sometimes difficult of detection ; but by examining the urine in a deep layer, or by adopting other precau- tions which for that particular case will suggest themselves, its band can always be seen. PIGMENTS OF BLOOD, BILE, AND URINE. 171 time we may believe that what have been supposed to be the true colouring matters of bile and of urine are not such, but probably a mixture of several sub- stances, owing to imperfect methods of separation. The spectroscope has suggested the connection, and when our knowledge of chemistry will be precise enough to allow of our being able to follow up the hints it has given, controversy will be no longer possible. Bilirubin is now generally supposed to be formed from haemoglobin, though actual proof has not yet been forthcoming. There is no doubt that haemo- globin in its passage through the liver is actually converted into bilirubin, and there is, according to some, an identity between haematoidin and biliru- bin (though this is denied by others). It has been affirmed by some that bile-pigments have appeared in the urine after the injection of haemoglobin solu- tion into the veins, or of any substance which is capable of dissolving the blood-corpuscles and setting free haemoglobin, such as water, bile acids, or ether; but whether this has been proved or not, there is no doubt that urobilin can be formed from bilirubin. E. Maly * has found that by dissolving bilirubin in dilute soda- or potash-ley, and adding sodium amalgam, the air being excluded, no hydrogen was given off, but the dark colour gradually lightened, and after two or three days' action the solution acquired a yellow or bright brown-yellow colour, and then gave off hydrogen. From this liquid hydrochloric acid sepa- * 'Ann. Ch. Pharm.,' clxi, 368; clxiii, 77. 172 UROCYAN1NE. rated a pigment, which appeared to be a weak acid, yielding with alkalis brown-yellow soluble salts, and with heavy metals insoluble compounds, which separated in red flakes. It was soluble in alcohol, slightly in water, readily in ammonia and fixed alkalies, in ether, liquid hydrocarbons, glacial acetic acid, and chloroform. Its spectrum is the same as that of urobilin, the band being intensified by acids and decreased by alkalis ; this has been called hydrobili- rubin, but it differs in no respect from urobilin. It is also found in fseces. Biliverdin can also be made to yield it in the same way. (See Thudichum's c Annals of Chemical Medicine.') The detection of blood, bile-pigment, and bile-acids in urine by means of the spectroscope has been already referred to. Sugar and albumen cannot be detected by this instrument, but we have already suffi- ciently delicate tests by means of which the smallest quantity of either can be detected with certainty. Urocyanine and urorubine. The urine from cholera patients in the early stage of reaction when cautiously boiled with nitric acid often gives a blue colour and sometimes a blue deposit. This deposit is soluble in alcohol, forming a dichroic purple-blue solution, giving a broad absorption band in yellow (" and green ?"), Chart III, Sp. 23 (Thudichum).* Lutein. u Luteine " is the name given by Dr * The absorption band described by Dr Moss in the * Medico-Chi- rurgical Transactions,' 1875, which he found in the urine of a case of cirrhosis of the liver is nothing but the band of urobilin. This spectrum is referred to in Beale's ' Microscope in Medicine/ last edition, p. 501, but the author of that book did not notice the error. LUTETN. 173 Thudichum* to a substance which he first discovered in the juice of the corpora lutea of mammals. To prepare it he dissected out the corpora lutea from the ovaries (of cows), pounded, warmed them, and pressed out the juice. The alcoholic solution of this dried residue gave the spectrum shown in Chart III, Sp. 10. This solution was examined by means of lime light. The etherial and chloroformic differ slightly in the position of the bands in their spectra from the alco- holic solutions. By boiling the yelks of eggs in 85 per cent, alcohol, filtering, and allowing to stand till clear, we get a solution giving nearly the same spectrum (this is ovo- luteine). Butyro-lutein is got by digesting butter in chloro- form and filtering, when we also get three bands nearly in the same place. Cysto-lutein. The same substance was discovered by Thudichum in ovarian cysts by means of the spectroscope, which showed bands in the blue, nearly in the same position as the bands of ovario-lutein got from cows' ovaries. (See p. 130.) Intestino-lutein was got from the fseces of sucking infants by mixing the faeces with alcohol, and filtering from the flakes of caseine. It gave a band between b and G and covering F. (Chart III, Sp. 13.) Sero-lutein in blood-serum and in pathologicsl fluids. By allowing blood to stand, decanting off * See Appendix and 'Chemical Physiology.' Piccoli and Lieben first described the yellow substance giving this spectrum according to Watts, 2nd Supp. 174 SPECTRA OF F^CES. the serum, allowing this to deposit, pouring off the supernatant liquid, and filtering repeatedly until clear, we get a fluid which gives one band at F and a very faint one between F and G. The importance of this to pathology is shown by the fact that I have found this absorption band at F in fluid removed from the pleural cavity by aspiration, and also in peritoneal fluid from a case of ascites due to chronic peritonitis. The spectrum of sero-lutein from dog's blood is shown in Chart II, Sp. 24, and in Chart III, Sp. 12, and the same band from the ascitic fluid in Chart II, Sp. 23. Perhaps the question may be asked, Was not the band due to urobilin ? If it had been it would have been intensified by acids and removed by alka- lies, but the latter seemed rather to darken it, as did also the addition of ammonium sulphide. The same band was also noticed in sero-purulent fluid removed from an abscess. Spectra yielded by faeces. Yaulair and Masius obtained urobilin from faeces, and I have myself noticed the band of this substance in meconium. The analysis of Zweifel* would lead us to expect its presence in the latter. It is probably formed in the intestine from bilirubin by the same process as it was prepared by Maly, inasmuch as abundance of hy- drogen is present in the intestine, and immediately exerts a hydrogenising action. By boiling faeces with sulphuric acid Thudichum obtained a substance, the spectrum of which is represented in Chart III, Sp. 14. This may be compared with Sp. 15, from treating a * ' Archiv. fiir Gynacologie.,' Band vii, p. 474. OTHER PHYSIOLOGICAL SPECTRA. 175 cholera stool in the same manner, and is very like sulphate of cruentin, shown in Chart I, Sp. 16. Other physiological and pathological spectra. The absorption spectra which have been described are those which will be found most useful to workers with the spectroscope ; there are three more which will require to be merely mentioned. Murexide, the purple colour produced from uric acid by the action of nitric acid and ammonia, gives a. broad absorption band extending from D to F. (Thudichum.) Fluopittine, a body obtained by Thudichum by the decomposition of albumen, gives the complicated spectrum shown in Chart III, Sp. 17. Liquor amnii gives a faint shadow at F, probably due to lutein. There is no doubt that when the spectroscope comes to be used more extensively than it has been hitherto, a great number of pathological absorption spectra will be discovered ; and it remains for those who have abundant clinical opportunities to do their share. By adding another exact physical method to their means of diagnosis, they will help to make medicine approach more nearly to the position which we all hope it will some day occupy that of an exact science. 1 76 WAVE-LENGTHS. APPENDIX I. WAVE-LENGTHS. MR SOEBY has, after having introduced a method of printing spectra in formulae, come to the conclusion that all results should be expressed in wave-lengths ; and he does this with his own microspectroscope by means of a complicated apparatus, composed of a plate of quartz placed between two NicoPs prisms, the thick- ness of the plate being such that the whole spectrum contains twelve dark bands. He prefers this method to reducing his readings to wave-lengths by means of the bright-point micrometer, as he maintains the latter method " has unfortunately several serious defects for expeditious practical working ;" but the adoption of Mr Sorby's apparatus is attended with even greater difficulties than any other. The photographed scale which I have had attached to my microspectroscope enables me to reduce the reading of any band to wave- lengths in a few seconds with the help of the curve I have described on p. 32, and after having used it for a considerable time I can recommend this method before any other. All we have to do (as stated before) is to take the reading of the centre of the band, find this number on the top line of the scale, and then its posi- WAVE-LENGTHS. 177 tion on the curve, the wave-length corresponding to the number will be found on the right-hand side of the scale. The importance of the wave-length method is further increased by the discovery made by Mr Sorby, that there is " a far more uniform connection between the wave-lengths of the centres of bands of the spectrum of a single substance containing a number of bands than there is between any other conditions" Thus, in many spectra having a series of bands whose centres are at wave-lengths a, b, c, and d, there is the same ratio between each consecutive two, so , i , a b c A that : Again, bed in the case of substances giving two or more well-marked bands, though the actual wave-lengths of the centres of the bands may vary with the conditions in which the substance occurs (solid or in solution), yet the ratio between the wave- lengths of the bands remains almost, if not quite, constant. Thus, in yellow xanthophyll, which may be taken as an example, we have :* Condition. Centre of the two bands. Ratio. In free state and solid .... In carbon bisulphide .... In absolute alcohol Combined with Canada balsam 501 469 498 467 471 442 488 457 1 : '936 1 : "937 1 : "938 1 : "936 * See the ' Monthly Microscopical Journal,' vol. xiii, p. 198. 12 178 WAVE-LENGTHS. EXAMPLE OF THE METHOD OP REDUCING EEADINGS TO WAVE-LENGTHS. An example will make the method of calculating wave-lengths by means of the curve shown opposite page 32 clear. Thus, some stale urine containing blood due to the breaking down of a carcinomatous growth in the bladder, gives three bands (those of methsemoglobin) . Now, on taking their readings on the scale attached to my microspectroscope, I find that the centre of the first band is at 17*5 ; I then find its place on the top line of the scale, and running my eye down the scale, I find where the line corresponding to this number and the curve intersect each other, and opposite to this point on the right-hand line of the scale I find the number 624. Therefore, the wave-length corresponding to the centre of the first band is 624. The scale reading of the centre of the next band is 27, and on proceeding as before, we find its wave- length is 574. The scale reading of the centre of the next band is 35'5, and its wave-length is therefore 540. It must be distinctly understood that the numbers at the top of the scale will differ according to the method of measurement adopted by each observer, and in this case they are those of my own scale ; but those at the right-hand line are constant. Having tried this method in every possible manner, I can recommend WAVE-LENGTHS. 179 its adoption to those who are anxious to reduce their readings to wave-lengths. In the lithographed scale the squares which represent those on the original are square centimetres, while the latter were square inches, the smaller divisions being square tenths of an inch. APPENDIX II. BIBLIOGRAPHY RELATING TO THE STUDY OF ABSORPTION SPECTRA, ETC.* ASKENAY, E. " Beitrage zur Kenntniss des Chloro- phylle und einiger dasselbe begleitender Farbstoffe." Botan. Zeit., 1867, Nos. 29 and 30. BENOIT, H. " Etudes Spectroscopiques sur le Sang." Montpellier. BREWSTER, SIR D. " On the Action of various Coloured Bodies on the Spectrum." Phil. Mag., 4th series, xxiv, p. 441. BRIDGE, H. G. " Mapping with the Microspectro- scope with the bright-point Micrometer." Month. Micro. Journ., vol. vi, 1871, p. 224. BROWNING, J. " On a Simple Form of Microspec- troscope." Month. Micro. Journal, vol. ii, p. 65. Ditto. " On a Method of Measuring the Position of Absorption Bands with a Microspectroscope." Month. Micro. Journ., vol. iii, 1870, p. 68. Ditto. " How to Work with the Spectroscope." London, N. D. CHURCH, PROF. " Microspectroscopic Investigation." A letter in Intellectual Observer, vol. ix, p. 291. Ditto. " Turacine : a New Animal Pigment con- * Other papers are referred to in the text. BIBLIOGRAPHY. 181 taining Copper." The Student and Intellectual Ob- server, April, 1868, p. 161. COHN, F. "Ueber den Farbstoff der Phycochro- maceon." Archiv fiir Mik. Anat., v. M. Schultze, iii, 6 (1867). COOKE, J. P. " On the Construction of Spectro- scopes." Sill. Journ., xl, 305 ; Phil. Mag. (4), xxxi, 110. CROOKES, W., F.R.S. "On a New Arrangement of Binocular Spectrum Microscope." See the Monthly- Microscopical Journal, vol. i, 1869, p. 371, and for the paper itself, Proc. Roy. Soc., 1869, p. 443. Ditto. " On Spectroscopes." Mech. Mag., June, 1861, p. 308. DBAS, F. " On Spectra formed by the Passage of Polarized Light through Double-refracting Crystals seen with the Microscope." Month. Micro. Journal, vol. vi, 1871, p. 135. DESCHANEL, A. P. " Elementary Treatise on Natural Philosophy." Translated by Everett. London, 1873. Pp. 973_979 5 981994, and 10121031. ESOFP, J. " Urobilin in Urine." Pfliiger's Archiv. f. Phys., xii, 50 53; and Journ. Chem. Soc., vol. ii, 1876, p. 108. GAMGEE, A., PROF. " Action of Carbonic Oxide on Blood." Medical Times and Gazette, 1866. Ditto. "Note on the Action of Nitric Oxide, Nitrous Acid, and Nitrites on Haemoglobin." Proc. Eoy. Soc. Edinb., 1867, No. 73, p. 108. Ditto. "On the Action of Nitrites on Blood." Philos. Trans., 1868, pp. 589625. 182 BIBLIOGRAPHY. GAUGE, C. " On the Spectroscopy of Blood-pig- ments." Deut. Chem. Ges. Ber., ix, 833 ; and Journ. Chem. Soc., vol. ii, 1876, p. 646. GAYER, E. J. " On a New Form of Microspectro- scope." Month. Micro. Journal, vol. ix, 1873, p. 1. GLADSTONE, J. H. " On the Use of the Prism in Qualitative Analysis." Chem. Soc. Journal, vol. x, 79 (1858). HERAPATH, W. B. (the late). "Memorandum of Spectroscopic Researches on the Chlorophyll of various Plants." Month, Micro. Journ., vol. ii, 1869, p. 131. Ditto. " On the Use of the Spectroscope and Micro- spectroscope in the Discovery of Blood-stains/ 5 Che- mical News, vol. xvii, pp. 113 123. HOFMANN. " On Cadaveric Phenomena." Yertel- jahrschrift, fur Gerich. Med., vols. xxiv and xxvi ; and Lond. Med. Record, Nov., 1878, p. 461. HOGG, JABEZ, F.L.S. " Microspectroscopy : Results of Spectrum Analysis." Month. Micro. Journal, vol. ii, 1869, p. 121. HOPPE, F. " On the Absorption Lines in the Blood Spectrum." Schmidt's Jahrbuch d. Ges. Med., cxiv (1862). HOPPE-SEYLER, F. " Ueber das Verhalten des Blut- farbestoffs im Spectrum des Sonnen-lichtes." Virchow's Archiv., xxiii, 446 ; xxiv, 233 (Z. f. Chem., 1865, 214). Ditto. " Handbuch der Physiologisch- und Patho- logisch-Chemischen Analyse," Berlin, 1870. Ditto. " Erkennung der Vergiftung mit Kohlen- oxyd." Ebenda, 1865, s. 52, 53. Ditto. " Weiters iiber die Optischen und Chemischen BIBLIOGRAPHY. 183 Eigenschaften des Blutfarbstoffes." Centralbl. f. d. Med. Wiss., 1864, Nos. 52 and 53. HUGGINS, W. " On the Prismatic Examination of Microscopic Objects." Quart. Journ. Micro. Sci., July, 1865. JADERHOLM, A. " The Colouring Matter of Blood." Zeit. f. Biologie, xiii, 193, 255; and Journ. Chem. Soc., March, 1878. JAFFE. " Identitat des Hamatoidins und Bilifulvins." Archiv f. Path. Anat. u. Physiol., 23 Band, pp. 192, 193 (1862). JONES, H. BENCE. " On the Rate of Passage of Crystalloids in and out of the Body." Proceed. Roy. Soc., vol. xiv, p. 400. Ditto. " On the Chemical Circulation in the Body." Proc. Roy. Inst., May 26th, 1865. Ditto. " Lectures on some of the Applications of Chemistry and Mechanics to Pathology and Thera- peutics," London, 1867, p. 12 et seq. KINGZETT, C. T. "Animal Chemistry," 1878. KOSCHLAKOFF and BOGOMOLOFF. " Wirkung des Am- moniaks, des Arsen- und des Antimon was sers toffs auf die Blut pigmente." Centralbl. f. d. Med. Wiss., 1868, pp. 609 and 627. KRAUS. " Chlorophyllfarbstoffe," Stuttgart, 1872. KUHNE. " Lehrbuch der Physiologischen Chemie," 1868. LANKESTER, E. RAT, PROF. "Abstract of a Report on the Spectroscopic Examination of certain Animal Substances, presented to the Brit. Assoc, at Exeter, 184 BIBLIOGRAPHY. 1869." Journ. of Anat. and Physiol., Nov., 1869, p. 119. Ditto. " On the Spectrum of a Dichroic Fluid." Month. Micro. Journ., vol. iv, 1870, p. 14. Ditto. " The Distribution of Haemoglobin in the Animal Kingdom." Proceed. Roy. Society, vol. xxi, No. 140; also Month. Micro. Journ., p. 171, vol. ix, 1873. Ditto. " On Blue Stentorin, the Colouring Matter of Stentor coeruleus." Quart. Journ. Micro. Sci., April, 1873. Ditto. " On Methaemoglobin." Quart. Journ. Micro. Sci., No. 5, 1870, pp. 402405. Ditto. " Observations with the Spectroscope." Journ. Anat. and Physiol., 1867, p. 114. Ditto. " Wirkung des Cyangases auf Hamoglobin." Archiv. f. d. Ges. Physiol./ 5 Bonn, 1869, p. 491. LETHEBY. " On Spectrum Analysis." Clinical Lec- tures and Eeports of the London Hospital, 1866. LIEBEEMANN, LEO. " On Choletelin and Bilirubin." Pfliiger's Archiv. f . Physiol., xi, 181 190 ; and Journ. Chem. Soc., 1876, vol. i, p. 407. LOCKYER, J. NORMAN, F.R.S. " The Spectroscope and its Applications." Nature Series, London, 1873. Ditto. " Studies in Spectrum Analysis," vol. xxiii. International Scientific Series, London, 1878. Ditto. " Solar Physics," London, 1874. LOMMEL. " Chlorophyll in its Relation to Light." Chem. News, Sept. 13, 1872. Ditto. " Optics and Light." Internat. Sci. Series, vol. xviii, 1875. BIBLIOGRAPHY. 185 MAO MUNN, C. A. " Studies in Medical Spectro- scopy." Dub. Journ. Med. Sci., 1877. Ditto. " Note on the Spectrum of Yenous Blood after Death." Brit. Med. Journ., Feb. 1st, 1879. Ditto. " A Delicate Spectroscopic Test for Blood in Urine." Brit. Med. Journ., July 19, 1879. Ditto. " A Spectroscopic Explanation of the Action of Nitrous Oxide." Dub. Journ. Med. Sci., Sept., 1879. MALASSEZ. " On the Estimation of Haemoglobin." Archiv. de Physiologic, Feb., 1877. Ditto. " Sur les Diverses Methodes de Dosage de PHsemoglobine et sur un Noveau Colorimetre." Labo- ratoire d'Histologie du College de France, Travaux de 1'Annee, 1876. MAXWELL. " Investigations on Colour-Blindness by means of the Spectrum." Philos. Mag., xxi, 1861, p. 145. MELDE, F. " On the Absorption of Light by Coloured Liquids." Pogg. Ann., cxxiv, 91 ; cxxvi, 264. MOSELEY, H. N. " On the Colouring Matter of various Animals, and especially of Deep-sea Forms, dredged by H.M.S. Challenger." Quart. Journ. Micros. Scien., Jan., 1877. PALMEE, T. " On a New Method of Measuring and Recording the Bands in the Spectrum." Month. Micro. Journ., vol. xvi, 1876, p. 277. Ditto. " On the Various Changes caused in the Spectrum by different Vegetable Colouring Matters." Month. Micro. Journ., vol. xvii, 1877, p. 225. Ditto. " An Introduction towards the Application 186 BIBLIOGRAPHY. of the Microspectroscope to the Study of Evergreens." Month. Micro. Journ., vol. xviii, 1877, p. 224. PIFFARD, H. G. " The Mode of Recording Absorp- tion Spectra." Being a letter in the Month. Micro. Journ., vol. xiv, 1875, p. 37. PREYER, W. " Quantitative Bestimmung des Farb- stoffs im Blute durch das Spectrum." Ann. d. Chem. u. Pharm., 1866, pp. 187200. Ditto. "Die Blutkrystalle," Jena, 1871. This book contains two beautiful charts of absorption spectra printed in chromo -lithography. PROCTOR, R. A. " The Spectroscope and its Work, 5 ' London, 1877. PURSER, J. M., PROF. " Lectures on the Blood- corpuscles." Irish Hosp. Gaz., 1873. QUINQUAND. " On the Distribution of Haemoglobin in the Animal Kingdom." Abstract in Month. Micro. Journ., vol. xi, 1874, p. 167. REYNOLDS, J. EMERSON, PROF. " Spectrum Analysis as applied to the Detection of Poisons, Adulterations, and Blood." Irish Hosp. Gaz., 1873. RICHARDSON, J. G., M.D. " Improved Method of Applying the Microspectroscopic Test for Blood- stains." Month. Micro. Journ., vol. xv, 1876, p. 30. ROOD, 0. N. " Modern Chromatics." International Scientific Series, vol. xxvii. ROSCOE, H. E., F.R.S. " Lectures on Spectrum Analysis," 3rd edit., London, 1873. SALKOWSKI. " Hamatoidin u. Bilirubin." Hoppes* Med.-Chem. Unters., 3 Heft, S. 436. SCHELLEN, H. " Spectrum Analysis." Translated BIBLIOGRAPHY. 187- by Jane and Caroline Lassel. Edited by Huggins. London, 1872. SCHULTZE'S, MAX, Arcliiv., May, 1871. In this paper, 7 Band, 3 Heft, is an article on the Spectro- scope in Microscopy. SOEBY, H. C., F.R.S. " On some Improvements in the Spectrum Method of Detecting Blood." Month. Micro. Journal, vol. vi, 1871, p. 9. Ditto. " On the Application of Spectrum Analysis to Microscopical Investigation, and especially to the Detection of Blood-stains." Chemical News, xi, pp. 186, 194, 282, 256. Ditto. " On some Technical Applications of the Spectrum Microscope." Quart. Journal Micro. Sci., ix, p. 358. Ditto. " On the Colouring Matters derived from the Decomposition of some Minute Organisms." Month. Micro. Journal, 1870, p. 229. Ditto. " On the Examination of Mixed Colouring Matters with the Spectrum. Microscope." Month. Micro. Journal, vol. vi, 1871, p. 124, et seq. Ditto. " On New and Improved Microscope Spectrum Apparatus, and on its Application to various Branches of Research." Month. Micro. Journ., vol. xiii, 1875, pp. 198208. Ditto. " On a New Method of Measuring the Position of the Bands in Spectra." Month. Micro. Journ., vol. xiv, 1875, p. 269. Ditto. " On the Evolution of Hsemoglobin." Quart. Journ. Micro. Science, 1876. Abstract in Month. Micro. Journ., vol. xv, 1876, p. 149. 188 BIBLIOGRAPHY. Ditto. " On a New Form of Small Pocket Spectro- scope." Month. Micro. Journal, vol. xvi, 1876, p. 64. Ditto. " On the various Tints of Autumnal Foliage." Quart. Journ. Micro. Sci., vol. i, p. 64. Ditto. " On some Compounds derived from the Colouring Matter of Blood." Quart. Journ. Micro. Sci., 1870, pp. 400402. Ditto. "On a Definite Method of Qualitative Analysis of Animal and Vegetable Colouring Matters by means of the Spectrum Microscope." Proc. Roy. Society, vol. xv, p. 433. Ditto. " On a New Microspectroscope, and on a New Method of Printing a Description of the Spectra seen with the Spectrum-microscope." Chem. News., vol. xv, p. 220. Ditto. "Colouring Matter of Birds' Eggs." Proceed. Zool. Soc., Lond., 1875, p. 351. STOKES, G. G. " On the Eeduction and Oxydation of the Colouring Matter of the Blood." Proc. Roy. Soc., vol. xiii, p. 353. Ditto. " On the Discrimination of Organic Bodies by their Optical Properties." Roy. Instit., March 4th, 1864. Phil. Mag., 4th series, xxvii, p. 388. Ditto. " On the Examination of Mixed Colouring Matters." Journ. Chem. Soc., June 2nd, 186^ (New Series), vol. ii, pp. 304318. STEICKER, S. " Researches with the Microspectro- scope." Archiv. fur d. Ges. Physiol., 1868, Bonn. STRUVE, H. " On the Existence in the Animal Organism of a New Compound exhibiting the Ab- sorption Spectrum of Blood." Deut. Chem. Ges. Ber., BIBLIOGRAPHY. 189 ix, pp. 623 627 ; and Journ. Chem. Soc., vol. ii, 1876, p. 318. See also Thudichum, Annals Chem. Med., infra, p. 103. STROGANOFF, N. " On the Process of Oxydation in Normal and Asphyxiated Blood." Pfliiger's Archiv., Band xii; Centralblatt f. d. Med. Wiss., No. 28. See Lond, Med. Rec., Dec. 15th, 1876, p. 539. SUFFOLK, "W. T. " On Spectrum Analysis applied to Microscopical Investigation." London, 1873. THUDICHUM, J. L. W. Tenth Report of the Medical Officer of the Privy Council, 1867 (published 1868). " Researches intended to Promote an Improved Che- mical Identification of Diseases." Ditto. " Annals of Chemical Medicine," vol. i, 1879, p. 93 et seq. (In this volume Thudichum shows that the colouring matter of birds' eggs is identical with cruentin.) Ditto. " Lutein." Centralblatt fur d. Med. Wiss., 1869. Ditto. "The Pathology of the Urine," London, 1877. Ditto. " Chemical Physiology," London, 1872. Ditto. " On some Reactions of Biliverdin." Journ. Chem. Soc., vol. ii, 1876, p. 27 et seq. THUDICHUM and KINGZETT. " On Hemin, Hematin, and a Phosphorised Substance contained in Blood- corpuscles." Journ. Chem. Soc., vol. ii, 1876, p. 255 et seq. VALENTIN, G. " Histologische und Physiologische Studien," Abth. ix. Zeitschrift fiir Biologie, vi. Ditto. " Einige neue Beobachtungen iiber die 190 BIBLIOGRAPHY. Erkenntniss des Blutes durch das Spectroskop." Arch. f. Pathol. Anatom. u. Physiol. u. Klin. Med., Bd. 26, S. 580585, 1863. VOGEL. "Recent Researches on Absorption Spectra." Nature, vol. xix, March 27th, p. 495. Ditto. " Spectroscopy of the Colouring Matters of the Blood." Deut. Chem. Ges. Ber., ix, 1472, 1473. WARD, F. H. ' ' Improvements in the Microspectro- scope." Trans. Roy. Micro. Soc., vol. i, 1878. WATTS, H. Dictionary of Chemistry, 1st Suppl., 1872; 2nd Suppl., 1875. Arts. "Blood, Bile, and Urine." WATTS, W. M. " Index of Spectra," London, 1872. WISKEMANN, MAX. " Spectroscopic Estimation of Haemoglobin." Zeitschr. f. BioL, xii, 434 447. Journ. Chem. Soc., March, 1878. ADDITIONAL NOTE. WHILE the last page of this book was passing through the press a small paragraph appeared in the ' Medical Times and Gazette ' (Dec. 6th, 1879), headed "Poisoning by Chlorate of Potash," from which it appears that, according to Dr. F. Marchand ('Virchow's Archiv,' Bd. 27, Heft 3), chlorate of potash in poisonous doses causes the blood to assume a chocolate colour, this change of colour being due to the conversion of haemoglobin into methsemoglobin, as evidenced by the spectrum. (Cf. the action of nitrites.) INDEX. A. PAGE ABSORPTION spectra ..... 5-7 mapping . . 1923,2729 method of observing . . .19 physiological . . . 48, et seq. Yogel's researches on . . 49-54 Acid hseniatin, see Hamatin. Acids, action of, on haemoglobin . . . 114, 115 Alkalis, action of, on blood .... 118, 119 and alkaline earths, spectra of . . 12-14,34,35 Alkaline hsematin, see Hamatin. Ammonia and alcohol, action of, on blood . . 102, 103, 119 gas, action of, on hsematin . . . H7 on haemoglobin . . . 100 Angle, refracting . . . . . .1 Angstrom . 3, 30, 31 Animal kingdom, distribution of haemoglobin in . 63, 64 Animals, bile spectra of . . . 156-160 Antimoniuretted hydrogen, action of, on hsematin . .117 on haemoglobin . . 101 Antimony, spectrum of . . . .38 Arsenic . . . . .37 Arseniuretted hydrogen, action of, on haematin . . 117 on haemoglobin . . 100 Ascitic fluid, spectrum of . . . . .174 Asphyxia, spectrum of blood in death from . . 74 } 75 192 INDEX. B. BILE of blackbird, spectrum of ofcat of chicken of crow of dog of duck ...... of frog of goose ,, of guinea-pig of hedgehog of lower animals, spectra of of mouse, spectrum of ofox of pig of rabbit of sheep of wild duck pigments described by Thudichum sketch of chemistry of spectrum of human . Bilicyanin . Bilifuscin Blood in urine, detection of spectra, easy method of procuring stains, detection of . effect of mordants on faint on leather Richardson on Sorby on Boviprasine Brewster . Bromine, action of, on blood PAGE . 159 . 157 . 159 . 159 . 157 . 159 . 159 . 159 . 158 . 159 156-160 . 158 . 158 . 157 . 158 . 158 . 159 154, 155 149-151 151. 152 152. 153 . 150 125, 126 118-123 130-148 . 137 136, 137 138-141 143-148 130-143 . 155 5 120, 121 0. CALCULI, bright-line spectra of Camera-lucida method of mapping . 34 21-23 INDEX. 193 PAGE Carbonic oxide, spectrum of blood after death from . 76-80 Cells and tubes for microspectroscope . . 26, 27 Charcoal fumes, death from, see Carbonic oxide. " Chemical circulation " . . . 39-47 ............... spectroscopes . . . 10,11 Chlorocruorin . . . - . . . . .64 Choletelin . . . . . 152,153 Cholocyanine ...... 154 .................. sulphate of . ... . . 154 Cholonematine . . . . , . 155 Cholothalline . . . . . .154 Colouring matters of blood, bile and urine, connection of 170-172 Cruentin, alkaline .... 107, 122 ............ hydrochloric product of neutral . . 108, 122 ............ neutral. .... 106,122 ............ reduced .... 107,108,122 ............ sulphate . . . ---. 106,121 Cyanhsematin, see Cyanides. Cyanides, action of, on blood . . .. 85-89, 123 Cyanosulphsem . . . . . .64 DIFFRACTION spectrum . Dispersion, irrationality of . ............... of light by prism Bonders . 29-31 29 et seq. .2 78 ELECTRIC lamp . F.ECES, spectra of Fluopittine, spectrum of Fraunhofer's lines E. F. G. GAMGEE, Prof., on the action of nitrites on the action of cyanides 19,20 174, 175 . 175 3,7-9 90-95 88 13 194 IXDEX. PAGE Gases, morbid, spectra of . . . . 38, 39 Gmelin's reaction, spectrum of . . 160-162 HJEMATIN, acid . . . . . . 103, 104, 120 action of ammonia, arsine, and stibine on . . 117 carbonic oxide on . . .' . 115 phosphorous chloride containing free phos- phorus . . . . . . 116 tin and hydrochloric acid on . . 116 alkaline . , . . 105,118,119 and hsemachromogen -.. . . . 101 five-banded . . . . . 103 general account of, and its reactions . 108-110 hydrochloride . 112-114 in ovarian and parovarian cysts . . 126-130 iron-free .... 110-112 reduced .... 106,119,120 Thudichum's researches on . . 102-106 Hsematinometer . . .* . . . 65 Hsematoin . . . . . .111 Haematolin . . . . . . Ill Hsematoporphyrin . . ,.. . Ill, 112 Hsematuria, paroxysmal, spectrum of urine of . . . 124 spectrum of urine in . . . 124-126 Hsemin . . . . . . 112-114 Hsemochromogen . . . . . . 101 Haemoglobin, action of carbonic oxide on . . 76-80 of various acids on . ,: 114,115 other reagents on, see these. distribution of, in animal kingdom . 63, 64 , ... estimation of . . . . 64,67 reduced .... 67-71 spectrum of, oxidized . . . 61-63 Hofmann . . . . . .73 Hoppe . . . . . . .67 Hoppe-Seyler . . .74, 83, 84, et seq. Human bile, see Site. INDEX. 195 PAGE Hydrobilirubin ..... 167, 171 Hydrobiliverdin ...... 171 Hyocceruline . ... 155 Hyoflavine . 155 INDUCTION coil . . . . . 14-17 Iodine, action of, on blood ..... 121 Irrationality of dispersion . . . 29 et seq. J. Ja/e ..... 161,162,166,167 K. Koeberle's test for paralbumin . . . . 129 Kosclilakoff . . . . . .117 and Bogomoloff . . . ^ ' 80,100 Krauss on hsemin crystals ..... 130 Kundt on absorption spectra . . . .50 L. Lankester on sulphsemoglobin . . . .81 on nitrites t . . . .95 chlorocruorin . . . . .64 Lead, spectrum of . . . . .38 Lines of Fraunhofer . . . . 3, 7, 9 Liquor amnii, spectrum of . . . . .175 Lutein in blood-serum and pathological fluids . . 173, 174 in butter ...... 173 (including sero-, butyro-, cysto-, and intestino-lutein) 172-174 in eggs . . . . . . 173 in faeces of infants ..... 173 in ovarian and parovarian cysts . . 130,173 M. Maty, Heynsius, and Campbell .... 152 Maty's hydrobilirubin . ... . 167, 171 Mapping spectra ..... 19-23,27-29 Medical jurisprudence, spectroscope in . . 130-148 Metals, heavy, spectra of . 14-19 196 INDEX. Metals of the alkalis and alkaline earths . . poisonous, spectra of Mercury, spectrum of Methaemoglobin . . Micrometer, bright-point Microspectroscope cells and tubes for Mixed colouring matters, Sorby on . Murexide, spectrum of , . . . N. Nawrocki . . < Newton's discovery of the prismatic analysis of light Nitric oxide, effect of, on blood * Nitrites, action of, on blood Nitrous oxide, effect of, on blood OVARIAN and parovarian cyst O. P. PAROVARIAN cysts Pettenhqfer's test, spectrum of Pleuritic fluids, spectra of . Podoliriki Poisonous metals, spectra of Popoffon. action of carbonic acids on hsematin Preyer on estimation of haemoglobin . on hsematoin on sulphsemoglobin . see Appendix II. Prism, action of, on light . definition of R. REFRACTING angle Refraction by prism Richardson, Dr. J. G., on blood stains Hitter on a blue bile pigment PAGE 12-14, 34, 35 35-38 . 38 98-100, 122 27, 28 23-26 26, 27 54^60 175 81, 87, et seq. . $.. . 80 90-9S 75, 76 126-130 126-130 165, 166 . 174 . 79 35-38 . 115 64-67 . Ill 82, 84 1 1 1 2 143-148 153, 154 INDEX. 197 s. PAGE 3 8 130-143 54-60 176 Simms introduces the collimating lens Sodium line, reversal of Sorby on " blood stains " on " mixed colouring matters " on wave-lengths .... see Appendix II. Spark condenser (Browning's) ... 15, 16 Spectra, bright- line . . . . 5, 12, 19 continuous . . . . . 5, 12 how to map, see Mapping. of alkalis and alkaline earths . . v. supra of blood, bile, urine, &c., see these. of heavy metals v. supra of poisonous metals v. supra stellar . . . . . .7 Spectrographs . . . . .21 Spectroscope, essential parts of chemical . . .4 micro- .... 23-26 miniature . . . . .61 Spectroscopes, chemical . . . . 10, 11 Spectrum, solar . . . . . 2, 3, 7 Stars, spectra of, see Spectra. Stokes, Prof. G. G. . . . . 67, 82 see Appendix. StoTcvis on a product of oxidation of bile pigments . 163, 164, 168 Sulphsemoglobin .... 80-84,123 Sulphuretted hydrogen, action on blood of, see Sulphaemoglobin. Sun, spectrum of, see Spectrum. T. Thudichum, Dr, on bilirubin on hsematin and cruentin on lutein . on other bile spectra see Appendix II. U. URINE containing bile acids, see Pettenkofer's test. pigment, see Gmelin's reaction. . 150 102-108 172-174 154, 155 14 198 INDEX. Urine containing blood, spectrum of diseased, spectra of . spectrum of Urobilin constantly present in urine . in bile of mouse, see Bile. in pink u rates Urocyanine in urine of cholera Urorubine V. VENOUS blood, spectrum of, after death Vogel on absorption spectra W. WAVE-LENGTHS, calculation of of Fraunhofer's lines remarks on Wollaston, Dr PAGE 125, 126 168-170 . 167 162, 163 166-168 . 168 . 172 172 71-74 49-54 29-33 31,32 176-179 3 Z. Zuntz Zweifel on meconium 78 174 The names of any authors omitted will be found in the Appendix. ERRATA. Page 8, second line from bottom, for "burned in an ron spoon," read " placed in an iron spoon." In Chart I, Sp. 19, there is some shading shown between the second and third bands ; this is not correct. Catalogue B] London, n, New Burlington Street November, 1882 S E L E C T I O N FROM J, & A. CHURCHILL'S GENERAL CATALOGUE COMPRISING ALL RECENT WORKS PUBLISHED BY THEM ON THE OF N.B.-As far as possible, this List is arranged in the order ii which medical study is usually pursued. A SELECTION FROM J. & A. CHURCHILL'S GENERAL CATALOGUE, COMPRISING ALL RECENT WORKS PUBLISHED BY THEM ON THE ART AND SCIENCE OF MEDICINE. N.B.- J. & A. Churchill 's Descriptive List of Works on Chemistry, Materia Medica, Pharmacy, Botany, Photography, Zoology, the Microscope, and other Branches of Science, can be had on application. Practical Anatomy : A Manual of Dissections. By CHRISTOPHER HEATH,. Surgeon to University College Hospital. Fifth Edition. Crown 8vo, with 24 Coloured Plates and 269 Engrav- ings, 155. Wilson's Anatomist's Vade- Mecum. Tenth Edition. By GEORGE BUCHANAN, Professor of Clinical Surgery in the University of Glasgow ; and HENRY E. 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Acton's Reproductive Organs, 14 Adams (W.) on Clubfoot. n Contraction of the Fingers, 1 1 Curvature of the Spine, n Allan on Fever Nursing, 7 Allingham on Diseases of the Rectum, 13 Anatomical Remembrancer, 4 Anderson (McC.) on Eczema, 13 Avelingon the Chamberlensand the Midwifery Forceps, 6 Influence of Posture on Women, 6 Balfour's Diseases of the Heart and Aorta, 9 Balkwill's Mechanical Dentistry, 13 Bantock on Rupture of the Female Perineum, 6 Barclay's Medical Diagnosis, 8 Barnes on Obstetric Operations, 5 on Diseases of Women, 5 Beale's Microscope in Medicine, 8 Slight Ailments, 8 Bellamy's Surgical Anatomy, 3 Bennet (J.H.) on the Mediterranean, 10 1 on Pulmonary Consumption, 10 on Nutrition, 10 Bentley and Trimen's Medicinal Plants, 7 Bentley's Manual of Botany, 7 Bigg (H. H.) on Orthopraxy, n Bigg (R. H.) on the Orthopragms of Spine, n Binz's Elements of Therapeutics, 7 Black on the Urinary Organs, 14 Bose's Rational Therapeutics, 7 Recognisant Medicine, 7 Braune's Topographical Anatomy, 3 Brodhurst's Anchylosis, n Orthopaedic Surgery, n Bryant's Practice of Surgery, 11 Bucknill and Tuke's Psychological Medicine, 5 Bulkley on Eczema, 13 on Diseases of the Skin, 13 Burdett's Cottage Hospitals, 5 Pay Hospitals, 5 Burnett on the Ear, 12 Burton's Midwifery for Midwives, 5 Butlin's Sarcoma and Carcinoma, 13 Buzzard's Diseases of the Nervous System, 9 Carpenter's Human Physiology, 4 Carter (H. V.) on Spirillum Fever, 8 Carter (W.) on Renal and Urinary Diseases, 14 Cayley's Typhoid Fever, 9 Charteris' Practice of Medicine, 8 Clark's Outlines of Surgery, 10 Clay's (C.) Obstetric Surgery, 6 Cobbold on Parasites, 14 Coles' Dental Mechanics, 13 Deformities of the Mouth, 13 Cormack's Clinical Studies, 8 Coulson on Stone in the Bladder, 14 on Syphilis, 14 on Diseases of the Bladder, 14 Courty's Diseases of the Uterus, Ovaries, &c., 5 Cripps' Cancer of the Rectum, 14 Curling's Diseases of the Testis, 13 Daguenet's Manual of Ophthalmoscopy, 12 Dalby's Diseases and Injuries of the Ear, 12 Dalton's Human Physiology, 4 Day on Diseases of Children, 6 on Headaches, 10 De Chaumont's Sanitary Assurance, 4 Dobell's Lectures on Winter Cough, 8 Loss of Weight, &c., 8 Mont Dore Cure, 8 Domville's Manual for Nurses, 7 Druitt's Surgeon's Vade-Mecum, n Duncan on the Female Perineum, 5 on Diseases of Women, 5 Dunglison's Medical Dictionary, 14 Ellis' s Manual for Mothers, 6 of the Diseases of Children, Emmet's Gynaecology, 5 Eulenburg and Guttmann's System of Nerves, 9 Fayrer's Climate and Fevers of India, 7 Observations in India, 7 Tropical Dysentery and Diarrhoea, 7 Fenwick's Chronic Atrophy of the Stomach, 8 Medical Diagnosis, 8 Outlines of Medical Treatment, 8 Fergusson's Practical Surgery, 10 Flint on Phthisis, 8 on Clinical Medicine, 8 Flower's Diagrams of the Nerves, 4 Foster's Clinical Medicine, 8 Fox's (C. B.) Examinations of Water, Air, and Food, 4 Fox's (G. H.) Photographs of Cutaneous Syphilis, 14 Skin Diseases, 13 Fox's (T.) Atlas of Skin Diseases, 13 Frey's Histology and Histo-Chemistry, 4 Fulton's Text-Book of Physiology, 4 Galabin's Diseases of Women, 6 Gamgee's Fractures of the Limbs, n Treatment of Wounds, 1 1 Gant's Diseases of the Bladder, 14 Gaskoin on Psoriasis or Lepra, 13 Gay on Hffimorrhoidal Disorder, 13 Gill on Indigestion, 10 Glenn's Laws affecting Medical Men, 14 Godlee's Atlas of Human Anatomy, 3 Gowers' Diseases of the Spinal Cord, 9 Epilepsy. 9 Medical Ophthalmoscopy, 9 Pseudo-Hypertrophic Muscular Paralysis, 9 Habershon's Diseases of the Abdomen, 9 Stomach, 9 Pneumogastric Nerve, 9 Hamilton's Nervous Diseases, 9 Hardwicke's Medical Education, 14 Harley on Diseases of the Liver, 9 Harris on Lithotomy, 14 Harrison's Surgical Disorders of the Urinary Organs, 14 Prevention of Stricture, 14 Heath's Injuries and Diseases of the Jaws, 10 Minor Surgery and Bandaging, 10 Operative Surgery, 10 Practical Anatomy, 3 Surgical Diagnosis, 10 Hemming on the Laryngoscope, 12 Higgens' Ophthalmic Out-patient Practice, n Hillis' Leprosy in British Guiana, 13 Hogg's Indian Notes, 7 Holden's Dissections, 3 Human Osteology, 3 Landmarks, 3 Holmes' (G.) Guide to Use of Laryngoscope, 12 Vocal Physiology and Hygiene, 12 Hood on Gout, Rheumatism, &c. , 9 Hooper's Physicians' Vade-Mecum, 8 Horton's Tropical Diseases, 7 Hutchinson's Clinical Surgery, n Rare Diseases of the Skin, 13 Huth's Marriage of Near Kin, 4 Ireland's Idiocy and Imbecility, 5 Irvine's Relapse of Typhoid Fever, 9 James on Sore Throat, 12 Jones' (C. H.) Functional Nervous Disorders, 9 Jones (C. H.) and Sieveking's Pathological Anatomy, Jones' (H. McN.) Aural Surgery, 12 Atlas of Diseases of Membrana Tympani, 12 Jones' (T. W.) Ophthalmic Medicine and Surgery, n Jordan's Surgical Enquiries, n Lancereaux's Atlas of Pathological Anatomy 4 Lane's Lectures on Syphilis, 14 Lee (H.) on Syphilis, 14 Leared on Imperfect Digestion, 10 Lewis (Bevan) on the Human Brain, 4 Liebreich's Atlas of Ophthalmoscopy, n Liveing's Megrim, Sick Headache, &c. , 10 Lucas's Indian Hygiene, 8 Macdonald's (A.) Chronic Disease of the Heart, 6 Macdonald's (J. D.) Examination of Water, 4 Macewen's Osteotomy: Knock-knee, Bow-leg, &c., n Mackenzie on Diphtheria, 12 on Diseases of the Throat and Nose, 12 Maclise's Dislocations and Fractures, n Surgical Anatomy, 3 MacMunn's Spectroscope in Medicine, 8 Macnab's Medical Account Books, 14 Macnamara's Diseases of Bones and Joints, n the Eye, 12 Madden's Principal Health-Resorts, 10 Marsden's Certain Forms of Cancer, 13 Martin's Military and State Medicine, 5 Mason on Hare-Lip and Cleft Palate, 12 on Surgery of the Face, 12 Mayne's Medical Vocabulary, 14 ' Notes on Poisons, 7 Therapeutical Remembrancer, 7 Mitchell (R.) on Cancer Life, 13 [Continued on the next /afe. I N DEX continued Mitchell's (S. Weir) Nervous System in Women, 6 Moore's family Medicine for India, 7 Health Resorts for Tropical Invalids, 7 Morris' (H.) Anatomy of the Joints, 3 Nettleship's Diseases of the Eye, 12 Nunn's Cancer of the Breast, 13 Ogston's Medical Jurisprudence, 4 Osborn on Diseases of the Testis, 13 on Hydrocele, 13 Parkes' Practical Hygiene, 5 Pavy on Diabetes, 10 on Food and Dietetics, 10 Peacock's Diseases of the Heart, 9 Pharmacopoeia of the London Hospital, 7 Phillips' Materia Medica and Therapeutics, 7 Pirrie's Principles and Practice of Surgery, 1 1 Pollock on Rheumatism, 9 Pridham on Asthma, 8 Purcell on Cancer, 13 Radford's Ca5sarean Section, 5 Ralfe's Morbid Conditions of the Urine, 14 Ramsbotham's Obstetrics, 6 Reynolds' (J. J.) Diseases of Women, 6 Notes on Midwifery, 6 Reynolds' (J. R.) Clinical Electricity, 10 Roberts' (C.) Manual of Anthropometry, 5 Roberts' (D. Lloyd) Practice of Midwifery, 5 Ross's Diseases of the Nervous System, 9 Roth on Dress : Its Sanitary Aspect, 5 Roussel's Transfusion of Blood, 8 Routh's Infant Feeding, 6 Royle and Harley's Materia Medica, 7 Sanderson's Physiological Handbook. 4 Sansom's Diseases of the Heart, 9 Antiseptic System, 9 Savage on the Female Pelvic Organs, 6 Sayre's Orthopaedic Surgery, n Schroeder's Manual of Midwifery, 6 Sewill's Dental Anatomy, 12 Sheppard on Madness, 5 Sibson's Medical Anatomy, 3 Sieveking's Life Assurance, 14 Smith's (E.) Wasting Diseases of Children, 6 Clinical Studies, 6 Smith's (Henry) Surgery of the Rectum, 14 Smith's (Heywood) Dysmenorrhea, 6 Gynaecology, 6 Smith (Priestley) en Glaucoma, 12 Southam's Regional Surgery, 10 Sparks on the Riviera, 10 Squire's Companion to the Pharmacopoeia, 7 Squire's Pharmacopoeias of London Hospitals, 7 Stille and Maisch's National Dispensatory, 7 Stocken's Dental Materia Medica and Therapeutics, 12 Sullivan's Tropical Diseases, 7 Swain's Surgical Emergencies, 10 Swayne's Obstetric Aphorisms, 6 Taft's Operative Dentistry, 12 Taylor's Medical Jurisprudence, 4 Poisons in relation to Medical Jurisprudence, 4 Teale's Dangers to Health, 5 Thomas on Ear and Throat Diseases, 12 Thompson's (SirH.) Calculous Disease, 13 Diseases of the Urinary Organs, 13 Diseases of the Prostate, 13 Lithotomy and Lithotrity, 13 Thompson's (Dr. H.) Clinical Lectures, 8 Thorowgood on Asthma, 8 on Materia Medica and Therapeutics, 7 Thudichum's Pathology of the Urine, 14 Tibbits* Medical and Surgical Electricity, 10 Map of Motor Points, 10 Tidy and Woodman's Forensic Medicine, 4 Tilt's Change of Life, 6, Uterine Therapeutics, 6 Tomes' (C. S.) Dental Anatomy, 12 (J. &C. S.) Dental Surgery, 12 Van Buren on the Genito-Urinary Organs, 14 Veitch's Handbook for Nurses, 6 Virchow's Post-mortem Examinations, 4 Wagstaffe's Human Osteology, 3 Walker's Ophthalmology, 12 Waring's Indian Bazaar Medicines, 7 Practical Therapeutics, 7 Warner's Guide to Medical Case-Taking, 8 Waters' (A. T. H.) Diseases of the Chest, 8 Waters (J- H.) on Fits, 9 Wells (Spencer) on Ovarian and Uterine Tumours, 6 West and Duncan's Diseases of Women, 5 Whistler's Syphilis of the Larynx, 12 Whitehead's (J. L.) Climate of the UnderclifF, 10 Whittaker's Primer on the Urine, 14 Wilks and Moxon's Pathological Anatomy, 4 Wilson's (Sir E.) Anatomists' Vade-Mecum, 3 Lectures on Dermatology, 13 Wilson's (G.) Handbook of Hygiene, 5 Healthy Life and Dwellings, 5 Wilson's (W. S.) Ocean as a Health-Resort, 10 Wise's Davos Platz, 10 Wolfe's Diseases and Injuries of the Eye, n Yeo's Contagiousness of Pulmonary Consumption, 8 The following CATALOGUES issued by J. & A. CHURCHILL will be forwarded post free on application : A. J. & A. Churchill's General List of about 650 'works on Anatomy, Physiology, Hygiene, Midwifery, Materia Medica, Medicine, Surgery, Chemistry, Botany, &c., &c., with a complete Index to their Subjects, for easy reference. N.B. This List includes B, C, & D. B. Selection from J. & A. Churchill's General List, comprising all recent Works published by them on the Art and Science of Medicine. C. J. & A. Churchill's Catalogue of Text Books specially arranged for Students. D. A selected and descriptive List of J. & A. Qiur chill's Works on Chemistry, Materia Medica, Pharmacy, Botany, Photography, Zoology, tfie Microscope, and other branches of Science. E. The Half-yearly List of New Works and New Editions published by J. & A. Churchill during the previous six months, together with Partictdars of the Periodicals issued from their House. [Sent in January and July of each year to every Medical Practitioner in the United Kingdom whose name and address can be ascertained. A large number are also sent to the United States of America, Continental Europe, India, and the Colonies.] AMERICA. J. & A. Churchill being in constant communication with various publishing houses in Boston, New York, and Philadelphia, are able, notwithstanding the absence of international copyright, to conduct negotiations favourable to English Authors. LONDON : NEW BURLINGTON STREET. Pardon &* Sons, Printers,} [Paternoster Row, London. UNIVERSITY OF CALIFORNIA LIBRARY This book is DUE on the last date stamped below. Fine schedule: 25 cents on first day overdue 50 cents on fourth day overdue . One dollar on seventh day overdue. MAY 5 1947 MAY 5 1947 INTER-LIBRARY LOAN LD 21-100j7i-12,'46(A2012sl6)4120