(P) with its rays parallel. In passing through the prism the rays are dispersed by unequal refraction, giving a spectrum. The spectrum thus produced is examined by the observer with the aid of the telescope (B). When the telescope is properly focussed for the rays entering it from the prism (P), a clear picture of the spectrum is seen. The length of the spectrum will depend upon the nature and the number of prisms through which the light is made to pass. For ordinary purposes a short spectrum is preferable for hæmoglobin bands, and a spectroscope with one prism is generally used. If the source of light is a lamp-flame of some kind, the spectrum is continuous, the colors gradually merging one into another from red to violet. If sunlight is used, the spectrum will be crossed by a number of narrow dark lines known as the "Fraunhofer lines"

A

B

FIG. 2.-Spectroscope: P, the glass prism; a, the collimator tube, showing the slit (s) through which the light is admitted; B, the telescope for observing the spectrum.

(see Pl. I., Frontispiece, for an illustration in colors of the solar spectrum). The position of these lines in the solar spectrum is fixed, and the more distinct ones are designated by letters of the alphabet, A, B, C, D, E, etc., as shown in the charts below. If while using solar light or an artificial light a solution of any substance which gives absorption bands is so placed in front of the slit that the light is obliged to traverse it, the spectrum as observed through the telescope will show one or more narrow or broad black bands, that are characteristic of the substance used and constitute its absorption spectrum. The positions of these bands may be designated by describing their relations to the Fraunhofer lines, or more directly by stating the wave-lengths of the portions of the spectrum between which absorption takes place. Some spectroscopes are provided with a scale of wave-lengths superposed on the spectrum, and when properly adjusted this scale enables one to read off directly the wave-lengths of any part of the spectrum.

When very dilute solutions of oxyhemoglobin are examined with the spectroscope, two absorption bands appear, both occurring in the portion of the spectrum included between the Fraunhofer lines D and E. The band nearer the red end of the spectrum is known as the "a-band;" it is narrower, darker, and more clearly defined than the other, the "B-band" (Fig. 3, and also Pl. I. spectrum 4). With a solution containing 0.09 per cent. of oxyhæmoglobin, and examined in layers one centimeter thick, the a-band extends over the part of the spectrum included between the wave-lengths λ 583

(583 millionths of a millimeter) and λ 571, and the B-band between 550 and À 532 (Gamgee). The width and distinctness of the bands vary naturally with the concentration of the solution used (see Pl. I. spectra 2, 3, 4, and 5),

FIG. 3.-Diagrammatic representation of the absorption spectrum of oxyhæmoglobin (after Rollett). The numerals give the wave-lengths in hundred-thousandths of a millimeter; the letters show the positions of the more prominent Fraunhofer lines of the solar spectrum. The red end of the spectrum is to the left. The a-band is to the right of D, the B-band to the left of E.

or, if the concentration remains the same, with the width of the stratum of liquid through which the light passes.

With a certain minimal percentage of oxyhemoglobin (less than 0.01 per cent.) the B-band is lost and the aband is very faint in layers one centimeter thick. With stronger solutions the bands become darker and wider and finally fuse, while some of the extreme red end and a great deal of the violet end of the spectrum is also absorbed. The variations in the absorption spectrum with differences in concentration are clear0.2 ly shown in the accompanying illus

0,6

0.5

0.4

0.3

0.1

0.

Gh

FIG. 4.-Diagram to show the variations in the ab. sorption spectrum of oxyhæmoglobin with varying

tration from Rollett' (Fig. 4); the thickness of the layer of liquid is supposed to be one centimeter. The numbers on the right indicate the percentage strength of the oxy

concentrations of the solution (after Rollett). The hæmoglobin solutions. It will be

numbers to the right give the strength of the oxyhæmoglobin solution in percentages; the letters give the positions of the Fraunhofer lines. To ascertain the amount of absorption for any given concentration up to 1 per cent., draw a horizontal line across the diagram at the level corresponding to the concentration. Where this line passes through the shaded part of the diagram absorption takes place, and the width

noticed that the absorption which takes place as the concentration of the solution increases affects the redorange end of the spectrum last of all. Solutions of reduced hæmo

of the absorption bands is seen at once. The diagram globin examined with the spectro

shows clearly that the amount of absorption increases as the solutions become more concentrated, especially the absorption of the blue end of the spectrum. It will be noticed that with concentrations between 0.6

into one.

scope show only one absorption. band, known sometimes as the

and 0.7 per cent. the two bands between D and E fuse "y-band." This band lies also in the portion of the spectrum included between the lines D and E; its relations to these lines and the bands of oxyhæmoglobin are shown in Figure 5 and in Pl. I. spectrum 6. The 1 Hermann's Handbuch der Physiologie, Bd. iv., 1880.

y-band is much more diffuse than the oxyhæmoglobin bands, and its limits therefore, especially in weak solutions, are not well defined; in solutions of blood diluted 100 times with water, which would give a hæmoglobin solution of about 0.14 per cent., the absorption band lies in the part of the spectrum included between the wave-lengths ▲ 572 and 542. The width

FIG. 5.-Diagrammatic representation of the absorption spectrum of hæmoglobin (reduced hæmoglobin) (after Rollett). The numerals give the wave-lengths in hundred-thousandths of a millimeter; the letters show the positions of the more prominent Fraunhofer lines of the solar spectrum. The red end of the spectrum is to the left. The single diffuse absorption band lies between D and E.

and distinctness of this band vary also with the concentration of the solution. This variation is sufficiently well shown in the accompanying illustration (Fig. 6), which is a companion figure to the one just given for oxyhemoglobin (Fig. 4). It will be noticed that the last light to be absorbed in this case is partly in the red end and partly in the blue, thus explaining the purplish color of hæmoglobin solutions and of venous blood. Oxyhæmoglobin solutions can be converted to hæmoglobin solutions, with a corresponding change in the spectrum bands, by placing the former in a vacuum or, more conveniently, by adding reducing solutions. The solutions most commonly used for this purpose are ammonium sulphide and Stokes's reagent.' If a solution of reduced hæmoglobin is shaken with air, it quickly changes to oxyhæmoglobin and gives two bands instead of one when examined through the spectroscope. Any given solution may be changed in this way from

a BC

D

Eb F

0.9

0.8

0.7

0.6

0.5

0,4

0.3

0.2

0.1

0.

G h

FIG. 6.-Diagram to show the variations in the absorption spectrum of reduced hæmoglobin with vary.

oxyhæmoglobin to hæmoglobin, ing concentrations of the solution (after Rollett). The

numbers to the right give the strength of the hæmoglobin solution in percentages; the letters give the positions of the Fraunhofer lines. For further directions as to the use of the diagram, see the description of

and the reverse, a great number
of times, thus demonstrating the
facility with which hæmoglobin Figure 4.
takes up and surrenders oxygen.

1 Stokes's reagent is an ammoniacal solution of a ferrous salt. It is made by dissolving 2 parts (by weight) of ferrous sulphate, adding 3 parts of tartaric acid, and then ammonia to distinct alkaline reaction. A permanent precipitate should not be obtained.

Solutions of carbon-monoxide hæmoglobin also give a spectrum with two absorption bands closely resembling in position and appearance those of oxyhæmoglobin (see Pl. I. spectrum 7). They are distinguished from the oxyhæmoglobin bands by being slightly nearer the blue end of the spectrum, as may be demonstrated by observing the wave-lengths or, more conveniently, by superposing the two spectra. Moreover, solutions of carbon-monoxide hæmoglobin are not reduced to hæmoglobin by adding Stokes's liquid, two bands being still seen after such treatment. A solution of carbon-monoxide hæmoglobin suitable for spectroscopic examination may be prepared easily by passing ordinary coal-gas through a dilute oxyhæmoglobin solution for a few minutes and then filtering.

Derivative Compounds of Hæmoglobin.-A number of compounds directly related to hæmoglobin have been described, some of them being found normally in the body. Brief mention is made of the best known of these substances, but for the details of their preparation and chemical properties reference must be made to the section on "The Chemistry of the Body."

Methæmoglobin is a compound obtained by the action of oxidizing agents on hæmoglobin; it is frequently found, therefore, in blood stains or pathological liquids containing blood that have been exposed to the air for some time. It is now supposed to be identical in composition with oxyhæmoglobin, with the exception that the oxygen is held in more stable combination. Methæmoglobin crystallizes in the same form as oxyhæmoglobin, and has a characteristic spectrum (Pl. I. spectrum 8).

Hemochromogen is the substance obtained when hæmoglobin is decomposed by acids or by alkalies in the absence of oxygen. It crystallizes and has a characteristic spectrum.

Hæmatin (CHN,FeO3) is obtained when oxyhæmoglobin is decomposed by acids or by alkalies in the presence of oxygen. It is amorphous and has a characteristic spectrum (Pl. I. spectra 9 and 10).

Hamin (C2H3N,FeО,HCl) is a compound of hæmatin and HCl, and is readily obtained in crystalline form. It is much used in the detection of blood in medico-legal cases, as the crystals are very characteristic and are easily obtained from blood-clots or blood-stains, no matter how old these may be.

18 2

Hæmatoporphyrin (C1HNO3) is a compound characterized by the absence of iron. It is frequently spoken of as "iron-free hæmatin." It is obtained by the action of strong sulphuric acid on hæmatin.

Hæmatoidin (C6H1N2O3) is the name given to a crystalline substance found in old blood-clots, and formed undoubtedly from the hæmoglobin of the clotted blood. It has been shown to be identical with one of the bilepigments, bilirubin. Its occurrence is interesting in that it demonstrates the relationship between hæmoglobin and the bile-pigments.

Histohamatins are a group of pigments said to be present in many of the tissues-for example, the muscles. They are supposed to be respiratory pigments, and are related physiologically, and possibly chemically, to hæmoglobin. They have not been isolated, but their spectra have been described.

Bile-pigments and Urinary Pigments-Hæmoglobin is regarded as the parent-substance of the bile-pigments and the urinary pigments.

Origin and Fate of the Red Corpuscles.-The mammalian red corpuscle is a cell that has lost its nucleus. It is not probable, therefore, that any given corpuscle lives for a great while in the circulation. This is made more certain by the fact that hæmoglobin is the mother-substance from which the bilepigments are made, and, as these pigments are being excreted continually, it is fair to suppose that red corpuscles are as steadily undergoing disintegration in the blood-stream. Just how long the average life of the corpuscles is has not been determined, nor is it certain where and how they go to pieces. It has been suggested that their destruction takes place in the spleen, but the observations advanced in support of this hypothesis are not very numerous or conclusive. Among the reasons given for assuming that the spleen is especially concerned in the destruction of red corpuscles, the most weighty is the histological fact that one can sometimes find in teased preparations of spleen-tissue certain large cells which contain red corpuscles in their cell-substance in various stages of disintegration. It has been supposed that the large cells actually ingest the red corpuscles, selecting those, presumably, that are in a state of physiological decline. Against this idea a number of objections may be raised. Large leucocytes with red corpuscles in their interior are not found so frequently nor so constantly in the spleen as we would expect should be the case if the act of ingestion were constantly going on. There is some reason for believing, indeed, that the whole act of ingestion may be a postmortem phenomenon; that is, after the cessation of the blood-stream the amoeboid movements of the large leucocytes continue, while the red corpuscles lie at rest-conditions that are favorable to the act of ingestion. It may be added also that the blood of the splenic vein contains no hæmoglobin in solution, indicating that no considerable dissolution of red corpuscles is taking place in the spleen. Moreover, complete extirpation of the spleen does not seem to lessen materially the normal destruction of red corpuscles, if we may measure the extent of that normal destruction by the quantity of bile-pigment formed in the liver, remembering that hæmoglobin is the mother-substance from which the bile-pigments are derived. It is more probable that there is no special organ or tissue charged with the function of destroying red corpuscles, and that they undergo disintegration and dissolution while in the bloodstream and in any part of the circulation, the liberated hæmoglobin being carried to the liver and excreted in part as bile-pigment. The continual destruction of red corpuscles implies, of course, a continual formation of new

It has been shown satisfactorily that in the adult the organ for the reproduction of red corpuscles is the red marrow of bones. In this tissue hæmatopoiesis, as the as the process of formation of red corpuscles is termed, goes on continually, the process being much increased after hemorrhages and in certain pathological conditions. The details of the histological changes will be found in the text-books of histology. It is sufficient here to state simply that a group of nucleated colorless cells, erythroblasts, is found in the red marrow.