iron is 04. The amounts of the other elements are variously given, but, roughly, they are the same as in the proteids. We know at present as little of the chemical structure of hæmoglobin as of the proteids generally.

On adding an acid or alkali to hæmoglobin, it is broken up into two parts-a brown pigment called hæmatin, which contains all the iron of the original substance, and a proteid of the globulin class called globin. Hæmatin shows different spectroscopic appearances in acid and alkaline solutions, and yields certain products, which will be more fully studied in Lesson XX. For the present we will be content with investigating two of these, called hæmin and hæmatoporphyrin.

Hæmin is of great importance, as the obtaining of this substance in a crystalline form is the best chemical test for blood. Hæmin crystals, sometimes called, after their discoverer, Teichmann's crystals,

FIG. 31. Hæmin crystals magnified. (Preyer.)

are from a chemical standpoint composed of the hydrochloride of hæmatin. They may be prepared for microscopic examination by boiling a fragment of dried blood. with a drop of glacial acetic acid on a slide; on cooling, triclinic plates and prisms of a dark brown colour, often in star-shaped clusters and with rounded angles (fig. 31), separate out.

In the case of an old blood-stain it is necessary to add a crystal of sodium chloride in addition. Fresh blood contains sufficient sodium chloride in itself.

The action of the acetic acid is (1) to split the hæmoglobin into hæmatin and globin; and (2) to evolve hydrochloric acid from the sodium chloride. The hæmatin unites with the hydrochloric acid, and thus hæmin is formed.

Hæmatoporphyrin is iron-free hæmatin; it may be prepared by mixing blood with strong sulphuric acid: the iron is taken out as ferrous sulphate. This substance is also found sometimes in nature; it occurs in certain invertebrate pigments, and may also be found in certain forms of pathological urine.

Hæmoglobin may be estimated (1) by the amount of iron in the ash, or (2) by colorimetric methods, which are more fully described in the Appendix.

Hæmoglobin forms at least four compounds with gases :

With oxygen

With carbonic oxide.
With nitric oxide.

J 1. Oxyhæmoglobin.

2. Methæmoglobin.

8. Carbonic oxide hæmoglobin.
4. Nitric oxide hæmoglobin.

These compounds have similar crystalline forms: they each probably consist of a molecule of hæmoglobin combined with one of the gases in question. They part with the combined gas somewhat readily; but they are arranged in order of stability in the above list, the least stable first.

Oxyhæmoglobin is the compound that exists in arterial blood. Many of its properties have been already mentioned. The oxygen linked to the hæmoglobin, which is removed by the tissues through which the blood circulates, may be called the respiratory oxygen of hæmoglobin. The processes that occur in the lungs and tissues, resulting in the oxygenation and deoxygenation respectively of the hæmoglobin, may be imitated outside the body, using either blood or pure solutions of hæmoglobin. The respiratory oxygen can be removed, for example, in the Torricellian vacuum of a mercurial airpump, or by passing a neutral gas like hydrogen through the blood, or by the use of reducing agents like ammonium sulphide or Stokes' reagent. The older observers estimated that 1 gramme of hæmoglobin will combine with 1.6 c.c. of oxygen.2

If any of these methods for reducing oxyhæmoglobin is used, the bright red (arterial) colour of oxyhæmoglobin changes to the purplish (venous) tint of hæmoglobin. On once more allowing oxygen to come into contact with the hæmoglobin, as by shaking the solution with the air, the bright arterial colour returns.

These colour-changes may be more accurately studied with the spectroscope, and the constant position of the absorption bands seen constitutes an important test for blood pigment. It will be first necessary to describe briefly the instrument used.

The Spectroscope.-When a ray of white light is passed through a prism, it is refracted or bent at each surface of the prism; the whole ray is, however, not equally bent, but it is split into its constituent colours, which may be allowed to fall on a screen. The band of colours beginning with the red, passing through orange, yellow, green,

1 Stokes' reagent must always be freshly prepared: it is a solution of ferrous sulphate to which a little tartaric acid has been added, and then ammonia till the reaction is alkaline.

2 Bohr has recently stated that oxyhæmoglobin is a mixture of several compounds of hæmoglobin with different amounts of oxygen in each.

blue, and ending with violet, is called a spectrum: this is seen in nature in the rainbow. It may be obtained artificially by the glass prism or prisms of a spectroscope.

The spectrum of sunlight is interrupted by numerous dark lines crossing it vertically called Frauenhofer's lines. These are perfectly constant in position, and serve as landmarks in the spectrum. The more prominent, lettered A, B, and C, are in the red; D, in the yellow; E, b, and F, in the green; G and H, in the violet. These lines are due to certain volatile substances in the solar atmosphere. If the light from burning sodium or its compounds be examined spectroscopically, it will be found to give a bright yellow line, or, rather, two bright yellow lines very close together. Potassium gives two bright red lines and one violet line; and the other elements, when incandescent, give characteristic lines, but none so

H

P

A.

H

FIG. 32.-Diagram of spectroscope.

simple as sodium. If now the flame of a lamp be examined, it will be found to give a continuous spectrum like that of sunlight in the arrangement of its colours, but unlike it in the absence of dark lines; but if the light from the lamp be made to pass through sodium vapour before it reaches the spectroscope, the bright yellow light will be found absent, and in its place a dark line, or, rather, two dark lines very close together, occupying the same position as the two bright lines of the sodium spectrum. The sodium vapour thus absorbs the same rays as those which it itself produces at a higher temperature. Thus the D line, as we term it in the solar spectrum, is due to the presence of sodium vapour in the solar atmosphere. The other dark lines are similarly accounted for by other elements.

The large forms of spectroscope (fig. 32) consist of a tube A, called the collimator, with a slit at the end S, and a convex lens at the end L. The latter makes the rays of light passing through the slit from the

source of light parallel they fall on the prism P, and then the spectrum so formed is focussed by the telescope T.

The third tube, D, seen in the next figure (fig. 33), carries a small transparent scale of wave-lengths, as in accurate observations the position of any point in the spectrum is given in the terms of the corresponding wave-lengths.

If we now interpose between the source of light and the slit S a piece of coloured glass (H in fig. 32), or a solution of a coloured substance contained in a vessel with parallel sides (the hæmatoscope of Herrmann, F in fig. 33), the spectrum is found to be no longer continuous, but is interrupted by a number of dark shadows, or

FIG. 33. Spectroscope: A, collimator with adjustable slit at one (left) end and collimating lens at the other (right) end; B, telescope moving on graduated arc divided into degrees; C, prism or combination of prisms; D, tube for scale; E, mirror for illuminating scale; F, vessel with parallel glass sides for holding fluid, shown with the flat side towards the reader; I, long spectroscope bottle for examining a deep layer of fluid; H, Argand burner; G, condenser for concentrating the light from H on the slit. (From a photograph taken by Dr. MacMunn, from McKendrick's Physiology.')

absorption bands corresponding to the light absorbed by the coloured medium. Thus a solution of oxyhemoglobin of a certain strength gives two bands between the D and E lines; hæmoglobin gives only one; and other red solutions, though to the naked eye similar to oxyhæmoglobin, will give characteristic bands in other positions.

A convenient form of small spectroscope is the direct vision spectroscope, in which, by an arrangement of alternating prisms of crown and flint glass, placed as in fig. 34, the spectrum is observed by the eye in the same line as the tube furnished with the slitindeed slit and prisms are both contained in the same tube.

Such small spectroscopes may be used for class purposes, and may

for convenience be mounted on a stand provided with a gas-burner and a receptacle for the test-tube (see fig. 35).

FIG. 35.-Stand for direct vision spectroscope: S, spectroscope; T, test-tube for coloured substance under investigation.

The next figure illustrates a method of representing absorption spectra diagrammatically. The solution was examined in a layer 1 centimetre thick. The base line has on it at the proper distances the chief Frauenhofer lines, and along the right-hand edges are percentages of the amount of oxyhemoglobin present in I, of hæmoglobin in II. The width of the shadings at each level represents the position and amount of absorption corresponding to the percentages.

The characteristic spectrum of oxyhæmogloblin, as it actually appears through the spectroscope, is seen in the next figure (fig. 37, spectrum 2). There are two distinct absorption bands between the D and E lines; the one nearest to D (the a band) being narrower, darker, and with better defined edges than the other (the ẞ band). As will be seen on looking at fig. 36, a solution of oxyhæmoglobin of concentration greater than 0.65 per cent. and less than 0.85 per. cent.

311