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The amount of iron in hæmoglobin is 0.4 per cent. Knowing this, hæmoglobin may be estimated quantitatively from the amount of iron present in the ash of an unknown amount of the hæmoglobin.

TO DEDUCE EMPIRICAL FORMULE FROM PERCENTAGE COMPOSITIONS

From the percentage composition the empirical formula can be calculated, provided that the combining weights of the elements are known. The actual size of the molecule and its constitutional formula are obtained by other methods.

The way in which an empirical formula is deduced may be most readily described by giving examples.

Example 1. Suppose starch has been subjected to elementary analysis and it was found to contain 4444 carbon, 6·17 hydrogen, and 49:39 oxygen per cent. Knowing that C = 12, H = 1, and 0=16, what is the empirical formula for starch? Divide the percentage numbers by the combining weights of the elements, and we obtain

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which gives us a rough guide to the formula; from this we can see that the hydrogen atoms are twice as numerous as the oxygen atoms, and the carbon atoms are also rather more numerous than the oxygen atoms. We must next find some common factor which will convert the above numbers into whole numbers; it is, however, generally impossible to do this exactly, and it will be found in the present instance that the number 1·62 is the smallest number which will give us approximately whole numbers, viz. :—

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The nearest whole numbers to these being taken, the simplest empirical formula for starch is CH1005.

Example 2. When we are dealing with a substance containing more than three elements, the arithmetical processes become more complicated. The example I will choose is that of mucin obtained from tendons. Loebisch found that the percentage composition of this material was C, 483; H, 6·44; N, 11·75; S, 0·81; O, 32.7. Divide each of these numbers by the combining weight of their respective element, and we obtain a guiding formula, viz. :

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It will be found that the lowest common factor which will convert these numbers most approximately into whole numbers is 39 75; this will give us—

C159-99

H255-99

N32-99

So-99

080-48

or approximately, C160 H256 N3 SO8o. The numbers do not correspond with equal exactness throughout; this is especially noticeable with regard to the oxygen. It must, however, be remembered that there are certain unavoidable small errors of analysis which have always to be allowed for. Moreover, in this particular instance the correspondence between the percentage calculated from the formula and that obtained by analysis is closer than in many other cases.

Such methods give us only an empirical formula; the true molecular weight

may be n times as great, and in substances in which the molecular weight can be ascertained by determination of vapour density, &c., the calculation is simplified. But with regard to the albuminous and starchy substances we have to deal with in animal chemistry, these methods are not available. The constitutional formula of any substance, i.e. the way in which the atoms are united to one another, must be determined by other methods also. Here, again, the substances we have chiefly to deal with in animal chemistry are those, in regard to the constitution of which, we are almost entirely in the dark at present.

CHAPTER IV

GAS ANALYSIS

THE gases with which the physiologist has to deal are those of the atmosphere, and those concerned in respiration, those present in the blood and other fluids, as well as those obtainable from the solid tissues of the body. In the greater proportion of cases, a physiologist has to investigate three gases or mixtures of these: viz.:-oxygen, nitrogen, and carbonic acid. Small quantities of carbonic oxide are also produced in the body, and in the alimentary canal, fermentation processes may give rise to hydrogen, marsh gas, and sulphuretted hydrogen. The reader is, however, referred to larger treatises dealing more especially with gas analysis, for the methods of investigating these more rarely occurring gases.

In examining the gases obtainable from either fluid or solid animal tissues, the methods adopted divide themselves naturally into three parts :-

1. The collection of the blood or other tissue in a suitable manner. 2. The extraction of the gases from this material.

3. The analysis of the gases so obtained.

1. METHODS OF COLLECTING MATERIAL INTENDED FOR

GAS ANALYSIS

a. Collection of blood. In some cases it is possible to lead the blood direct from the blood-vessel of the animal, by a tube into the vacuum chamber of an air-pump. The gases can be then pumped from it forthwith. The vacuum chamber can be weighed before and after the entrance of blood into it; the increase of weight giving the weight of blood used.

In other cases it is advisable to collect and measure the blood in a separate vessel before introducing it into the air-pump. It must then be collected over mercury, and the following apparatus, as described by Gamgee,' answers this purpose admirably. A long graduated tube ab is filled with mercury, and placed in connection with a reservoir of

1 Physiol. Chem. p. 181.

a

mercury e by an india-rubber tube; the stopcock at a is closed; a narrow elastic tube leading from the blood-vessel is filled with blood, and slipped over the free end a, and the stopcock is opened; b is also open; the mercury will fall and is replaced by blood. When sufficient blood has been collected, the two stopcocks are closed; the tube is released from the clamp and from the india-rubber tubes at either end, and inverted repeatedly. When blood and mercury are shaken together in this way, fibrin separates from the blood in a very fine state of subdivision. The tube is then laid in a trough containing ice, until the blood within it is wanted for analysis.

b. Collection of other fluids. Here again care must be exercised in obtaining them as fresh as possible, and free from air by collecting them over mercury. The blood-serum, for instance, must be obtained from blood allowed to clot over mercury; the urine, bile, saliva, chyle, &c., are obtained by inserting a cannula into the duct or vessel, as the case may be, and then leading the fluid thence for collection in some such apparatus as that just described for blood.

c. Methods of obtaining solid organs. In the analysis of the gases of a solid organ, such as muscle, a great difficulty is met with at the outset; for the muscles cannot be transferred to the vacuum, without preliminary exposure to air or indifferent fluids. They must be as speedily as possible freed from blood, and plunged instantly into a large volume of boiling salt solution; they will be coagulated en masse, and

FIG. 13.

die without undergoing the change known as rigor mortis. The scalded muscle is at once covered with a beaker filled with the hot

saline solution, and any gases that escape are at once collected. The temperature is then lowered, the muscle is minced, and (still contained in salt solution) is introduced into the boiling flask attached to the air-pump. In other cases the muscle is kept from undergoing rigor by being frozen. In other cases, again, the muscle is introduced quickly over mercury into a vessel containing a known volume of air; the changes in the composition of the gases in this closed chamber can be subsequently investigated.

2. THE EXTRACTION OF THE GASES FROM THE MATERIAL UNDER INVESTIGATION

The materials which have been most often investigated are the blood and muscle. It will, therefore, be more convenient to speak of these two, the first an instance of a liquid, the second of a solid tissue. The gases are extracted by means of a mercurial air-pump.

The earliest forms of pump were made by Ludwig and his pupils, Setschenow and A. Schmidt.2 The best-known pump is probably Pflüger's; but improvements have been introduced by Alvergniat and others.

The principle of all these pumps may be explained by the diagram in fig. 14, in which the parts of Pflüger's pump are reduced to their simplest elements.

7 is a large glass bulb filled with mercury; from its lower end a straight glass tube, m, about 3 feet long, extends, which is connected by an india-rubber tube, n, with a reservoir of mercury, o, which can be raised or lowered as required by a simple mechanical arrangement. From the upper end of the bulb, 7, a vertical tube passes; above the stopcock, k, this has a horizontal branch, which can be closed by the stopcock, f. The vertical part is continued into the bent tube, which dips under mercury in the trough, h. A stopcock, j, is placed on the course of this tube. Beyond ƒ the horizontal tube leads into a large double glass bulb, ab; a mercurial gauge, e, and a drying-tube, d, filled with pieces of pumicestone moistened with sulphuric acid being interposed. a is called the bloodbulb, and the blood is brought into it by the tube, c; the gases, as they come off, cause the blood to froth, and the bulb, b, is called the froth-chamber, as it intercepts the froth, preventing it from passing into the rest of the apparatus.

The pump is used in the following way: 7 is filled with mercury, the level in and o being the same; k is closed; o is then lowered, and when it is 30 inches lower than the stopcock, k, the mercury in falls also, leaving that bulb empty; j being closed and ƒ open, k is then opened, and the air in a, b, d, &c., rushes into the Torricellian vacuum in 7; ƒ is closed and j opened; the reservoir, o, is raised; the mercury in 7 rises also, pushing the air before it, and it bubbles out into the atmosphere through the mercury (the tube, h, is not at this stage in position). When is full of mercury, k and j are once more closed and o is again lowered;

1 Zeitsch. f. rat. Med. 3rd ser. x. 112.

2 Ber. d. k. sächs. Gesellsch. d. Wiss. Leipsig (1867), xix. 33.

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