ELEMENTARY COURSE

LESSON I

THE ELEMENTS CONTAINED IN PHYSIOLOGICAL COMPOUNDS

1. TAKE a fragment of meat about the size of a pea and place it in a porcelain crucible over a Bunsen flame. Note that it chars, showing the presence of carbon, and that it gives off the unpleasant odour of burning flesh, which is due to the fact that it contains the nitrogenous substances called proteins. In course of time the organic material is completely burnt up, and a small amount of white ash or inorganic material is left behind.

2. Repeat the experiment with a pure organic substance like sugar. Note that no ash is left. Charring, as before, indicates the presence of carbon, but there is no characteristic smell of burning nitrogenous substances (absence of nitrogen).

3. The tests for carbon depend on the fact that when this element is oxidised it gives rise to carbon dioxide; the test for hydrogen depends on the fact that when this element is oxidised it gives rise to water. If all the carbon dioxide and water formed by oxidation from a weighed amount of any organic substance under examination are collected and estimated, the amount of carbon and hydrogen respectively which it contains can be easily calculated. The following exercises, however, deal only with the qualitative detection of these elements.

4. Tests for Carbon.—The following tests can be carried out with sugar. (a) When burnt in the air it chars and subsequently the carbon entirely disappears, passing off in combination with oxygen as carbon dioxide (carbonic acid gas).

(b) Mix some of the powdered sugar in a dry mortar with about ten times the quantity of cupric oxide (which has been freed from water by previous heating); place the mixture in a dry test-tube provided with a rubber cork perforated by a bent glass tube which dips into either lime water or baryta water. Heat the tube over a Bunsen flame, and as the carbon of the sugar becomes oxidised carbon dioxide comes off and causes a white precipitate of calcium or barium carbonate, as the case may be.

5. Test for Hydrogen.—In the experiment just described (4 b) note that drops of water due to oxidation of hydrogen condense in the colder parts of the test-tube.

6. Tests for Nitrogen.—The greater number of tests for this element are due to the circumstance that on the breaking up of organic substances which contain it, it is given off as ammonia. If the ammonia is all collected and estimated, the amount of nitrogen can be easily calculated. Kjeldahl's method for carrying out this quantitative analysis is described in the Appendix. The following exercises, however, are qualitative only.

(a) The characteristic odour of burning flesh, horn, hair, feathers, &c., has been already noted, and, though only a rough test, is very trustworthy.

(b) Take a little dried albumin and mix it thoroughly in a mortar with about twenty times the amount of soda-lime and heat in a test-tube over a Bunsen flame. Ammonia comes off in the vapours produced, and may be recognised by (i.) its odour; (ii.) it turns moistened red litmus paper (held over the mouth of the tube) blue; (iii.) it gives off white fumes with a glass rod (held over the mouth of the tube) which has been dipped in hydrochloric acid. (c) Mix some dried albumin with about ten times its weight of a mixture of equal parts of magnesium powder and anhydrous sodium carbonate. A small quantity of the mixture-such as would lie on the end of a penknife— is then carefully heated in a dry test-tube and finally heated more strongly for about half a minute to red heat. Dip the tube while still glowing into a beaker containing a few c.c. of distilled water; the tube will break and its contents mix with the water. Filter and label the filtrate A; add to this filtrate a little strong solution of potash, one or two drops of cold saturated solution of ferrous sulphate and a drop of ferric chloride solution. Bring the mixture to boiling point, then cool and acidify with hydrochloric acid. The fluid becomes bluish green, and gradually a precipitate of Prussian blue separates out. This test is due to the fact that some of the nitrogen is fixed as sodium cyanide, and this gives the Prussian blue reaction with the reagents added.

7. Tests for Sulphur.-(a) In the foregoing test (6 c) the sulphur of the albumin combines with the sodium to form sodium sulphide. This may be detected by taking some of the filtrate A and adding freshly prepared solution of sodium nitro-prusside; a reddish violet colour forms.

(b) Test for loosely combined Sulphur.-Add two drops of a neutral lead acetate solution to a few c.c. of caustic soda solution. The precipitate of lead hydroxide which is first formed soon dissolves. Heat a small portion of the albumin with this alkaline solution. The mixture turns black in consequence of the formation of lead sulphide, part of the sulphur present in albumin in the unoxidised form having been split off from it by the caustic soda as sodium sulphide.

(c) Take some dried albumin and fuse with a mixture of potash and potassium nitrate. Cool; dissolve in water and filter. The filtrate will give the following tests for sulphates :-Acidulate with hydrochloric acid and add barium chloride; a white precipitate of barium sulphate is produced.

(d) Take some solution of albumin and heat in an open dish in a fume cupboard for at least an hour with large excess of fuming nitric acid, renewing the acid from time to time as necessary. The resulting fluid will give the test for sulphate as in c.

8. Test for Phosphorus.--The two tests just described (7 c and d) may be repeated with some substance (such as caseinogen, nucleoprotein, or lecithin) which contains phosphorus in organic combination; or the organic matter may be more conveniently destroyed by Neumann's method, which consists in heating it with a mixture of sulphuric and nitric acids. The resulting fluid in each case gives the following test for phosphates:-Mix it with half its volume of nitric acid; add ammonium molybdate in excess and boil; a yellow crystalline precipitate falls.

The reactions described in the foregoing exercises show how the processes of pure chemistry may be employed for the detection of some of the most important elements that occur in substances of physiological importance, and thus form a fitting introduction to a study of physiological chemistry.

ELEMENTS CONTAINED IN PHYSIOLOGICAL COMPOUNDS 11

They show, in the first instance, how the substances with which we have to deal fall under the two main categories of organic and inorganic. In some of the tissues of the body, like bone and tooth, the inorganic or mineral material is in excess, but in the softer portions of the organism the organic compounds are in great preponderance.

Organic chemistry is sometimes defined as the chemistry of the carbon compounds; carbon is in all cases present, and is usually the most abundant element.

The most important of the nitrogenous substances are the proteins, as already explained in the introductory chapter, and the detection and estimation of nitrogen are thus exercises of the highest interest.

All the proteins contain a small amount of sulphur; keratin, or horny material, contains more than most of them do.

Phosphorus is another element of considerable importance, being present in nuclein and nucleo-proteins, and also in certain complex fats, of which lecithin may be taken as a type. Iodine occurs united to protein material in the colloid substance of the thyroid gland; iron in the pigment of the blood called hæmoglobin; sodium, calcium, potassium, and other metals in the inorganic substances of the body. It would, however, lead us too far into the regions of pure chemistry to undertake exercises for the detection of these and other elements which might be mentioned, and have been already commented upon. The teacher of physiological chemistry is bound to assume that the students who come before him have already passed through a course of ordinary chemistry.

The main interest of the exercises selected as types lies in their physiological application. As a rule an element is detected by breaking up or oxidising the more or less complex molecule in which it occurs into substances of simpler nature, and then performing tests for these simpler products. Thus carbon is identified by the formation of carbon dioxide, nitrogen by the formation of ammonia, and so forth.

A great many reactions which can be performed in the test-tube imitate those which are performed in the body. Reactions in vitro and in vivo, to use the technical phrases, often, though not always, run parallel. Life, from one point of view, is a process of combustion or oxidation; the fuel is supplied by the food; this becomes assimilated, and so forms an integral part of the living substance of the body; it is then burnt up by the oxygen brought to it by the blood-stream, giving rise to animal heat and other manifestations of

energy; and finally the simple products of oxidation or chemical breakdown are carried to the organs of excretion (lung, skin, kidney, &c.), where they are discharged from the body.

A candle consists principally of carbon and hydrogen; when it is burnt the products are carbonic acid gas and water; the former may be detected by means of lime water, the latter, by holding a dry beaker upside down for a few moments over the burning candle, when the moisture will condense on the cold glass.

The body is more complex than a candle, but so far as its carbon and hydrogen are concerned the main products of combustion are the same. The carbon dioxide is discharged by the expired air, as may be proved by blowing it into lime water. The water finds an outlet by several channels, lungs, skin, and kidneys. The presence of nitrogen in the body is perhaps the most striking chemical distinction between it and a candle, and here again the process of metabolism runs a course analogous in some degree to our experiments in vitro. Here again the most important and abundant substance which contains the waste nitrogen is the simple material ammonia, but ammonia is only discharged as such to a very small extent in health. It unites with carbon and oxygen to form the body called urea (CON,H1), which finds its way out of the body via the urine. The urine also contains the sulphates, due to the oxidation of the sulphur of the proteins, and the phosphates due to the similar oxidation of the phosphorus of such substances as lecithin and nuclein. Some of the salts of the urine, however, in particular the chlorides, come directly from the food. This we shall discuss at the proper place when we come to the study of the urine.

LESSON II

THE CARBOHYDRATES

1. NOTE the general appearance of the specimens of grape sugar or dextrose, cane sugar, dextrin, and starch which are given round.

2. Put some of each into cold water. Starch is insoluble; dextrose, cane sugar, and dextrin dissolve after a time, but more readily in hot water.

3. Trommer's test.-Put a few drops of copper sulphate solution into a test-tube, then solution of dextrose, and then strong caustic potash. On adding the caustic potash a precipitate is first formed, which, owing to the presence of the sugar, rapidly redissolves, forming a blue solution. On boiling this a yellow or red precipitate (cuprous hydrate or oxide) forms.

4. Fehling's test.-Fehling's solution is a mixture of copper sulphate, caustic soda, and Rochelle salt of a certain strength. It is used for estimating dextrose quantitatively (see Lesson XII.). It may be used as a qualitative test also. Boil some Fehling's solution; if it remains clear it is in good condition; add to it an equal volume of solution of dextrose and boil again. Reduction, resulting in the formation of cuprous hydrate or oxide, takes place as in Trommer's test.

5. Moore's test.-Add to the dextrose solution about half its volume of 20-per-cent. potash and heat. The solution becomes yellowish brown. Add to this some sulphuric acid (25 per cent.) and the odour of caramel becomes apparent.

6. Fermentation test.—Add a fragment of dried yeast to the dextrose solution in a test-tube; fill the test-tube up with mercury, and invert it over mercury in a trough. Place it in an incubator at body temperature for 24 hours. The sugar is broken up into alcohol and carbon dioxide; the latter gas collects in the upper part of the test-tube.

7. Cane Sugar. (a) The solution of cane sugar when mixed with copper sulphate and caustic potash gives a blue solution. But on boiling no reduc tion occurs.

(b) Take some of the cane-sugar solution and boil it with a few drops of 25-per-cent. sulphuric acid. This converts it into equal parts of dextrose and levulose. It then gives Trommer's or Fehling's test in the typical way.

(c) Boil some of the cane-sugar solution with an equal volume of concentrated hydrochloric acid. A deep red solution is formed. Dextrose, lactose, and maltose do not give this test.

8. Starch. (a) Examine microscopically the scrapings from the surface of a freshly cut potato. Note the appearance of the starch grains with their concentric markings.

(b) On boiling starch with water an opalescent solution is formed, which, if strong, gelatinises on cooling.

(c) Add iodine solution. An intense blue colour is produced, which disappears on heating, and if not heated too long reappears on cooling. N.B.-Prolonged heating drives off the iodine, and consequently no blue colour returns after cooling.