of 1·34, and dilute with water to 500 c.c. These solutions should be kept in well-stoppered bottles, and before using equal volumes of A and B mixed together. The result is a dark blue solution, the Rochelle salt holding the cupric hydrate in solution. The solution should be freshly made, because tartaric acid tends to become converted into its isomeride, racemic acid; and racemic acid itself reduces cupric salts like sugar. One should always ascertain that it is absent by boiling the Fehling's solution, which should remain unaltered by this treatOn adding a little solution of sugar and boiling, a red precipitate of the cuprous oxide or hydrate occurs. ment. 3. Böttger's test.'-Take 5 grammes of basic nitrate of bismuth, 5 grammes of tartaric acid, and 30 c.c. of distilled water. To this add slowly, and with constant stirring, some strong caustic soda solution, until a clear solution is obtained. To a little of this add some solution of dextrose, and boil. A black precipitate of metallic bismuth separates. Or the test may be performed as follows:-The solution of dextrose is mixed with an equal volume of sodium carbonate solution (1 part to 3 of water); a few fragments of bismuth subnitrate added, and the mixture boiled. A grey or black precipitate of metallic bismuth is thrown down. 4. Silver test.-Add ammonia in excess to a little strong solution of silver nitrate; add some dextrose and boil, metallic silver is deposited as a mirror at the bottom of the tube. Aldehyde and tartaric acid behave like sugar in this test. 5. Moore's test.2-Heat the solution of dextrose with a solution of caustic potash. The mixture becomes yellow, and, on exposure to the air, brown from the formation of melassic and glucic acids. 6. Picric acid test. Heat the solution of dextrose with a few drops of solution of picric acid, and heat. Add a little caustic potash, and a brown-red solution is obtained. 7. Indigo-carmine test.-A solution of dextrose is rendered faintly blue with indigo-carmine, and faintly alkaline with sodium carbonate. It is then heated to boiling without agitation; it turns violet, then yellow, but if it is shaken the blue colour is restored. 8. Fermentation test.-A test-tube is half filled with solution of dextrose and a little dried German yeast added. Invert the tube over mercury, and leave it in a warm place for 24 hours. The sugar will undergo fermentation; carbonic acid gas accumulates in the tube, and the liquid gives the tests for alcohol. A control experiment should be made with yeast and water in another test-tube, as a small yield of carbonic acid is often obtained from impurities in the yeast. 1 Böttger, Journ. prakt. Chem. lxx. (1857), p. 432. Nylander, Zeit. physiol. Chem. viii. (1884), p. 175. 2 Moore, Lancet, 1844, ii.; Heller, Arch. f. mikr. Chem, vol. i. 1844, p. 292. 9. The Phenylhydrazine test.-This test is applied in testing for minute quantities of dextrose, especially in urine. Add a pinch of sodium acetate and a little solution of phenylhydrazine hydrochloride to a solution of dextrose; a yellow precipitate of phenylglucosazone crystals occurring both singly and in bundles forms in a few minutes if the mixture is kept in the water bath at 100° C. ; and in a dilute solution of dextrose the crystals should be searched for microscopically (v. Jaksch).' 10. The Saccharimeter test.—A solution of dextrose rotates the plane of polarised light to the right. In testing for dextrose, as many tests should be tried as possible, as many other substances give certain of the above tests; for example, reduce copper salts, or rotate the plane of polarised light. The best tests will be found those numbered 2, 3, 8, 9 and 10; and the best of all is 8. Quantitative determination of Dextrose.-1. By the Saccharimeter. This instrument is a polarimeter, and the instruments used and methods adopted are the same as that employed in polarimetric processes generally (see pp. 40, 53). 2. The Fermentation process. When mixed with yeast about 95 per cent. of the dextrose in solution is converted into carbonic acid and alcohol. Small quantities of amyl alcohol, glycerine and succinic acid are formed at the same time. The dextrose originally in solution may be estimated either from the loss of weight of the apparatus from the evolution of the gas, or from the gain in weight of an absorption tube containing caustic potash connected with the escape pipe, and which absorbs the carbonic acid. 1 part of CO2=2045 parts of sugar. Sir William Roberts devised a simpler process, especially applicable to sugar in urine, in which the sugar present is estimated from the loss of specific gravity a solution undergoes during fermentation. The specific gravity of the solution is accurately taken: yeast is added and after remaining 24 hours in a warm place the specific gravity is again taken. The number of degrees of density lost indicates the number of grains of sugar per ounce; and the percentage is obtained by multiplying the degrees of density by a constant factor. This constant factor is according to Worm-Müller 0-23; according to Manassein 0.219. Thus in a urine whose specific gravity before fermentation is 1040, and afterwards 1010, the degrees of density lost 30, and accordingly 30 grains of sugar are present per ounce, or 30 × 0.23=6.9 per cent. This method, however, is found practically to give very inaccurate = Zeit. klin. Med. xi. 20, see also note on p. 110. results. The reason of this is, that a constant factor is an impossibility, and, in fact, increases as the percentage of sugar diminishes. For the mathematical demonstration of this fact see Budde (Pflüger's Archiv, xl. 137). 3. Fehling's method.-10 c.c. of Fehling's mixture (see p. 95) corresponds to 0.05 gramme of sugar. The solution to be tested should not contain more than about 0-5 per cent. of dextrose. It will be found necessary to dilute strong solutions, and most diabetic urines,' with 9 times the amount of water; this must be allowed for in the subsequent calculation. The solution of dextrose is placed in a burette; and 10 c.c. of Fehling's mixture diluted with 40 c.c. of water, in a white porcelain, dish. The Fehling's mixture is kept constantly boiling, and the sugar is run into it from the burette gradually. The cuprous oxide is thrown down as a red precipitate, and the blue colour of the solution gets less and less, and finally disappears. When the blue colour has gone, the burette is read, and the quantity of solution of sugar used, is that which contains sufficient sugar to reduce 10 c.c. of Fehling's mixture, i.e. 0·05 gramme sugar. of Suppose 9.5 c.c. of the solution reduced 10 c.c. of Fehling's mixture (=0.05 gramme sugar); then the percentage of sugar = 0.05 x 100 = 5 9.5 =0.526; and if the solution, or the urine had been previously diluted 10 times, the percentage of dextrose in the original solution=0·526 × 10 =5.26. In order to insure accuracy it is always advisable to make a second observation, using the first only as an indication, and proceeding more cautiously. Beginners often find it difficult to determine exactly the point at which the blue colour has completely disappeared. In such a dilemma, a little of the hot fluid should be quickly filtered through a thick filter paper, the filtrate acidulated with acetic acid, and a drop of potassic ferrocyanide added. If copper is present a brown colour or precipitate is produced; in this case, more of the sugar solution must be added, and the operation continued until the filtered hot fluid gives Flückiger2 recommends that a small quantity no reaction for copper. of calcium chloride should be added before filtering, in order to prevent the mechanical suspension of finely divided cuprous oxide in the solution, and Hagemann3 has pointed out that in the case of urine, it is necessary to test the first two drops of the filtrate; for by the time 1 If the urine is albuminous, the albumin must be first separated by acidulating with dilute acetic acid, boiling, and filtering. 2 Zeit. physiol. Chem. ix. 335. 3 Pflüger's Archiv, xliii. 501. the third drop comes through, oxidation of the cuprous oxide has taken place, and cupric oxide is in solution. Such rapid reoxidation does not occur however in solutions of pure dextrose. Hagemann further states that by this method as good results are obtained as by Allihn's method, which is one for determining the amount of copper in the precipitate. In making these determinations in urine, it must be borne in mind that other substances may be present which reduce alkaline solutions of copper salts, such as uric acid, creatinine, pyrocatechin, and compounds of glycuronic acid. None of these substances, however, give the fermentation test. 4. Other Methods.-Knapp's method is a volumetric one, in which a standard solution of alkaline mercurie cyanide is used. (10 grammes of mercuric cyanide, caustic soda (of sp. gr. 1·14) 100 c.c. made up to a litre with water; 40 c.c. corresponds to 0-1 gramme of sugar). The solution is kept hot, sugar solution run in from a burette, and metallic mercury is deposited. The end of the reaction is the absence of mercury in the fluid; this is ascertained by placing a drop of the clear supernatant fluid on a piece of fine filter-paper, and exposing it to the vapour of ammonium sulphide; when the drop remains unblackened mercury is absent. Sachsse's method is very similar; the standard solution is mercuric iodide 18 grammes, potassium iodide 35 grammes, caustic potash 80 grammes, water to 1000 c.c.: 40 c.c. corresponds to 0.15 gramme of sugar. The end of the reaction is ascertained by means of drops of a solution of stannous chloride supersaturated with caustic soda, placed on a porcelain dish; as long as the mercuric salt is present, the addition of a drop of the clear supernatant fluid gives with one of these drops a brown colour, or grey precipitate. Vogel's method is a colorimetric one, and depends on the intensity of the colouration produced by boiling the solution of dextrose with caustic potash. This is compared with a standard solution similarly treated. Dr. George Johnson has also devised a colorimetric method, depending on the depth of the tint produced by boiling a solution of dextrose with caustic potash and a saturated solution of picric acid, as compared with the tint of a standard. Pavy's and Gerrard's methods are modifications of Fehling's, and being especially applicable to urine, will be described under that head (see Chapter XLV.). LEVULOSE When cane sugar is treated with dilute mineral acids, it undergoes a process known as inversion, i.e. it takes up water, and is converted into a mixture of equal parts of dextrose and levulose. Similar hydration changes are produced by ferments, such as the invert ferment of the intestinal juice. Levulose has been discovered in blood, urine, and muscle. It is uncrystallisable, very soluble in water and in alcohol; it gives the same tests as dextrose, except that it has a powerful lavorotatory action on polarised light. («)=-106°. 1 Zeit, anal. Chem. vol. xxii. 248. p. 2 Annal. d. Chem. vol. cliv. p. 252. We have seen that dextrose is regarded as an aldehyde; by some, levulose is regarded as the corresponding ketone. Pure levulose may be obtained by neutralising with lime the mixture of glucoses obtained by the action of sulphuric acid on cane sugar. The levulose lime compound is a solid, while that of dextrose is liquid. By decomposing the lime compound with oxalic acid, pure levulose is obtained. GALACTOSE Galactose is formed by the action of dilute mineral acids, or inverting ferments, on lactose or milk sugar. It is dextrorotatory. (a)p= +83·3°. Nitric acid oxidises it to mucic acid. Galactose is directly fermentable with yeast; it also reduces Fehling's solution. INOSITE Inosite is a glucose which is found in muscle, kidney, liver, nervous tissues, and several other organs of the body. It has also been separated in small quantities from the blood, traces exist in most diabetic urines, in the urine of certain cases of Bright's disease, and according to Choetta, Gallois, and Külz,3 in normal urine too. It is also obtained from peas, beans, lentils, potato, asparagus, dandelion, foxglove, and many other plants. Preparation. From beans. A watery extract is evaporated to a syrup, and precipitated with alcohol; the precipitate is dissolved in water, and the inosite allowed to crystallise out. From muscle or other tissues. An aqueous extract is freed from albumin by acidulation, boiling and filtering; from phosphates by the addition of baryta water and filtering. The filtrate is concentrated, and creatine crystallises out. The mother liquor is boiled with four times its volume of alcohol, and the precipitate so formed is removed. The clear liquid is set aside for twenty-four hours, and crystals of inosite often separate; if not, ether is added, and the mixture shaken, inosite then separates in lustrous leaflets. It is purified by recrystallisation. From urine. Take several litres of urine, and add neutral then basic lead acetate. Collect the precipitate produced by the latter; decompose it with sulphuretted hydrogen; filter; evaporate the filtrate to a syrup, and add alcohol and ether. Inosite crystallises out. Properties.—It forms large colourless monoclinic prisms, often 1 Ann. Chem. Pharm. xcix. p. 289. 5 Centralbl. f. Med. Wiss. 1875, p. 933. 4 Boedeker, Ann. Chem. Pharm. vol. cxvii. De l'inosurie, Thesis, Paris, 1864. P. 118. |