by both the optical and the reducing properties of the invert-sugar formed.

Inversion of Cane-Sugar with Acid.-Dissolve an accurately weighed quantity of about 10 grms. of dry cane-sugar in 50 c.c. of water, and transfer the solution without loss to a 100 c.c. flask, taking care that the volume of the solution and wash water does not exceed 90 c.c. Add 5 c.c. of strong hydrochloric acid, and make up the volume of the solution nearly to the 100 c.c. mark. Heat the flask in a boiling water-bath for thirty minutes, when inversion of the cane-sugar should be complete. Cool the solution, and make up the volume to 100 c.c.

(1) Determine the rotatory power of the solution at a temperature of 20° (68° F.), and calculate the amount of invert-sugar present in 100 c.c. of the solution from the known specific rotation of invert-sugar, [a] 19.6° (at 20°).

(2) Determine the cupric oxide reducing power of the solution, using about 15 c.c. for reduction, and calculate the amount of invert-sugar present in 100 c.c. of the solution from the weight of reduced copper obtained.

Acid inversion of cane-sugar does not give such satisfactory quantitative results as inversion with yeast (described below), as the levulose formed is slowly acted upon and decomposed by the acid employed.

Inversion of Cane-Sugar with Yeast.-The inverting power of yeast is due to the enzyme inver

tase contained in the yeast cells. Two methods of experiment may be adopted when yeast is employed as an inverting agent :

(1) Prepare a solution of a known weight of cane-sugar in a 100 c.c. flask as in the previous experiment, but in place of hydrochloric acid, add 1 grm. of washed pressed yeast, and digest the solution in a water-bath at 50° (122° F.) for six hours. A temperature of 50° is employed because the fermentative power of yeast is arrested at this temperature, but its power of inversion is still retained. When the digestion is completed, raise the solution to the boiling point and cool to 155° (60° F.). Add a little alumina cream to facilitate clarification, make up to 100 c.c., and filter. Determine the rotatory power of the solution as in the previous experiment, and calculate the percentage yield of invert-sugar from cane-sugar which has been obtained. Compare with the calculated amount according to the equation for the inversion of canesugar (p. 82).

(2) Measure 50 c.c. of a solution of cane-sugar of known concentration into a flask doubly graduated to 50 and 55 c.c. and add 0·5 grm. of pressed yeast and three or four drops of chloroform. Cork the flask tightly, place it in an incubator at a temperature of about 30° (86° F.), and allow it to remain overnight. Chloroform is added because it arrests the fermentative power of yeast but does not influence its power of inversion. Remove

the cork, place the flask in a boiling water-bath and allow it to stand there until free from the smell of chloroform. Removal of the chloroform is necessary, as it reduces Fehling's solution and would affect the estimation of the invert-sugar by the reduction method if it was allowed to remain in the solution. Cool the solution, add a little alumina cream to facilitate clarification, and make up the volume to the 55 c.c. mark with water. Filter, and determine the reducing power and rotatory power of the solution, as before. When making the requisite calculations, note that a correction for the dilution of the solution from 50 c.c. to 55 c.c. is necessary.

Determine the Amount of Cane-Sugar present in Malt by Inversion with Yeast.-Weigh out 50 grms. of finely ground malt, introduce it into a flask, and add 200 c.c. of water. Make the mixture very faintly alkaline with caustic soda, and allow it to stand over night. The solution is made slightly alkaline in order to prevent the invertase which may be present in the malt from acting on the canesugar during the process of extraction. Filter 100 c.c. of the solution and render it very faintly acid with dilute acetic acid. This is done in order to restore the solution to a condition in which invertase is active. Observe the reading of the solution in the 100 mm. tube of the polarimeter. Now place 50 c.c. of the solution in a 50-55 c.c. flask, add 0.5 grm. pressed yeast and a few drops of chloro

form, and proceed according to the second yeastinversion method described above. Make a second reading of the solution in the 100 mm. tube, and after correcting for the dilution of the solution (50 c.c. to 55 c.c.), calculate from the readings made before and after inversion the amount of cane-sugar in 100 grms. of the malt.

Levulose (C6H12O6).-Pure levulose may be obtained from Schering's levulose (originally prepared from invert-sugar by means of the insoluble calcium compound of levulose), by recrystallisation from absolute alcohol. Pure levulose may also be obtained by the hydrolysis of inulin with dilute acid. (For method of preparation see abstract of paper by A. Wohl in Journ. Chem. Soc., 1890, vol. lviii., p. 1087.)

Specific Rotation of Levulose.-[a] - 92.0° at 20° (68° F.). Dissolve about 5 grms. of pure levulose in 80 to 100 c.c. water, heat to boiling (see p. 78), cool and determine the concentration of the solution by means of its specific gravity. (See Table I. for solution factors for levulose.) Determine the rotatory power of the solution in the 200 mm. tube at the different temperatures of 15·5° (60° F.), 20° (68° F.) and 30° (86° F.), and calculate the [a] of levulose for the three temperatures. These observations will demonstrate that the optical activity of levulose is very sensitive to alterations of temperature, and that it is necessary when determining the rotatory power of solutions of levulose and invert-sugar to take special care with regard to the temperature of the solutions at the time of observation. Determination of the Cupric Oxide Reducing Power of Levulose.-Determine the reducing power of about 2 c.c. of the solution of levulose according to standard conditions, and compare the result with the reduction figures for pure levulose given in Table III.

Preparation of the Osazone of Levulose.-Dilute about 20 c.c. of the levulose solution to 50 c.c. with water, and heat in

the usual way with 2 grms. of phenyl-hydrazine and acetic acid (p. 81). An osazone separates during heating with the characteristics of glucosazone. Under the microscope the crystals appear similar to those of glucosazone, and in fact the osazone of levulose is identical with glucosazone. Study the constitution of levulose and dextrose with regard to the formation of the same osazone from both.

PART II.

The Following Course of Experiments Constitutes a Study of the Hydrolysis of Starch by Diastase, and of the Products of Hydrolysis.

Preparation of Cold-water Malt Extract.-A cold-water malt extract supplies a solution of active diastase for the experiments described below. Mix one part by weight of finely ground pale dried malt with two and a half parts of cold water, allow the mixture to stand four or five hours, and filter. When cold-water malt extract is referred to in the following studies it is intended to be prepared by this method unless otherwise stated.

Study the Hydrolysing Action of Diastase on Starch.-Prepare 200 c.c. of a 3 per cent. starch paste (see p. 66). Cool to 60° (140° F.), and add 10 c.c. of cold-water malt extract, the temperature of the starch paste being kept constant. Observe the rapid liquefaction of the starch paste due to its conversion into soluble starch.