unless the activity of the original invertase solution greatly exceeds a k value of 0.1 and it is desirable to conserve the invertase. In this case, dilute to a k value of 0.1, which is done in the same manner as diluting other solutions to a standard strength. The activity of the invertase preparation required for rapid inversion is the same as that needed for overnight inversion, but the proportion of invertase preparation used in the former case is twice that used in the latter.

(b) PURIFICATION OF INVERTASE BY ADSORPTION

Yeast invertase, in common with other enzymes, can be absorbed on various suspended solid materials. Willstätter and Racke, whose starting point was the autolysate according to Hudson, purified the invertase by separating out impurities with kaolin which, in the crude mixture, did not adsorb invertase. They then fractionally adsorbed the enzyme with alumina in acid reaction with the addition of acetone. The eluted and purified enzyme was then found to be adsorbable by kaolin.

Adams and Hudson [41] have given in detail a method of adsorption by bentonite and subsequent elution. The following procedure is typical: A fractional autolysis of baker's yeast was prepared by treating 430 g of yeast (time value, 34.3) at 30° C with 43 ml of ether, adding after the yeast had liquified 43 ml of toluene, 430 ml of water, and 3.2 g of sodium carbonate, and 4 hours after the addition of the ether, filtering through Filter-cel, with 80 g of Filter-cel added to the mixture before filtration. The filtrate, which contained only 7.7 percent of the invertase, was discarded. To the residue was added 43 ml of toluene and 430 ml of water and autolysis was continued for 5 days at 20° C. After filtration, this autolysate was dialyzed immediately in Visking sausage casings. To a mixture of 80 ml of 0.5 percent bentonite suspension and 27 ml of a solution at pH 4.1, prepared by mixing 1 N acetic acid and 1 N sodium hydroxide was added 265 ml of the dialyzed autolysate, which contained 7.53 units of invertase per 100 ml and had a time value of 2.24 minutes. The bentonite was separated by centrifuging, washed by stirring with 200 ml of distilled water, and again centrifuged. Ninety-two percent of the invertase was adsorbed. Elution was effected by shaking gently with 3 portions, 40, 30, and 20 ml, respectively, of an acetate solution at pH 5.7, prepared from mixtures of 0.1 N acetic acid and 0.1 N sodium hydroxide solutions. The three extracts represented 57.8, 13.2, and 3.6 percent of the invertase in the original autolysate, and after dialysis had time values of 0.216, 0.215, and 0.278 minute, and contained 10.5, 2.26, and 0.64 units, respectively. The relatively small change in pH between adsorption and elution requires careful adjustment of the buffer solutions by colorimetric or electrometric

methods.

Richtmyer and Hudson [42] have described an alternative adsorption method with the use of zinc sulfide precipitated directly in the invertase solution. They give in detail a typical preparation.

A baker's yeast of relatively high invertase content was allowed to autolyze fractionally, in the manner described in the process, with bentonite and the first fraction discarded. The main autolysate was dialyzed in Visking sausage casings and then represented 60 percent. of the original invertase in the yeast. To 1,940 ml of this solution,

containing 110.2 invertase units, was added 1,940 ml of water, 43.5 ml of a 10-percent zinc acetate solution, 160 ml of a buffer solution at pH 4.5 (made by mixing 2 N sodium hydroxide and 2 N acetic acid), and 450 ml of a 10-percent sodium chloride solution (to prevent the zinc sulfide from becoming colloidal). Hydrogen sulfide was bubbled through the solution, and the zinc sulfide separated by centrifuging; the supernatant liquid had a pH of 4.4 and contained only 6 percent of the invertase. The zinc sulfide was washed by shaking with 1,500 ml of a 1-percent sodium chloride solution and again centrifuged. The invertase was eluted by shaking with 400, 200, and 100-ml portions, respectively, of a solution containing 1 percent of sodium chloride and 1-percent of mono- and dibasic ammonium phosphate until it had a pH of 6.1. The combined extracts, after dialysis, contained 77.6 invertase units, and had a time value of 0.20 unit.

Zinc sulfide has been used in similar fashion in purifying the dialyzed autolysates of brewer's yeast of relatively low invertase content. With these solutions a fractional adsorption with zinc sulfide is necessary, 15 to 20 percent of the invertase being discarded in the first portion. Adsorption and elution, as described, then produced invertase solutions with time value 0.21 to 0.22 minute.

(c) INVERTASE DIVISOR

Paine and Balch [43] determined with care the values of the Clerget divisor when pure sucrose is inverted with invertase. The basic value, as determined, was found to be in essential agreement with that previously measured by Zerban [44], and the values at other concentrations agreed with those of Ogilvie and of Hudson. Invertase from both top and bottom yeasts was purified by the Reynolds method [38]. Measurements were made at widely varying concentrations of sugar. The relation between sucrose concentration and the divisor is expressed by the equation

Divisor 131.17+0.073c,

in which c is the number of grams of sucrose in 100 ml.

The basic value of the divisor thus becomes 132.12 at 20° C. Zerban found experimentally 132.10. Their concentration coefficient 0.072 is considerably at variance from the 0.082 determined by Jackson and McDonald (see p. 134).

4. DETAILED ANALYTICAL PROCEDURE

(a) CONSIDERATIONS GOVERNING THE CHOICE OF METHODS

The choice of a method of Clerget analysis depends largely upon the purpose of the analysis, the nature of the material, and upon the precision required. In general, it must be recognized that the only methods completely free from limitations are those in which the sucrose and raffinose are hydrolyzed by enzymes (see p. 159), and if high precision and perfect selectivity are required, these methods alone can insure a correct analysis. However, for the purely practical considerations of the analyst's time and the expense of the enzymes, these methods are confined largely to research projects.

The acid methods may be divided into two groups, the plain acid methods (p. 154 and 155) and the compensation methods. The compensation methods were devised, first, to eliminate errors in the analysis of products high in invert-sugar content arising from the increased rotation of the original invert sugar in the presence of acid, and second, to eliminate errors caused by the change of rotation of amino acids when highly acidified. Thus for the analysis of relatively pure samples high in invert sugar Jackson and Gillis method IV (p. 155) is to be recommended. For the analysis of samples containing both invert sugar and amino acids Jackson and Gillis method II (p. 154) is available.

There is, however, in many products of the sugar industry a group of disaccharides or oligosaccharides which are not sucrose or raffinose but are hydrolyzable by acids. The hydrolysis of these substances introduces errors into the Clerget analysis which are obviously insurmountable except by the enzyme methods.

(b) REVISED METHODS OF JACKSON AND GILLIS

(1) GENERAL METHODS OF INVERSION.-The following methods of inversion were designed to avoid destruction of invert sugar subsequent to the completion of the inversion. There is, however, a variable amount of destruction during the process of inversion which is unavoidable. Each method, therefore, requires the use of its appropriate value of the Clerget divisor. These methods, which were determined by inversion of pure sucrose, are in general applicable to all products which do not contain considerable quantities of inorganic salts of weak acids. They can be applied to cane molasses but not to beet molasses. The latter should be inverted in a bath at 70° C, as described on page 155.

(a) Pipette 50 ml of the solution into a 100-ml flask, add 20 ml of water and 10 ml of hydrochloric acid (d20° 1.1029 or 24.85° Brix at 20° C). Immerse in a water bath at 60° C. Agitate the solution continually for about 3 minutes, and allow it to remain in the bath for a total time of 9 minutes. Cool quickly. Basic value, 133.18.

(b) Pipette 70 ml of the solution into a 100-ml flask, add 10 ml of hydrochloric acid (do 1.1029) and proceed as in (a). Basic value,

133.18.

(c) Pipette 75 ml of the solution, add 10 ml of hydrochloric acid (d20° 1.1029) and proceed as in (a), but allow the solution to remain a total time of 9.5 minutes. Basic value, 133.18.

(d) Pipette 50 ml and add 20 ml of water, or pipette 70 ml of the solution, into a 100-ml flask, add 10 ml of acid, d20° 1.1029, and allow to remain 30.8 hours at 20° C, 14.6 hours at 25° C, or 7.1 hours at 30° C. Basic value, 133.28.

(e) Walker method. Transfer 50 ml and add 25 ml of water or transfer 75 ml of the solution to a 100-ml flask and heat in a water bath to 65° C. Remove from bath, add 10 ml of hydrochloric acid (d20° 1.1029), allow to cool spontaneously for 15 minutes, and cool to the temperature of polarization. In the case of low-grade products containing an excess of basic lead acetate, add previous to heating, 1 ml (or 2 ml if excess of lead is large) of the acid used for inversion. Basic value, 133.18 (tentative).

(2) METHOD I.-(Applicable to pure sucrose, or to sugar mixtures in which the impurities are unaffected optically by hydrochloric acid.)

Reagents. Hydrochloric acid (d20° 1.1029 or 24.85 Brix).

Prepare a normal solution of the sample or a solution of such fractional normality as the nature of the substance will permit. Defecate, if necessary, with basic lead acetate in the usual manner, making to volume at the temperature at which the observations are to be made, and filter.

(If desired, the excess of lead may be removed at this point by the addition of pulverized potassium or sodium oxalate. However, it is necessary that the whole filtrate be treated by the deleading reagent, since the latter exerts an effect upon the rotation of invert sugar which should be as far as possible offset by a similar effect on sucrose.) Polarize the solution to obtain the direct reading and, if necessary, correct to the value which would have been obtained if 26 g of the sample were taken in 100 ml of solution.

To obtain the invert polarization, invert as described on page 153. make to a volume of, 100 ml at the temperature at which the observations are to be made, and polarize. Correct the observed polarization to that of a normal solution. The algebraic difference between the two polarizations corrected for dilution gives the value P-P'. Determine by refractometer the dry-substance concentration of the original sample and calculate the weight of solids, m, taken for the invert polarization.

Calculate the percentage of sucrose by the formula

S=

P-P'

Basic value +0.0794 (m-13)-0.53(t-20)'

The basic value is 133.18 for inversion at 60° C or 133.28 for roomtemperature inversion.

(3) METHOD II.-(General method applicable to all products.) Reagents. Hydrochloric acid (do 1.1029 or 24.85 Brix); ammonium hydroxide solution, 5 to 6 N; solution of ammonium chloride containing 226 g per liter; pulverized potassium or sodium oxalate.

Ascertain by at least three concordant titrations in the presence of methyl orange the volume of the ammonia solution required to neutralize 10 ml of the hydrochloric acid.

Prepare a normal solution of the sample or a solution of such fractional normality as the nature of the substance will permit. Defecate, if necessary, with basic lead acetate in the usual manner, making to volume at the temperature at which the observations are to be made. Filter.

(If desired, the solution may at this point be freed from lead; but if this is done, the deleading reagent must be added to the whole filtrate. Finely pulverized potassium oxalate in minimum quantity is added until precipitation is complete. Filter. If this procedure is omitted, the lead is precipitated satisfactorily by the chlorides added later.)

Pipette into two 100-ml flasks two equal volumes of the filtrate. (50, 70, or 75 ml).

For the direct polarization, add to 1 portion 15 ml of the ammonium chloride solution or 3.392 g of dry ammonium chloride. Make to volume at the temperature at which the observations are to be made; filter, if necessary, and polarize.

For the invert polarization, add hydrochloric acid to the other portion and invert by one of the methods described on page 153. Cool quickly. After the solution has become quite cold add from a burette during continual shaking, the precisely determined volume of ammonia required to neutralize the acid. Adjust the temperature, make to volume, filter, if necessary, and polarize at carefully controlled temperature. Correct both polarizations to the normal weight of sample.

Calculate the percentage of sucrose by the formula

S=

P-P'

Basic value +0.0794(m-13)-0.53 (t-20)'

in which m is the weight of dry substance taken for invert polarization. The basic value of the divisor is 133.27 for 60° C inversion or 133.37 for room-temperature inversion.

(4) METHOD IV.-(Applicable in the presence of invert sugar, but inapplicable in the presence of optically active non-sugars which change rotation with acidity. In principle sodium chloride equalizes the effect of hydrochloric acid on invert sugar present as an impurity.) Prepare a normal solution of the sample or a solution of such fractional normality as the nature of the substance will permit. Defecate, if necessary, with basic lead acetate in the usual manner, making to volume at the temperature at which the observations are to be made. Filter.

(If desired, the excess of lead may be removed at this point. Add pulverized potassium or sodium oxalate to complete precipitation of lead. The deleading reagent should be added to the whole filtrate. If the deleading is omitted, the lead is satisfactorily removed by the chlorides subsequently added.)

Pipette two 70-ml (or 50 ml+20 ml of water) portions of the clear filtrate into two 100-ml flasks. If preferred, 75-ml portions may be taken.

To 1 portion add 2.315 g of sodium chloride or 7.145 ml of a saturated sodium chloride solution or 10 ml of a solution containing 231.5 g per liter; make to volume at the temperature at which the observations are to be made and polarize.

To the other portion add hydrochloric acid and invert by one of the methods described on page 153. Cool and make to volume at the temperature at which the observations are to be made. Polarize. Correct both rotations to the normal weight of sample.

Calculate the percentage of sucrose by the formula

S=

=

P-P'

Basic value +0.0794(m-13)-0.53(t—20)'

in which m is the weight of dry substance taken for the invert polarization. The basic value is 132.56 for 60° C inversion or 132.66 for room-temperature inversion.

(c) ACID METHODS OF THE ASSOCIATION OF OFFICIAL AGRICULTURAL CHEMISTS

(1) DETERMINATION OF SUCROSE BY POLARIZATION BEFORE AND AFTER INVERSION WITH HYDROCHLORIC ACID. (In the presence of much levulose, as in honeys, fruit products, sorghum sirup, cane sirup,