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5. Add 2 drops of nitrazine yellow solution and neutralize with 0.1 M sodium hydroxide. Then add exactly (buret) 5 cc. excess of 0.1 M sodium hydroxide, and add immediately 5.00 cc. of 0.025 M standard iodine solution. It should not take longer than 2 to 3 minutes for neutralization, addition of excess alkali and addition of iodine. Set in a dark place at 25’ to 30” d. for 45 minutes. It is convenient to run the samples at intervals of 2.5 to 3 minutes as suitable to the individual analyst. 6. Forty-five minutes 1 minute after addition of iodine, add 5 cc. of concentrated hydrochloric acid, shake, then titrate immediately with 0.025 M thiosulfate. If the back-titer of thiosulfate is less than 3 cc., the resulting alkali-labile value will be low and 7 cc. instead of 5 cc. of iodine should be added for the oxidation of the next sample, so that the final back-titer is at least 3 cc. Some samples give a red color at the end point, so a little starch indicator has t o be added; otherwise the stable amylose material left in the solution will act as the iodometric indicator without added starch paste. Calculate the number of milligrams of iodine consumed, divide by the weight of the sample, and multiply by 100. The result is the alkali-labile value.
way. I n samples low in electrolytes the alcohol will not bring about precipitation until a small amount of salt is added when there will be copious flocculation. For this purpose a few drops of 0.1 M barium chloride have been found to be most effective. Although better precipitants, such as acetone and ethyl alcohol, give more complete yields and do not cause fractionation of the amylose, they cannot be used because they consume iodine in the iodometric titration. Long drying in a vacuum oven will not remove the last traces of alcohol (6), so only methanol should be used. Starch as well as cellulose acts in this way. Free chlorine, hypochlorites, peroxides, borax, and sulfur dioxide, as well as any other reagents that react with iodine or iodide ion, must be absent. However, the allowable limit of sulfur dioxide of 0.005 per cent is equivalent to only 0.02 alkali-labile value and may be neglected.
The average deviation among the results from several determinations on one sample will run *0.5 alkali-labile unit, except when the sample is very nonuniform (some pastes) or in certain whole starches where gelatinization is slow and incomplete. Two operators making independent determinations will check to about 1.5 alkali-labile units. This should be considered satisfactory. Pastes and other excessively wet samples may be treated with anhydrous, acetone-free methanol which when used in large quantities will precipitate out the solids. These can be dried and the alkali-labile value determined in the usual
(1) Fales, “Inorganic Quantitative Analysis,” pp. 350-60, New York, Century Co., 1925. (2) Hall, A. J., “Cotton Cellulose,” p. 216, London, Ernest Benn, 1924: J. Textile Inst., 115, 27 (1924). (3) Hirst, Plant, and Wilkinson, J. Chem. SOC.,2379 (1932). (4) Klein and Acree, Bur. Standards J. Research, 5, 1063 (1930). (5) Koelichen, Z. phys. Chem., 33, 177 (1900). (6) Mease, IND.ENG. CHEM.,Anal. Ed., 5, 317 (1933). (7) Taylor and Beckmann, J. Am. Chem. Soc., 51, 294 (1929). (8) Taylor and Salzmann, Ibid., 55, 264 (1933), (9) Wenker, IND.ENQ.CHEM.,26, 350 (1934).
Literature Cited
RECEIVED June 5, 1935.
Estimation of the Saccharifying Power of
Malt Diastase Adaptation of the Hagedorn and Jensen Method H. C. GORE AND H. K. STEELE, The Fleischmann Laboratories, 810 Grand Concourse, New York, N. Y.
N
0 LINTNER method now exists which adequately fills the need for a short accurate process for the measure-
ment of the saccharifying activity of diastase of plant origin suitable for general use in the analysis of malt, malt extracts, and similar products. Such a method should require only apparatus that is generally available in laboratories and should give concordant results in the hands of analysts working in different laboratories. The classical Lintner method ( l a ) needs no description here. While still retained as standard by English brewing chemists ( 7 ) ,it has been adverseIy criticized by Sherman (16) and by Browne (a), especially when applied to very active malts. The Sykes and Mitchell method (IS)is probably the most precise Lintner method when provided with pH control, but is too long for control purposes, especially as the filtration frequently is extremely slow. The operator has to contend with not only this delay, but, as has been noted by Hanes (6), with the possibility of back-oxidation of the reduced copper. Moreover, according to Blish ( I ) , methods for estimation of reduced copper in Fehling’s solution fail to give concordant results in the hands of collaborators in different laboratories. The Sherman, Kendall, and Clark method (16) is one of the most accurate of those involving use of Fehling’s solution. Its improved technic consists of adding the starch solution rapidly to the malt infusion, thus reducing initial timing
errors to the minimum. Digestion takes place a t 40’ C. and a special scale has been developed for the expression of the diastatic activity. Like the Sykes and Mitchell method, however, it is too long for control purposes. One of the standard methods used by English brewing chemists (7) is a form of the Lane-Eynon (11)method. This method is not sufficiently precise for application to highly active malts, and measurement of the initial reducing powers of the malt infusion and starch solution is difficult. Moreover, the Lane-Eynon method in general does not appear well suited for routine malt analysis because maltose acts upon Fehling’s solution much more slowly than dextrose or levulose and it is necessary to boil for about 2 minutes after the addition of each portion of sugar solution before the full effect of the latter is manifest. The titration thus becomes decidedly cumbersome. I n the method of Windisch and Kolbach (17), adopted by continental brewing laboratories, the reducing substances formed in the substrate solution are oxidized by alkaline hypoiodate. The method as formulated is inadequate in dealing with highly active malts, while the hypoiodate technic as ordinarily used has been adversely criticized by Kline and Acree (9, I O ) . The polarimetric method (3) requires a special starch solution, and is operated a t a higher concentration of substratenearly 5 per cent-than used in other Lintner methods. It
SEPTEMBER 15, 1935
ANALYTICAL EDITION
is the most rapid of all Lintner methods and substantially as accurate as methods involving the weighing of the copper oxide. Hanes (6) described an exact titrimetric method for estimation of reducing substances formed by malt diastase, estimated as maltose, based on the Hagedorn and Jensen (6) micromethod for estimation of sugar in blood. For convenience and accuracy his method leaves little to be desired. Blish and Sandstedt (1) likewise used the Hagedorn and Jensen method as the basis for a n excellent method for estimating the reducing substances formed upon diastasis. Their method was applied to the study of saccharogenesis in flour suspensions. They discussed the results of the extended collaborative testing of the diastatic activity of flour, extending over a period of several years, and reported poor concordance of results when gravimetric copper reduction methods were used and when a picric acid method was tested. Excellent collaborative results were obtained, however, by using the principles of the Hagedorn and Jensen method. They worked out and thoroughly tested complete specifications for a procedure based on the above method and adapted to the measurement of diastatic activity in flour suspensions. They found the method t o be superior from the standpoint of accuracy, reliability, simplicity, and convenience to the technician, and expressed the opinion that "it should be conveniently possible to adapt the HagedornJensen method to advantage in the estimation of Lintner values." This suggestion has been carefully tested by the present authors, who find the procedure based upon the Hagedorn and Jensen technic superior to any existing Lintner method. It is fully as precise as the most exact of the maltose estimation methods, which use Fehling's solution, is shorter, and is far more convenient. Because the Blish and Sandstedt modification permits larger amounts of maltose to be estimated than the Hanes method, the reagents specified by Blish and Sandstedt were selected as most readily adapted for the measurement of Lintner activity. It remained for the authors t o work out the relationship of the reducing substances formed, estimated as maltose hydrate, to the alkaline ferricyanide consumed, and to fit the method to the analysis of malt and similar diastatic plant materials; since, as stated b y Blish and Sandstedt, the table given by them is applicable only t o solutions containing the special clarifying agents used in the measurement) of diastasis in flour suspensions. The method applied t o the analysis of malt, described below, depends on the reduction of potassium ferricyanide in slightly alkaline solution, followed by acidification and titration of the ferricyanide remaining by standard thiosulfate.
Reagents The following reagents are required, and are prepared according to the directions of Blish and Sandstedt (1): Buffer Solution: Walpole's 4.6 acetate buffer consisting of a mixture of 102 cc. of N acetate acid and 98 cc. of N sodium acetate diluted to 1 liter. 0.4 N Sodium Hydroxide. 0.4 N Hydrochloric Acid. Alkaline Ferricyanide Solution: 16.5 grams of pure dry potassium ferricyanide and 22 grams of anhydrous sodium carbonate, dissolved in water and diluted to 1 liter. This solution should be kept in a dark-colored bottle away from the light. The ferricyanide normality is 0.05. 0.05 N Sodium Thiosulfate Solution: 12.41 grams of sodium thiosulfate, dissolved in boiled-out distilled water and diluted to 1 liter. This solution may be checked against iodine solution which in turn is standardized against sodium arsenite, or the iodate titration method described by Hanes (6) may be used. Acetic Acid Reagent: 200 cc. of glacial acetic acid, 70 grams of potassium chloride, and 20 grams of zinc. sulfate (ZnSOr7H20), dissolved in water and made up to 1 Mer.
325
Potassium Iodide Solution: 50 grams of potassium iodide dissolved in water and diluted to 100 cc. It contains 1 drop 01 concentrated sodium hydroxide to prevent deterioration on standing. Soluble Starch Indicator Solution: 1 per cent solution of Lintner's soluble starch in 30 per cent salt solution. Suspend the soluble starch in water and pour into boiling hot water, cool, add the salt, and make up to volume. This solution keeps for several months.
Procedure The 10-cc., 25-cc., and 50-cc. pipets should be calibrated, or, preferably, certified by the Bureau of Standards. PREPARATION OF MALTINFUSION. Digest 25 grams of finely ground malt, prepared preferably by grinding 25 grams plus several extra kernels in a Seck mill, with 500 cc. of water at room temperature for 1.5 hours with occasional mixing. Filter, rejecting the first 50 cc. of filtrate. Repeated tests on many different samples of malt by Rteele show that the 1.5-hour digestion is amply long. The reason for the rejection of the first 50 cc. of filtrate is the known adsorption of malt diastase by filter paper, shown by Schultz and Landis (14). In case of malt sirups, mashes, etc., a multiple or fraction of a 5 per cent solution should be used, depending on the diastatic activity. PREPARATION OF SOLUBLESTARCH SOLUTION.Heat about 750 CC. of distilled water nearly to boiling, add 22 grams of Lintner's soluble starch suspended in water, heat to boiling, cool, add 50 CC. of Walpole's 4.6 acetate buffer, and dilute to 1 liter a t room temperature. The starch used preferably should not contain more than 2 per cent of reducing substances estimated as maltose hydrate. Such starch can be readily prepared from high-grade potato starch, using the directions given by Lintner (IS): The starch is mixed with 7.5 per cent hydrochloric acid and let stand for 6 days at 17" to 20" C. Then it is washed, suspended in water, and neutralized with a little sodium bicarbonate, thus neutralizing the last of the acid which clings persistently to the starch. I t is then washed and dried in a gentle current of warm dry air. The directions call for 22 grams per liter, 80 that when diluted by the addition of 10 per cent by volume of diastase infusion, the substrate concentration shall be 2 grams of air-dry starch per 100 cc., many tests having shown that more precise control of the starch concentration is unnecessary. DIGESTIONOF MALT INFUSION WITH STARCHSOLUTION. Dilute 25 cc. of the filtered infusion to 250 cc., transfer 10 cc. of the dilute infusion to a 200-cc. volumetric flask, and place in a water bath maintained at 20' C. At a noted time deliver 100 cc. of the starch solution also at 20" C. from a fast-delivery 100-cc. pipet, counting time from the moment when the starch solution reaches the infusion. At the end of 0.5 hour, add 20 cc. of 0.4 N sodium hydroxide from a pipet, mixing the first of the alkali instantly with the starch solution, thus abruptly checking the diastatic action. Neutralize the alkali by the addition of 20 cc. of 0.4 N hydrochloric acid, complete the volume to 200 cc., and mix well. The sodium hydroxide used for terminating the diastatic action is neutralized with hydrochloric acid to prevent the possible injurious effect of the alkali on the oxidation of the reducing substances by the mild alkaline ferricyanide solution. The effect on the reaction of the salt formed was found to be negligible. The increase in Lintner activity per l a C. is about 8 per cent, so that it is necessary to keep the temperature at exactly 20" C., using a thermometer of known accuracy and graduated, preferably in tenths of a degree. Transfer 25 cc. of the starch solution to a 500-cc. TITRATION. Erlenmeyer flask, and add 50 cc. of the alkaline ferricyanide. Mix well, and heat the flask and its contents for exactly 15 minutes in a boiling water bath, keeping the contents completely under the surface of the boiling water. Cool to room temperature, add 125 cc. of the acetic acid reagent and 5 CC. of the potassium iodide solution, and titrate with the standard 0.05 N thiosulfate. DETERMINATION OF BLANK. Mix 10 cc. of the diluted infusion in a 200-cc. volumetric flask with 20 cc. of the 0.4 N sodium hydroxide. Then add 20 cc. of the 0.4 N hydrochloric acid and 100 cc. of the starch solution, dilute to 200 cc., mix, and measure the reducing power of a 25-cc. aliquot by mixing it in a 500-cc. Erlenmeyer flask with 50 cc. of the alkaline ferricyanide, heating, cooling, acidifying, and titrating as above described. EVALUATION.Calculate the net volume of 0.05 N ferricyanide consumed by subtracting the volume of 0.05 N thiosulfate required from the 0.05 N equivalent of the alkaline ferri-
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cyanide solution, and correct by subtracting the 0.05 N ferricyanide value of the blank. Degrees Lintner are then computed by the formula L = 8.092 F , where L is degrees Lintner and F is the net volume of 0.05 N ferricyanide consumed. Table I shows analyses of a malt, a malt sirup of about 60 Lintner, and a malt sirup of about 300 Lintner. TABLEI. ANALYSES Dilutions of 5% Infusion cc
.
Net 0.05 N 0.05 N 0.05 $7 ThioFern- Ferrisulfate cyanide cyanideu
cc.
cc.
Factor
Degrees Lintner
cc.
Malt 25 t o 2 5 0 30.24 19.81 17.39 8.092 140.7 Blank 25 to 250 47.63 2.42 ... Malt sirup 50 to 250 29 4 20.65 13:90 8,'OBZ X 0.5 56.2 6 85 Blank 50 to 250 43.2 296:6 Malt sirup 10 to 200 28.47 21.58 18:33 8.'092 X 2 10 to 200 46.8 3.25 ... ... Blank a Fifty cc. of the reputed 0.05 N alkaline ferricyanide were equivalent to 50.05 cc. of 0.05 N thiosulfate. I,.
Titration Hanes observed that the end point in the titration is extremely sharp. The authors find that the estimation of hundredths of a cubic centimeter can be approximated. The ratio of cubic centimeters of 0.05 N ferricyanide consumed to maltose oxidized was determined experimentally. Fifteen minutes' heating in a boiling water bath was found to be preferable to 20 minutes' heating as recommended by Blish, who presumably worked a t a slightly lower alkalinity. The maltose hydrate used was recrystallized from alcohol and carefully dried. Its specific rotatory power (5 grams dissolved in water, heated to go', cooled, diluted to 100 cc. at 20' C., polarized at 20' C. at 37.7" V.) was a = 130.6. A series of solutions was prepared in calibrated 200-cc. flasks and 25-cc. aliquots were mixed with 50-cc. portions of the alkaline 0.05 N ferricyanide solution, heated for 15 minutes in a boiling water bath, cooled, and titrated as above described, using Bureau of Standards calibrated pipets and buret.
TABLE 11. DETERMINATION O F RATIO Maltose Hydrate Mg./?26ec. 25 50 75
87.6
Alkaline 0.05 N Ferricyanide Reduced
cc. 13.06 26.64 39.57 46.0
Ratio of Maltose Hydrate .to Ferricyanide Consumed Mo./cc. 1.91 1.88 1.89 1.90 Av. 1 . 9 0
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power to 408.3 mg. of maltose hydrate, are formed under standard conditions by 50 mg. of malt of 100 Lintner. In the above method 25 cc. of the digestion mixture correspond t o 6.25 mg. of original sample. An infusion of 6.25 mg. of a malt of 100 Lintner acting on starch solution under standard conditions at 21" C. would produce 51.04 mg. of reducing substances per hour computed as maltose hydrate. If acting at 20" instead of 21" C., 6.25 mg. of a malt of 100 Lintner would produce only 92 per cent of the above amount of reducing substance, or 46.96 mg. per hour, equal to 23.48 mg. per half hour.
If we accept Kjeldahl's law of proportionality (8) as operative during the reaction, we may write the following formula 100 m L = - -23.48 9 where L is the Lintner value of the malt and m is the amount in mg. of reducing substance reckoned as maltose hydrate. We can substitute for m its value 1.90 F where F equals cubic centimeters of alkaline 0.05 N ferricyanide reduced. We then have the expression L = 8.092 F. The value 92 per cent is subject to revision, as it is only approximately correct. When redetermined, the value of the factor 8.092 probably will be slightly affected. When we examine the classical papers of Lintner ( I Z ) , we realize that the above definition of the Lintner scale can be accepted only if certain conventions are agreed upon. Thus Lintner seems to have specified room temperature only, and not 21" C. Again, while we may assume that he measured diastatic power within the optimum range (4) of its p H curve, we have no specific knowledge that this was done; indeed, he specified neutrality as the optimum reaction. The writers believe that the Lintner activity should be considered to be a specific property of the diastatically active plant product as above defined. As a convenience the Lintner value may be measured a t some other temperature (or pH) and the activity then calculated to true Lintner, provided that the temperature (or pH) coefficient is known. I n the method described above 20" C. is specified as the digestion temperature for greater convenience in malt analysis, since a water bath maintained a t 20" C. is required in t h e estimation of extract. Discussion The above method introduces a technic of precision replacing methods based on Fehling's solution. It is offered tentatively for trial by those interested. Acknowledgments The authors wish to thank C. N. Frey for his constant support and encouragement, and G. E. Miller and W. R. Johnston for their helpful advice.
Literature Cited Evaluation The evaluation formula is derived from the standard definition of the Lintner malt scale. Thus Browne (2) states, "a malt is given a diastatic value of 100 on Lintner's scale when 0.1 cc. of the filtered 5 per cent extract just reduces 5 cc. of Fehling's solution under the above conditions." I n other words, a standard infusion of a malt of 100 Lintner is of such activity that 1 cc. of filtered extract corresponding to 50 mg. of sample produces sufficient reducing substances, reckoned as maltose hydrate, completely to reduce 50 cc. of Fehling's solution upon acting on a 2 per cent solution of soluble starch a t 21" C. for 1 hour. According t o Browne (8) there are in 50 cc. Fehling's solution 438 mg. of copper, which are equivalent to 493.1 mg. CUZO. By extrapolation from the Munson and Walker table 493.1 mg. CUZO are equivalent to 408.3 mg. maltose hydrate. Thus, reducing substances consisting largely of maltose, equivalent in reducing
Blish and Sandstedt. Cereal Chem.. 10. 189 (1933). Browne, C. A., "Handbook of Sugar Anaiysis,;' New York, John Wiley & Sons, 1922. Gore, IND. ENQ.CHPIM., 20,865 (1928). Gore, J. Am. Chem. Soc., 47, 281 (1925). Hagedorn and Jensen, Biochem. Z.,135,46 (1923). Hanes, Biochem. J.,23, 99 (1929). Inst. Brewing, J . Inst. Brewing, 39, 518 (1933). Kjeldahl, Carlsberg Lab., 1879; 2. ges. Brauw., N. F., 3, 49,
84,123,149, 179,222 (1880). Kline and Acree, Bur. Standards J. Research, 5, 1063 (1930). Kline and Acree, IND. ENQ.CHEM.,Anal. Ed., 2, 413 (1930). Lane-Eynon, Chemistry & Industry, 42, 32 (1933). Lintner, J. prakt. Chem., (2)34, 378 (1886); 36, 481 (1888). Lintner, 2. ges. Brauw.. 31, 421 (1908). Schultr and Landis, J . A m . Chem. Soc., 54, 211 (1932). Sherman, Kendall, and Clark, Ibid., 32, 1073 (1910). Sykes and Mitchell, Analyst, 21, 122 (1896). Windisch and Kolbach, Wochschr. Brau., 42, 139 (1925). R ~ C E I V EJune D 7, 1935. Presented before the Division of Agricultural a n d Food Chemistry at the 89th Meeting of the American Chemical Bociety, Nay York, N. Y., April 22 to 26, 1936.
