Determination of the Alkali-Labile Value of Starches and Starch Products T. C. TAYLOR, H. H. FLETCHER, A N D M . H. ADAMS Department of Chemistry, Columbia University, New York, N. Y.
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From experience the more important variables have been found to be:
N A PREVIOUS paper (8) it was shown that by meas-
uring with hypoiodite the amount of material in a starch, amylose, or starch product that is attacked by hot aqueous alkali, a number could be obtained that was an index to the peculiar make-up of the sample in hand. Slight changes in the starch or starch product that cannot be detected by such well-known determinations as viscosity, specific rotation, color with iodine, and initial reducing value, are, however, immediately reflected in a change in the stability of the product to the action of hot aqueous alkali. While this has been used in a qualitative way (3) by others, the subsequent quantitative determination by iodometric titration of the extent of attack by the aqueous alkali has made the method of interest to starch chemists. Every starch and starch product contains some material which is very quickly attacked by hot aqueous alkali and a part which is relatively slowly attacked by the same reagent. The latter part can be recovered substantially unchanged after the alkali has acted. The two parts have been called, respectively, alkali-labile and alkali-stable, the one being the antithesis of the other. Various pretreatments of the starches or their amyloses cause changes in the amount of this alkali-susceptible portion. By titrating iodometrically the alkali-digested solution of noncarbohydrate breakdown material having its origin in the alkali-labile portions, a number, expressed in milligrams of iodine per hundred milligrams of sample may be obtained. This is called the alkali-labile value. A somewhat analogous procedure, called the coppernumber determination (d), has been used to some extent in cellulose chemistry for a somewhat similar purpose. Both methods depend on reactions that have for their point of departure the attack of free or easily available aldehydic groups in certain parts or fractions of complex carbohydrates. The copper-number method is not, however, useful for application to the starches or starch products, one reason being that the hot aqueous alkali that causes the decomposition and the copper that measures it are both present during the operation. I n the alkali-labile determination, on the other hand, the alkali is allowed to act, the solution is neutralized, and the effect of the hot aqueous alkali measured separately by hypoiodite solution, volumetrically. The small amount of cold aqueous alkali used in the iodometric determination produces itself no further alkali-labile material. The method under discussion is a semi-micro one and very sensitive to changes in technic. After being applied to several thousand samples by several analysts, certain discrepancies in results made their appearance. Therefore all apparent variables were investigated one a t a time and a modified method was evolved which, while giving slightly higher results than the older method, gives much greater precision. The values are still admittedly empiric, but with care and a little practice they can be duplicated and for comparative purposes serve very well.
PARTI. (1) period of digestion in the hot 0.1 M alkali (This molarity was fixed when the original work was done. A dearture from it will also cause a slight variation in samples of gigh alkali-labile value; otherwise there will be very little difference. The more dilute alkali gives the higher' results during digestion.); (2) the elapsed time after digestion and before iodometric titration: and (3) the alkalinity or acidity during this period. PART11. (1) period of contact with the hypoiodite solution before the thiosulfate titration: (2) amount of excess alkali present during treatment with iddine and (3) amount of iodine in excess present during the hypoiodite treatment, In these experiments a sample of dry cornstarch ground in a ball mill for 168 hours (7) was used as a standard. The iodometric titrations (4) were made at first by a slight modification of the Taylor and Salzmann (8) technic. I n this first revision 5.00 cc. of 0.1 M alkali were added at one time and then enough iodine (as determined by a trial run) so that there would be about 0.75 cc. of iodine unused a t the end of the operation. The values given in the first part of this paper are therefore on this basis. Subsequently it was found desirable, in the light of better understanding of the variables, to modify again this part of the analysis. The old and new values on this ground starch, however, bear a fixed relationship to one another, so any curves drawn with values for the former iodometric method will illustrate the argument but should not be taken as the best index values for a permanent record. It became evident soon after the experiments were started that the alkali-labile values taken immediately a t the end of the digestion in hot aqueous alkali were high but fell rapidly as the solution cooled, reaching an approximately steady state, the rate being dependent on the hydroxyl-ion concentration and the temperature while standing. The higher the alkalinity the shorter the time it takes for the solutions to reach a state of slow change a t room temperature. If the alkaline-digestion mixture is chilled immediately after completion of the digestion and made acid, the high value may be fixed, so that the drift to a lower value is very slow. These results are shown graphically in Figure 1 where I shows drift in alkali-labile values in a solution neutralized to phenolphthalein (therefore still alkaline), I11 shows changes when excess acid was added after chilling immediately a t end of digestion, and I1 after a sample was neutralized to phenolphthalein, allowed to undergo changes a t this lower alkalinity for a time, and then over-acidified with hydrochloric acid and fixed at the lower value. When the sample was left, on the other hand, in the concentrated alkali used in the digestion, the alkali-labile value dropped from 28.2 to 21.8 in 15 minutes at 25' C., but only ' C. When kept at 25' C. to 23.0 in the same interval a t 4 for longer periods the alkali-labile value drifted slowly down, until at the end of a 6-hour interval it reached 17.0. Because of the rapid drop in alkali-labile value in the early part of the interval after the hot aqueous alkaline digestion has been terminated by cooling, and even partial neutralization (to phenolphthalein), it is necessary, if the high initial value is to be preserved, not only to chill quickly but to make the mixture definitely acid. By adding excess acid immedi-
Development of Method The method may be divided into two parts-that which has to do with the hot alkaline digestion and subsequent partial or complete neutralization, and that which has to do with concentrations of iodine and alkali in the iodometric titration. 321
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ately upon chilling the hot alkali-digested sample, the alkalilabile value is fixed and no longer drifts as it did when left on the alkaline side. While it is possible to over-acidify after a longer interval in the cooled alkaline mixture and stop the relatively rapid drift in alkali-labile values that in fact would go on even at phenolphthalein neutrality, these rather arbitrary values are not easily reproducible. Indeed, the
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ous alkali is used twice in the determination, once in excess a t 100" C. for the digestion, and second in the iodometric determination to make hypoiodite. I n the latter instance i t is used, of course, in the cold and only in small amounts where its role is totally different from that in the first instance. Too large an excess of alkali converts all the iodine to iodate.) To this end the excess acid used in the first step t o quench the alkali of the first hot digestion mixture is neutralized to 30 some fixed point and an accurately measured amount of carefully standardized alkali added. Then the iodine is added as in the usual procedure and the mixture 28 allowed to react. Because phenolphthalein even in methyl alcohol uses 9 26 iodine and because its turning point is back in the alkaline regibn where the solution begins to drift again, the new indicator nitrazine yellow (9) is employed (made by the Wenker Chemical Co., 616 Jackson Ave., Elizabeth, N. J.). This not only gives a sharper and 22 more reproducible color change in the brown solutions but the change takes place very near pH7. Further, the indicator does not itself react with iodine. A careful PO back-titration of the excess acid with this indicator gives solutions which can be made uniformly properly alkaline for the subsequent iodometric technic. /8 2 3 4 5 6 7 8 0 / 9 The time interval of reaction with the alkaline hyHours poiodite also affects the results. As Figure 4 shows, CHANGE OF ALKALI-LABILE VALUES WITH TIME, FIGURE1. the previously specified 15-minute period comes when AFTER DIGESTION IN BOILING ALKALI I. Solution neutralized only to phenolphthalein the rate of change is high. Therefore a 45-minute 11. Effect of excess acid in stopping change interval was decided upon, for in this part of the curve 111. Effect of excess acid added immediately to cooled digestion mixture the rate of change is very slow and the results therefore are easier of duplication with precision. Below 20" C. the action of iodine and aqueous alkali (hyprincipal reason for lack of concordance among the results poiodite) on the digested samples proceeds so slowly that it from various analysts is now easily explicable for, if the iodohas not reached a steady state a t the end of 45 minutes. metric determination is made a t different periods after the Concurrently iodate is formed in these solutions also and the hot alkaline digestion, the values will be different and to a active hypoiodite is removed thereby, so that the latter canlarge extent a function of the personal habits of the analyst. not function as it should with the decomposition products of Except for this part of the determination, all other periods the amylose. Between 25" and 30' C. under the conditions of treatment, concentration of reagents, and temperatures were selected so that the transformations involved at the par25 ticular points in the procedure had reached a steady state. I Turning to the cause for the drifting in alkali-labile value, $24 it is probable that the behavior of the products from the alkaline decomposition of the carbohydrate material is due R in part a t least to aldol-type reactions which are known to be $23 reversible with temperature in many cases (6) and catalyzed by hydroxyl ion. Apparently the materials when in equilib0 rium in the hot alkali may be kept approximately at that $22 point by rapid chilling and slight over-acidification. Indeed, if the solution is made even slightly alkaline the alkali-labile 2/ values drift much more rapidly again to a new approximately 85 30 35 40 45 50 56 60 steady state, temperature also having an influence. 002.5/? ZWne /h cc. If the determination must be interrupted, it is best t o do FIGURE2. EFFECTON ALKALI-LABILE VALUEOF VARIAthis after cooling and quenching with acid after the digestion TION OF AMOUNT OF IODINE ADDEDBEFORE BACKTITRATION (Figure 1,111), for now the change is slow. Values represent excess of reagent over that consumed if the old I n the iodometric part of the determination, it was found method of digestion in the previous step is used. Relatjons are same in revised teohnic, but amounts of iodine are pro ortionately that the amount of iodine in excess of that which will be used higher. In each case 5.00 0 0 . of 0.1 M alkali were addelto form the in the reaction does not affect the results as much as it was hypoiodite. first thought to do. Keeping the amount of alkali used in the hypoiodite treatment constant at 5.00 cc. of 0.1 M sodium given, the speed of reaction of the hypoiodite on the amylose hydroxide but varying the amount of excess 0.025 M iodine decomposition products is much faster and in 45 minutes has solution, the alkali-labile value increases slowly and then reached a point beyond which there is substantially no change decreases again. This is shown in Figure 2. (Figure 4). During this time at the higher temperature the When, however, the amount of 0.025 M iodine is kept conamount of iodate formed at the expense of the hypoiodite is stant at 5.00 cc. and the amount of 0.1 M aqueous alkali is apparently negligible and so does not affect the completeness progressively increased, the alkali-labtle values increase a t a of the reaction. The optimum conditions for the iodometric high rate. This is shown in Figure 3. part of the determination were therefore set with these conIt is very important, therefore, to have the amount of siderations in mind. alkali fixed at a known point before the sample is allowed to An iodometric method for determining reducing sugars has react with the iodine. (It should be borne in mind that aque-
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SEPTEMBER 15. 1935
ANALYTICAL EDITION
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7
8
9
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0
20
O./N NaON in cc.
40
50
60
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FIGURE4. CHANGE IN ALKALI-LABILE VALUEWITH TIMEINTERVAL DURING WHICH HYPOIODITE WASALLOWED TO ACT BEFORE BACK-TITRATION OF EXCESS IODINEWITH THIOSULFATE SOLUTION
FIGURE 3. EFFECTON ALKALI-LABILE VALUEOF VARIAmoN OF AMOUNT OF ALKALI PRESENTDURING TREATMENT OF DIGESTED MIXTUREWITH IODINE In each caee 5.00 CD. of 0.025 Y were added.
been used in the past (4) and the iodometric part of this procedure is a modification of that original method. If the new adaptation is used to determine reducing value-that is, when the titration with hypoiodite is made without the preceding hot alkali digestion of glucose-the stoichiometric oxidation to gluconic acid is not complete. Were it complete, the reducing value in terms of milligrams of iodine per 100-mg. sample would be 141, whereas by this method it is 80 and the alkali-labile value a t the end of 60 minutes is 66. In the last value the iodine consumed, of course, has reacted with the noncarbohydrate decomposition products, principally through the iodoform reaction. There is, therefore, no special significance to this so-called initial reducing value except in an empiric sense. Maltose monohydrate has an alkali-labile value of 67.1 (calculated anhydrous alkali-labile value of 70.7). It may be said, in general, however that for the amylose and dextrins the initial values are always much less than the alkali-labile values, while with glucose and maltose the initial values are always greater than the alkali-labile values, all determinations being made by the same iodometric procedure peculiar to the method. A good grade of air-dried commercial cornstarch had an alkali-labile value of 22.8 and a commercial tapioca flour a value of 14.3. A certain thin-boiling starch, on the other hand, had a high characteristic value of 60, while several canary torrefaction dextrins had values a t or near 20. It is interesting to note that the initial reducing values expressed also in milligrams of iodine per 100-mg. sample of the three above-mentioned materials were substantially the same, about 9.0. These few data show the range of the alkalilabile values for widely differing products in contrast to the relatively constant initial values. Small amounts of glucose admixed with these starches and dextrins will contribute largely to the initial reducing value but hardly a t all to the alkali-labile value. The theoretical aspect of the alkali-labile value and its use in interpreting various transformations is the subject of work t o be published in the near future.
30
On the starches the values by the new method are about 10 units higher than the old.
Procedure The following solutions are required: 1. 0.1 M sodium hydroxide, slightly more than 4 grams per liter, diluted t o proper molarit . 2. 0.1 M hvdrochloric a c i l 8.5 cc. of reagent hvdrochloric acid made up t6 1 liter with water. 3. Concentrated hydrochloric acid, reagent. 4. 0.025 M sodium thiosulfate, 6.2 grams of NazSz03.5H20 made up t o 1 liter with water (I). 5. 0.025 M iodine, 6.3 grams of iodine plus 35 grams of Dotassium iodide made UKJt o 1 liter with water. 6. Phenolphthalein, 605 gram made up to 100 cc. of 50 per cent alcohol. 7. Nitrazine yellow, 0.1 gram made up to 100 cc. with water
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The absolute strength of iodine and thiosulfate need not be exactly 0.025 M , but should be standardized carefully ( 1 ) . The sodium hydroxide, however, should be accurately standardized at 0.1 M (within 3 to 5 parts per thousand) with potassium acid phthalate, using phenolphthalein indicator. The hydrochloric acid need not be standardized unless it is desired to know how much sodium hydroxide is used during the digestion. PROCEDURE. It is convenient t o run 12 determinations (6 samples) at B time. Each sample is run in duplicate. 1. Weigh approximately 50-mg. samples of the starch product into large Pyrex test tubes (20 X 2.5 cm., 8 X 1 inch). Weigh t o 0.1 mg. 2. Add 10.00 cc. of exactly standardized 0.1 M sodium hydroxide to each test tube from a buret. The test tubes are loosely stoppered and floated in actively boiling water for 1 hour. Enough water should be used so that the tubes will float throughout the hour. 3. After the digestion, remove the test tubes from the bath, hold under running cold water for about 30 seconds, and then immediately acidify with 10 cc. of 0.1 A4 hydrochloric acid (use buret), with thorough shaking. It should not take over 1 minute from removal from bath t o complete cooling and acidification. 4. Transfer the contents of the test tubes to 250-cc. Erlenmeyer flasks with two washings of 10 cc. each of distilled water. The time interval before the next step is not important. TWO hours may elapse if necessary.
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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. In 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.
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0 LINTNER method now exists which adequately fills the need for a short accurate process for the measurement 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. In 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
