SEPTEMBER 15, 1935
ANALYTICAL EDITION
minutes, from 0.4 to 0.6 mg. per ml. was the smallest amount detectable. Sodium in amounts up to 50 mg. per ml. seemed without effect on the sensitivity. Solutions containing both sodium and potassium nitrates were treated with zinc cobaltinitrite until all the potassium was removed and then tested for sodium by zinc uranyl acetate reagent prepared according to Reedy (2). After removal of 50 mg. of potassium per milliliter 3 mg. of sodium per milliliter were detectable by the formation of a distinct yellow-green precipitate. When the amount of potasium was 10 mg. per milliliter, 1 mg. of sodium could be detected in the filtrate.
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Procedure To the solution from the alkaline earth group, which contains no barium, arsenate, phosphate, or ammonium, add an equal volume of zinc cobaltinitrite solution and let stand for 15 minutes. A yellow precipitate of potassium cobaltinitrite indicates potassium. Filter and to the filtrate add zinc uranyl acetate solution. Sodium is indicated by the formation of a crystalline
greenish yellow precipitate.
Literature Cited (1) Kolthoff, I. M., 2.anal. Chew., 70, 397 (1927). (2) Reedy, J. H., “Elementary Qualitative Analysis,” 2nd ed., New York, MoGraw-Hill Book Go., 1932. RECEIVED July 15, 1935.
The Estimation of Starch J. T. SULLIVAN, Purdue University Agricultural Experiment Station, Lafayette, Ind.
T
HE estimation of starch is of interest to those engaged in many industries, to those concerned with the enforcement of food laws, and to plant chemists and physiologists in the investigation of botanical, horticultural, and agronomic problems. The objects to be attained by the various analysts, the degree of accuracy required, the methods which are most suitable, and the particular technical difficulties involved, vary. I n the analysis of plant products for any constituent, a separate problem may arise for each type of material. This is true of starch analysis and each field of activity has its favored methods which are believed to be more suitable in their particular circumstances. Starch is usually accompanied by other carbohydrate substances, such as cellulose, hemiculluloses, pentosans, gums, mucilages, glucosides, pectins, and tannins, and its own hydrolytic products, sugars and dextrins. It is the problem, of course, to separate starch from these substances, if possible, and, if not, to hydrolyze it to sugars in their presence under such conditions as will cause the least interference by these other materials.
Acid Hydrolysis The simplest method of converting starch t o sugar is the acid hydrolysis method. Approximately 90 parts of starch yield 100 parts of glucose. I n practice, this yield, popularly considered theoretical, has not been obtained, What is the theoretical yield? To the starch manufacturer, “starch” is everything in the granule (17). It has been customary, however, with those in other fields to consider starch as only that which on hydrolysis yields simpler carbohydrates. With the latter viewpoint the theoretical yield may be calculated from the organic structure of starch. If starch consists of a chain of glucose units linked together with the loss of water at each linkage, on hydrolysis there will be added to its weight one molecule of water for each glucose unit, less one. If the chain contains six, twelve, or twenty-four glucose units, the glucose starch conversion factors are, respectively, 0.917, 0.907, and 0.904. If the chain is longer, the conversion factor approaches more closely 0.9. If the chain is closed t o form a loop, the factor is exactly 0.9, regardless of the size of the molecule. Recent contributions of organic chemists (16, 18, dW, and others) indicate that the chain is very long and the correct factor is therefore very nearly 0.9. However, the work of Noyes (36) indicates that 100 per cent recovery cannot be obtained by acid hydrolysis, and he accordingly suggests the
factor 0.93. Others have adopted this and other factors. Noncarbohydrate constituents, moisture excepted, such as ash, protein, fat, and fiber, are rarely present in excess of 1 per cent in a good grade of starch, although these constituents and also moisture are not easily and accurately determined. It has been demonstrated that glucose is not injured by heating with acid unless excessive concentrations of acid or duration of heating are used (5,8). Nevertheless there have been formed from glucose by the action of acid and there also occur in the mother liquor of starch hydrolyzates in some industrial processes, disaccharides which are probably derived from glucose and have less reducing power than glucose. These factors probably account for the failure to obtain complete recovery of starch unless the larger factor, for which there is no theoretical basis, is used. Using the factor 0.9, the author has recovered, as measured by the reducing power of the glucose, 97 per cent of the starch. Because of the susceptibility of other carbohydrates to attack by boiling acids, the use of this method has limited application. It has been discontinued by workers in most plant fields. The only recent attempt to utilize this method is that of Fraps (15) who used a very dilute acid (0.02 N ) and completed the hydrolysis with stronger acid after the insoluble matter had been removed and corrected for pentosan dissolved.
Enzymatic Hydrolysis When carbohydrate substances other than starch are present, it has been customary to resort to enzymatic hydrolysis. Diastatic, as well as other enzymic preparations, are notoriously impure and contain other enzymes which attack many other substances. Moreover these preparations do not hydrolyze starch into a single product, but rather into mixtures of sugars, or of sugars and dextrins. The relative proportions of these products are not constant but depend upon every possible variation in the conditions of hydrolysis. As a result of this situation, the estimation of starch by diastatic means has been resolved into three types of methods : (1) the mixed products obtained are estimated by two different methods, and each product may be calculated by the use of simultaneous equations; (2) empirical methods, by which the products are obtained under rigid conditions of hydrolysis and are found to have certain definite reducing or rotational values; and (3) methods in which the diastatic action is supplemented by a subsequent hydrolysis with mineral acids to yield a single product, glucose.
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Barley and Malt Diastases
Takadiastase
It is not possible here to consider all the diastatic methods. Granular starch consists mainly of two substances, a-amylose or amylopectin and /3-amylose, or “amylose.” Diastatic enzymes have been differentiated into two amylases, called a-and p-, according to the stereochemical form of the maltose produced (27). The a-amylase presumably converts the amylopectin into dextrins and is referred to as the liquefying or dextrinogenic amylase. P-Amylase converts the amylose into maltose and is known as the saccharogenic amylase (37). Both enzymes are present in malt but only the p- in the ungerminated grain. Later work, however, indicates that both enzymes will produce both dextrin and maltose but at different rates (39, and that both enzymes are present in ungerminated grains, but in different amounts (44). Ling, Nanji, and Harper (SO) have suggested the use of the diastase of ungerminated barley. When this acts on the cereal starches the maltose formed is derived wholly from the P-amylose. Amylopectin yields a hexa-amylose (31) and a third constituent present in some starches, amylohemicellulose, is unattacked. The relative amount of maltose produced from the p-amylose is determined by a control experiment, using potato starch. Assuming that the ratio of amylose to amylopectin in cereal starches is fixed-namely, 2 to 1-the starch content may be calculated from the reducing power. However, other ratios of amylose to amylopectin have been reported ($6,41, 42). The use of germinated barley is more common. Containing both types of amylases, it has both dextrin-forming and sugar-forming activity. Both amylose and amylopectin are attacked. The fate of amylo-hemicellulose is unknown. The products of this action are mainly maltose and in lesser degree dextrin, of varying composition. Under standard conditions the amount of maltose formed depends upon a group of factors referred to as the diastatic power of the malt. According to Ling (WQ), if a series of malt preparations which vary from 20 to 100 on his scale of diastatic power act upon barley and wheat starches, the amount of maltose which they will produce under identical conditions varies from 80 to 87.5 per cent of the weight of the starch. Therefore in making a determination on cereal products the yield of maltose obtained is compared with that theoretically produced by the same malt. Ling and others have contributed to quantitative methods based on this principle. Another procedure involving the use of malt diastase and one apparently more in use in this country is that of heating the products of enzymatic hydrolysis with acids. By this means the maltose and dextrins are converted into glucose. The advantage of this procedure is that since glucose is the only final product arising from starch, it may be easily estimated. Also the extremely uniform conditions of hydrolysis required when a mixed product is produced are rendered less necessary. The chief disadvantage, as pointed out before, is that acid hydrolysis is not specific for starch or its hydrolytic products, maltose and dextrin. Other substances present may also be hydrolyzed, although the conditions of hydrolysis in this case are not as severe as those in the direct acid hydrolysis method. To avoid in part this disadvantage, various means have been employed to remove some of these contaminating substances. Simple filtration removes insoluble matter. Pectin material, but not dextrins, are p r e cipitated by alcohol of a 60 per cent concentration. The Walton and Coe method (&), using this step, has been adopted as official by the Association of Official Agricultural Chemists (3). Herd and Kent-Jones (19) have recently published a criticism and comparative study of some of these methods.
Takadiastase, the enzyme of the fungus Aspergillus oryzae, is widely used among workers in the field of plant physiology. Commercial preparations of this enzyme are mixtures of many enzymes (34), the pertinent ones being a-amylase and maltase. Davis and Daish (6) introduced it as a quantitative reagent and referred to Hill (21) as the authority that its products are maltose and glucose and that it does not give rise to resistant dextrins. Though the publications of Hill do not furnish any definite proof of such products, the statement has been repeated in many publications. Davis and Daish followed the course of the hydrolysis of starch and made periodical determinations of the optical rotation and the copper reducing power. Six hours from the start no dextrins were present. Assuming that the products were glucose and maltose only, they calculated that at 6 hours the ratio between the two was 0.1, and this ratio gradually increased to 6.0 at 72 hours. After 72 hours no further hydrolysis of maltose to glucose took place. The sum of the two constituents at any time was practicalry equivalent to the amount of starch originally present. Horton ($3) was unable to duplicate these results and believed dextrins were present. However, the.use of takadiastase has persisted and many methods have been proposed to determine the amounts of each of the sugars present. Thomas (43) found that at 24 hours the ratio between glucose and maltose was 2 to 1, but Widdowson (47) found higher proportions of glucose. Horton’s data had previously shown that an increased concentration of enzyme gave a higher proportion of glucose, but Collins (4) pointed out the necessity of a high concentration of enzyme which, together with a sufficient length of time and the proper hydrogen-ion concentration, gave complete hydrolysis to glucose. Collins bases her conclusions on the fact that both takadiastase and acid gave a recovery of 93 per cent of the dry weight of the starch. Acid hydrolysis will give, however, a higher yield than 93 per cent, if the factor 0.9 is used to convert glucose to starch. If the factor 0.93 is used as she suggests, a recovery of nearly 100 per cent should be obtained. Denny (7) and Lehmann (28) report complete recovery of starch by this method. If both maltose and glucose are present during the hydrolysis, and if present in the proportions that have been reported, it should be possible to detect the maltose. According to Davis and Daish, there were ten times as much maltose as glucose present at 6 hours, three times as much a t 12 hours, and one-fourth as much at 48 hours. According to Thomas, at 24 hours, there was one-half as much maltose as glucose. Working with mixtures of pure glucose and maltose, the author has been able to identify the osazones of maltose when i t was mixed with three parts of glucose. The total sugar concentration of the solution was 10 per cent. With lower concentrations or with higher proportions of glucose, the osaaones of maltose could not be detected in the presence of those of glucose. When these tests were made upon the hydrolyzate from starch, no maltose was detected at any time, even at the early stages of hydrolysis when the proportion of it should be high. The enzyme was killed and the solution concentrated before making the test so that the conditions were comparable to those with pure sugars. Precipitation of the enzyme material with alcohol did not alter the results. Hill (20) described a method for the isolation of maltose when mixed with glucose by the use of maltase-free yeast, but he did not report having used it on the products of takadiastase action. Englis, Pfeifer, and Gabby (11) suspected that some other disaccharide than maltose was present in the hydrolytic products. The failure of the attempt to identify maltose does not prove its absence nor would the proof of its absence invalidate the use of takadiastase. If, however, the
SEPTEMBER 15, 1935
ANALYTICAL EDITION
intermediate product were some carbohydrate other than maltose and could be identified, means might be found to hasten its hydrolysis. The use of takadiastase is widespread among plant workers and thus deserving of attention. The preparations, however, contain many enzymes which attack other insoluble carbohydrates than starch. Thus after enzymatic hydrolysis, in addition to maltose and glucose, there are many other substances in solution. Some of these are readily removed by clearing agents. The maltose may then be hydrolyzed by acid to glucose, the wisdom of which step was pointed out before, or each sugar may be determined separately by many combinations of methods which have been proposed. Denny (7) has recently published detailed directions for converting starch to glucose quantitatively without the necessity of acid treatment. The author has used this method on a number of different types of plants and obtained lower results than when the takadiastase was supplemented with acid hydrolysis. This difference in results is probably not due to the incomplete hydrolysis of the starch by either of the methods. It is probably due to the high results obtained when acid hydrolysis takes place on substances not derived from starch. However, even the lower of these results is undoubtedly higher than the true starch content.
Chemical and Physical Methods There are other types of methods which have appeared for many years in isolated cases but which only recently have obtained recognition. These methods involve the isolation of the hydrolyzed starch by making use of some of its chemical and physical properties. Starch is dissolved by many reagents with a greater or less alteration in its physical properties, but apparently with little hydrolysis. Reagents which have been used for this purpose are various organic and inorganic acids, alkalies, salts, and glycerol. The solubility of starch in cold and relatively concentrated hydrochloric acid is the basis of a method by Rask (38). The starch is dispersed into a clear or slightly opalescent but filtrable solution, out of which it can be precipitated quantitatively by alcohol. According to Rask, “Starch seems to undergo no deep-seated changes in the process of acid dispersion, followed by alcoholic coagulation. It still gives the characteristic blue color with iodine and it has no reducing action on Fehling’s solution. The only noticeable changes in the starch are that the original morphology or identity of the grains has been destroyed and that the starch itself has been rendered water-soluble.’’ The starch which is precipitated by alcohol is collected on a filter and weighed. The method has received considerable attention in the analysis of whole wheat, flour, and bran, and has been adopted as a tentative method by the Association of Official Agricultural Chemists (1). Collaborative work in this association indicates that this method needs considerable study and perhaps modification. Conflicting statements appear as to the relative yields obtained by this method in comparison with various diastatic methods (19, 24, $9). Ling and Salt (32) believe that hydrolysis of starch occurs. Denny (8) presents data which indicate that the method tends to give low results by incomplete extraction of the starch from flour and also high results by inclusion of nonstarch substances in the alcoholic precipitate. He proposes that, after gelatinization with water, four extractions be made with acid to dissolve the starch and that the precipitate obtained later by alcohol be hydrolyzed with takadiastase. He also applied the procedure to leaf material, using both hydrochloric and sulfuric acids as the starch solvents. The author attempted to apply the Rask procedure to the analysis of starch in woody material containing large amounts
313
of hemicelluloses, although Rask made no claims as to the adaptability of the method to such material. The acid caused the evolution of such a large number of gas bubbles, presumably of carbon dioxide from the decomposition of uronic acid groups, that the making of the solution to volume with any degree of accuracy was impossible.
Precipitation of Starch Iodide The large number of methods which have appeared for starch analysis is alone evidence of the unsuitability of any one of them for more than limited use. The field of plant analysis probably presents the most difficult problems for in the leaves, stems, and seeds of plants occur a variety of forms of carbohydrates. Many plant workers customarily check their quantitative results for starch with the qualitative iodine test and hope for but do not always obtain some correlation. The iodine test is fairly specific for starch in that few other substances giving this test are present in plants. The complex formed by the absorption of iodine by dissolved starch may be salted out of solution. If this precipitate could be collected free from other carbohydrate material and estimated by some appropriate method, the results would of course be readily correlated with the iodine test. This type of method, first proposed by Kaiser (ab) received impetus from the work of von Fellenberg ( l a , 13, 1 4 , who dissolved starch with hot calcium chloride solution and precipitated it from this solution with iodine. A number of similar methods applied to cereal products have since appeared (2, 10, 33, 40, 46, and others). Denny (7, 9) applied the method to plants and determined the amount of the starch precipitated first by titration of the iodine and later by hydrolyzing the starch to glucose with takadiastase. The author has attempted to apply this method to the analysis of woody plants. There are two main steps in the isolation of the starch-namely, the extraction of the starch from the ground woody material and the precipitation of the starch from solution with iodine. There are two methods of obtaining a calcium chloride extract of the starch. One procedure is to extract the sample successively with small amounts of the salt solution until experience has shown that no more starch is removed. This probably ensures complete extraction but is tedious. Another procedure is to make one extraction with calcium chloride solution, to dilute the solution to a definite volume, and by removing the insoluble residue, obtain a clear extract. An aliquot of this can then be used for further work. It was found that this latter procedure would give results similar to the one of successive extractions only if the concentration of calcium chloride was sufficiently high. Long boiling was necessary in coarsely ground samples but not in those finely ground. The drastic conditions which are necessary to extract all the starch from woody plants may not be necessary in more succulent plants or in cereal products. The second step in the method is the precipitation of the starch from the solution with iodine. This had been done directly in the calcium chloride extract, the calcium chloride acting as the salting agent. However, in using an extract of woody plants, this precipitation was found to be unreliable, and the precipitate, if obtained, had a tendency to run through the filter. If, however, the starch is precipitated from solution with alcohol, in which calcium chloride is soluble, it may be redissolved in water and again precipitated by iodine in the presence of some other salt. Ammonium sulfate was found to be very favorable for this precipitation, giving starch iodide particles which settle rapidly and may be easily retained on a filter. This precipitate may then be hydrolyzed by acid to glucose. The extra step involved in precipitating the starch twice does not lengthen the method, as
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time is saved a t other points. The double precipitation removes sufficient impurities from the starch so that the usual preliminary extraction of the plant to remove sugars and the usual final treatment with clearing agents are unnecessary. This method gives lower results in all types of plants than other starch methods and is believed to give a more accurate value for the true starch present.
Summary The large variety of methods which exist for starch estimation bring out the fact that few of them are reliable even in limited cases, Neither acids nor diastatic enzymes are sufficiently specific for accurate results. The development of highly purified and specific enzymes may solve the problem. Methods based on other chemical and physical properties of starch, such as its solubility in salts and acids, and its insolubility in iodine and salt solutions, deserve closer study and no doubt many contributions will be made in the future along such lines.
Literature Cited Assoc. Official Agr. Chem., Official and Tentative Methods of Analysis, 1930. Chinoy, J. J., Edwards, F. W., and Nanji, D. R., Analyst, 59, 673 (1934). Coe, M. R., J. Assoc. OficiaZ Agr. Chem., 9, 147 (1926). Collins, I. D., Science, 66, 430 (1927). Davis, W. A., and Daish, A. J., J . Agr. Sci., 5, 437 (1913). Davis, W. A., and Daish, A. J., Ibid., 6, 152 (1914). Denny, F. E., Contrib. Boyce Thompson Inst., 6, 129 (1934). Denny, E'. E., Ibid., 6 , 381 (1934). Denny, P. E., J. Assoc. OficiulAgr. Chem., 6 , 175 (1922). Eckart, H., Chem. Zelle Gewebe, 12, 243 (1925). Englis, D. T., Pfeifer, G. T., and Gabby, J. L., J . Am. Chem. SOC.,53, 1883 (1931). Fellenberg, Th. von, Mitt. Lebensm. Hyg.,7, 369 (1916). Ibid., 8, 55 (1917).
VOL. 7, NO. 5
Ibid., 19, 51 (1928). Fraps, G. S., J. Assoc. Oficial Agr. Chem., 15, 304 (1932). Freudenberg. K., J. SOC.Chem. Ind., 50, 288T (1931). Hall, E. H.;Ibid., 50, 4291' (1931). Haworth, W. N., Ibid., 53, 1059 (1934). Herd, C. W., and Kent-Jones, D. W., Ibid., 50, 15T (1931). Hill, A. C., Proc. C h m . SOC.,17, 45 (1901). Ibid., 17, 184 (1901). Hirst, E. L., Plant, M. M. T., and Wilkinson, M. D., J . Chem. SOC.,1932, 2375. Horton. E.. J . Am. Sci.. 11. 240 (1921). Jones, L,, J. Assic. Oficiul Agr. Chem ,'Xi, 582 (1932). Kaiser, A., Chem.-Ztg., 26, 180 (1902). Klinkenberg, G. A. van, 2. physiol. Chem., 212, 173 (1932). Kuhn, R., Ann., 443, 1 (1925). Lehmann, O., Plantu, 13, 575 (1931). Ling, A. R., J . Inst. Brewing, 28, 838 (1922). Ling, A. R., Nanji, D. R., and Harper, W. J., Ibid., 30, 838 (1924). Ling, A. R., and Nanji, D. R., J . C h m . SOC.,123, 2666 (1923). Ling, A. R., and Salt, F. E.,J . Inst. Brewing, 37, 595 (1931). Long, W. S., Trans. Kansas Acad. Sci., 28, 172 (1916). Nishimura, S., Chem. ZeZZe Gewebe, 12,202 (1925). Nordh, G., and Ohlsson, E., 2. physiol. Chem., 204, 89 (1932). Noyes, W. A,, Crawford, G., Jumper, C. H., Flory, E. L., and Arnold, R. B., J. A m . C h m . Soc., 26, 266 (1904). Ohlsson, E.,2.phyeiol. Chm., 189, 17 (1930). Rask, 0. S., J . Assoc. Oficial Agr. Chem., 10, 108 (1927). Ibid., 10, 473 (1927). Small, J. C., J . Am. Chem. Soc., 41, 107 (1919). Taylor, T. C., and Iddles, H , A,, IND.ENQ.CHEM,, 18, 713 (1926). Taylor, T. C., and Walton, R. P., J. Am. Chem. Soc., 51, 3431 (1929). Thomas, W., Ibid., 46, 1670 (1924). Waldeschmidt-Leitz, E., Reichel, M., and Purr, A., Naturwissenschujten, 20, 254 (1932). Walton, G. P., and Coe, M. R., J . Agr. Research, 23, 995 (1923). Weiss, H., 2.ges. Brauw., 45, 122 (1922). Widdowson, E. M., Biochem. J., 25, 863 (1931). RIWBIVE~D May 23, 1935. Presented before the Division of Agricultural and Food Chemistry at the 89th Meeting of the American Chemical Society, New York, N. Y., April 22 to 26, 1936.
Determination of Alkalies in Feldspars A Modified Hydrofluoric Acid Method E. W, KOENIG, Consolidated Feldspar Corporation, Erwin, Tenn.
M
UCH time and effort have been expended upon investigations of the possibility of replacing the timehonored J. Lawrence Smith (8) method for the disintegration of silicates, preliminary to the determination of the alkali metals, sodium and potassium, by a more expeditious procedure. Because of the ease of initial decomposition of the silicate sample by means of hydrofluoric acid, possible modifications of the original Berzelius (1) method-in the direction of the elimination of the undesirable conversion of sulfate to chloride by means of barium chloride-have long intrigued the analytical chemist. Low (6) and later Krishnayya (4) modified some portions of the method but did not successfully eliminate the particularly objectionable sulfate-chloride conversion. Scholes (7) described an excellent modification which eliminated most of the undesirable features of the original method and yet took full advantage of the hydrofluoric acid disintegration. The method gave results of good accuracy and precision with a considerable reduction in the amount of time necessary for analysis. Unfortunately, the procedure required a prohibitive amount of direct supervision and has. therefore never become popular for routine determinations. The method devised by Knowles and Redmond (S), wherein
the alkali metals are isolated as mixed chlorides after removal of aluminum and calcium as quinolate and oxalate, respectively, is productire of excellent results. The only objectionable feature of the method is the fact that, in materials with as high a percentage of aluminum as feldspar, the removaI of this component with 8-hydroxyquinoline is extremely burdensome unless a relatively small sample-0.1 to 0.2 gram-is used. The use of a small eample does not appreciably affect the accuracy of the total chloride determination, if reasonable attention is paid to the details of the method. However, the ultimate separation of the sodium and the potassium, when working with small samples, is uncertain by most of the gravimetric methods now available, unless an unreasonable amount of time is expended in their recovery. This is true regardless of the method of separation-perchlorate, chloroplatinate, or triple acetate-because of the solubility factors affecting one or the other of the alkali salts, depending on the method chosen for the separation. After a thorough investigation of each of the foregoing methods, a further modification of the Berzelius method was evolved. Volatilization of silica as tetrafluoride is unnecessary if complete removal can be accomplished by other means. This eliminates the necessity for the introduction of sulfuric
