INDUSTRIAL AND ENGINEERING CHEMISTRY
502
VOL. 28. NO. 4
2 =a Let K K1
For other oxygen concentrations the appropriate value of the constant may be substituted.
Rearranging,
Literature Cited Clement, J. K., Adams, L. H., and Haskins, C. N., Univ. Ill. Expt. Sta., Bull. 30 (1909).
Kinney, 5. P.,Bur. Mines, Tech. Paper 454, 66 (1930). Kreisinger, H.,Augustine, C. E., and Harpster, W. C.,Ibid., 207 (1919).
Kreisinger, H.,Ovitz, F. K., and Augustine, C. E., Ibid.. 137 (1916).
Substituting a for K z / K , in Equation 13 and rearranging,
Mott, R. A., and Wheeler, R. V., “Coke for Blast Furnaces,” p. 130 and Table XLV, Colliery Guardian Co. Ltd., 1930. (6) Perrott, G. St. J., and Kinney, 9. P., Trans. Am. I n s t . Mining Met. Engrs., 49,543-84 (1923).
Sherman, R.A., Iron Age, 115,1043-5 (1925). Sherman, R. A., and Blizard, J., Trans. Am. I n s t .
iManing Met. Engrs., 49,526-42 (1923). Tu, C . M., Davis, H., and Hottel, H. C., IND. ENG.CHBM.,26, 749-57 (1934).
and preceding equations The ‘Onstant o‘20 in refers to the proportion of oxygen in the original gas stream.
RECEIVED September 28, 1935. Presented before the Division of Gas and Fuel Chemistry at the 90th Meeting of the American Chemical Society, Ban Frmoisco, Calif., .4ugust 19 to 23, 1936.
Granule Disintegration of Cornstarch T. C. TAYLOR AND JOHN C. KERESZTESY Columbia University, New York, N. Y.
HEN the viscosity of pastes made from air-dried cornstarch that had been ground for various periods of time in a ball mill under specified conditions (7‘) falls to substantially that of the soluble corn p-amylose of the same concentration, the ratio of the insoluble fatty-acid-bearing a-amylose to the &amylose as determined by an electrophoretic separation is approximately 15 to 85. With the precision for this type of measurement the ratio corresponds to the one obtained by Taylor and Iddles (9) in pastes where the granule disintegration was effected by the use of concentrated solutions of ammonium thiocyanate followed by separation in an electrophoretic cell or on an ultrafilter. For cornstarch the stopping point in the grinding of the dry starch occurs after 168 hours have elapsed at the particular ball load, size of mill, speed of revolution, etc., that had been selected. If we grind beyond that point, no great changes in viscosity of the subsequently made pastes occur as they do in the early part of the operation. It is necessary, therefore, to look to other variable and determinable factors in order to search for significant effects in this region; that exploration has been made and the results are reported here. To follow the changes, use was made of the alkali-labile determination ( l a ) , estimation of yield of a-amylose, and determination of combined fatty-acid content of a-amylose as a function of period of grinding. For reference, some samples were analyzed in the same way after treatment with cold acid according to the method of Lintner (4). Th’is was done because cold acid so affects the starch that the granules disintegrate more readily in water and give lumped dispersions which are similar in many respects to those from ground starch.
The a-amylose was recovered in each case after migration and washing the solid deposited on the positive membrane of the electrophoretic cell. The combined fatty acids were determined after acid hydrolysis of the residue, and the alkalilabile determinations were made on the samples by the recently revised method (8).
Alkali-Labile Value The latter apparently gives a sensitive measure of the change in available reducing groups during the treatment, for it is here that the attack by alkali on an amylose chain begins. It serves, therefore, as a guide to the progress of certain types of transformation that take place during soluble starch formation. The simple reducing value of samples without the alkaline treatment does not change sufficiently to be significant. Briefly, the method depends on the ready attack of hot aqueous alkali on certain fractions of the amylose or the starch in contrast to the slowness of attack on other parts
I
I n making soluble starch, certain changes take place that can be followed by determination of the ’alkali-labile” value. Dry-grinding cornstarch produces changes similar to those produced by the Lintner acid treatment when alkali-labile value is taken as a criterion. Corn alpha amylose loses combined fatty acids and amyloid material becomes soluble, but the insoluble residue has a higher fatty acid content than the material from which it came. A working hypothesis is discussed briefly in connection with interpretation of the results.
APRIL. 1936
1NDUSTRIAL AND ENGINEERING CHEMISTRY.
after the amyloid material has been subjected to a given treatment. That portion which is attacked by the alkali is converted into material which consumes iodine, the latter being conveniently estimated volumetrically. The amount of iodine used is expressed in mg. per 100 mg. of sample and is called the “alkali-labile” value. The alkali-labile value as a function of period of grinding of cornstarch is given in the drawing. It is interesting to note that the alkali-labile value rises rapidly a t first, then changes very slowly, and reaches an approximately steady state as the grinding is continued. Even after the 1848-hour period the material is still decidedly starchy and its color with iodine test solution is a bluish red. The greatest change in the alkali-labile value comes always during the initial break up in the ball mill of the organized granules. Similar changes take place when cornstarch is treated with cold hydrochloric acid. In this case, however, many granules still retain their general form but the amyloses are so modified that during subsequent paste formation disintegration takes place without marked swelling, and the viscosity of the gelatinized material is low. To compare the two methods for making soluble starch, cornstarch was treated by the Gore modification (2) of the Lintner method, but the starch was left in contact with the acid for varying periods of time, was then well washed with water and dilute ammonia to neutralize the acid, and sucked dry on a suction filter. Under these conditions the alkalilabile value of a dry sample after 5 hours’ contact with the acid is that of 168-hour ground starch, after 28 hours, that of 672-hour ground starch, and after 120 hours of acid pretreatment beyond that, of 1848-hour ground starch.
503
Hours of Grhdinq
FIGURE1.
EFFECTOF GRINDINGON ALKALI-LABILE VALUE OF CORNSTARCH
As an independent check on the ratio of a- to @-amylose, some of the ground starch was gelatinized in cold aqueous alkali and the a-amylose was precipitated by careful neutralization according to the method of Taylor and Morris ( 1 1 ) . Retrograded @-amylosewhich is insoluble in water would in the electrophoretic method find its way into the a-amylose fraction, whereas in the new method this does not happen. The values from the two methods allow us to estimate the extent of the contamination in this direction (Table I, columns 2 and 3). Alpha-amylose fractions are contaminated with insoluble silica from abrasion of balls in the ball mill. The values given, however, are corrected to an ash-free basis. The column headed “By precipitation” gives values taken by the Taylor and Morris method (11). There is a progressive loss of insoluble material as the starch is ground for an increasing period of time. A comparison of the ratio of a- to @-amylosein the acid-treated soluble starch shows that, after 5 hours under the conditions described, TABLEI. ALPHA-AND BETA-AMYLOSE FRACTIONS RECOVERED the value is the same as that in the pastes from the 168-hour FROM CORNSTARCH ground material. Not only is soluble material being formed Period -a-Amylose Fraction5-Am,ylose through disorganization of the granule and the liberation of of BY BY Fraction by Grinding electrophoresis precipltation Electrophoresis 0-amylose but some soluble material is coming also from the Hours % % a-amylose, for the latter slowly disappears. 168 336
18.6 14.6
15 6
86 0
The shape of the curve showing the relation between the alkali-labile value and time of acid pretreatment to form soluble starch is the same as that for the dry grinding. There is, however, one outstanding difference and that is the loss of highly soluble material in the wash water, particularly in the case of the longer acid treatment. While negligible after the 5-hour treatment, it becomes 14 per cent of the weight of the starch after the 28-hour treatment, and 38 per cent after the 120-hour treatment. Neither cornstarch nor any other starch investigated in this laboratory has ever had a zero alkali-labile value. However, samples of raw starches other than commercial cornstarch have been found to have somewhat lower alkali-labile values. The initial alkali-labile values reflect in part, probably, the effect of agents which have a solubilizing effect during the manufacture of the starch but more especially natural properties inherent in that particular starch.
Yield of a- and P-Amylose Turning now to the yield of a- and /3-amylose after electrophoretic separation of these materials from pastes made of the various ground samples, some interesting data are found. These are recorded in Table I.
Combined Fatty Acid Content Analysis for combined fatty acids, as a distinguishing characteristic of the insoluble fraction, shows that fatty acids are liberated as soluble amyloid material is formed. The results are given in Table 11. TABLE11. EFFECT OF GRINDING ON COMBINED FATTYACID CONTENT OF WAMYLOSE FRACTIONS Period of Grinding Hours
0 168 336 672 1344
a
Fraction
% 18:6
14.5 8.1 6.2
Combined Fatty Acid in a-Fraction Per cent 0.64 (orig. starch) 3.3 4.0 6.3 7.3
Calcd. Loss of Total Combined Fatty Acid Per cent
5:s
8.8 a
14.4 29.2 ~~
It is apparent from these data that although both soluble amyloid material and fatty acid residues are released from this insoluble fraction during grinding, there is more carbohydrate lost than there are fatty acids liberated. This is shown by the fact that the small insoluble residues contain a higher percentage of still combined fatty acids. Again this is in accord with the findings of Taylor and Sherman (13) who used amyloclastic enzyme and other hydrolytic agents to reduce the a-amylose fraction and indicates that the effects are not unique in the case of dry grinding.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Tentatively Taylor and Sherman (IS) suggested that some of the three different fatty acid groups (IO) attached to the a-amylose were glucosidically held and liberated a t a greater rate than those in other positions. The alkali-labile values of each amylose fraction were higher than the ground starch from which it was separated, while the /?-amylose fraction was slightly lower in the corresponding separation. If the alkali-labile value measures available aldehyde groups, these results are to be expected. Certainly as the corn a-amylose is disintegrated, it loses not only carbohydrate but some fatty acid radicals. In this respect it differs from insoluble and still-organized /?-amylose and also retrograded amylose both of which are likewise insoluble. In an attempt to study the possible effect of the pretreatment on the properties of /?-amylose, we have no fatty acid factor to help serve as a guide to changes. Yet the @-amylose material may be heterogeneous especially when isolated from samples that have been ground for a longer time or given extensive treatment with the cold dilute acid as in making soluble starch. The alkali-labile value does serve, however, to give useful information, for, as the amylose aggregate is simplified the number of easily available and readily attackable aldehyde groups increases rapidly, and, if fractional precipitation with methanol precedes the determination, it is possible to tell by the magnitude of the alkali-labile value of the fractions approximately what has been accomplished. Further, as the solubility in water of a starch or amylose increases, so does the alkali-labile value, and the more soluble the amyloid material, the more precipitant is required to bring it out of solution. Likewise, as the amylose is simplified, the color with iodine test solution, as is well known, changes from blue toward red. Rough fractionation’ was found to take place, indeed, when clear @-amylosesolutions taken from the electrophoretic cells were treated with methanol, if the starch from which the /?-amylose was separated had previously been ground for a long period of time or had remained for an extended time in contact with the dilute acid. Under the conditions described in the section on “Experimental Procedure,” noticeable fractionation was evident in the @-amyloseafter grinding the starch from which it came a t least 672 hours. The first fraction from this and another sample which had been ground 1344 hours was approximately 95 per cent of the total solid @-amylosepresent in the solution. Both gave a characteristic blue color with iodine test solution and had an alkali-labile value of 44.8 for the former and 46.6 for the latter sample. The second fraction obtained after concentration by evaporation of the filtrate i n vacuo and addition of large excess of methanol in each case was, respectively, 5.0 and 2 per cent; the former still gave a blue color and had an alkali-labile value of 47.1, the latter gave a red color with iodine and an alkali-labile value of 54.2. After contact with acid for 5 days another sample of cornstarch after washing gave a /?-amylose which could be fractionated to yield 80 per cent of blue coloring material with an alkali-labile v a h e of 49.9 and 17 per cent of a red coloring material with an alkali-labile value of 61.1. It is not pretended that a sharp fractionation has been effected; the r e s u l t s a r e g i v e n merelv to show that in certain solubie starches the &amylose is definitely heterogeneous. C r i t i c i s m of t h e method of evaporating and concentrating p1 Molecular weight and particle size determination on these fractions are being attempted at this time, but until more aignificant results are obtained it is impossible to use them
VOL. 28, NO. 4
amylose solutions may be raised on the ground that hydrolytic scission may take place here to a larger extent than during the solubilizing action on the whole granule. That criticism is not justified, for experiments show that during the evaporation of neutral dispersed amylose solutions there is no change in the alkali-labile value. Indeed this behavior illustrates the difference between organized starch granules and the amyloses after disorganization. Changes in viscosity and alkali-labile value take place rapidly during the disorganizing process but much less rapidly after the granule organization has been broken down either directly by mechanical grinding or potentially by treatment with cold dilute mineral acid. The principal criterion is the appearance of vulnerable aldehyde groups toward the attack of hot aqueous alkali.
Discussion of a Working Hypothesis If a working hypothesis is set up on the basis of current chemical concepts, it must explain these changes. There seem to be two definite ways in which available aldehyde groups may be produced from organized starch granules. While more evidence substantiating this will be published shortly, it may suffice here to give a brief outline of the interpretation that fits the already reported data. The simple /?-amylose would be envisaged as a chain of n glucopyranose rings linked through the usual glucosidic oxygen bridges ( 3 ) . This would place a free aldehyde group at the terminus of every chain. Fixed rigidly in the usual stereo-configuration indicated by optical properties are hydrogen atoms and hydroxyl groups attached to the carbon skeleton. Each oxygen atom is a donor (6) and each hydroxyl hydrogen atom is an acceptor by which a coordinate link can be formed. By virtue of the multiplicity of these links many amylose chains are held together parallel-wise ( 5 ) . By an elaboration of this pattern and a possible dovetaillike fitting end to end of one bundle with another, it is possible to see how a granule could be organized and how aldehyde groups, although primarily chemically free, might be occluded and shielded. Reducing value as reflected in the alkali-labile value could increase after a given treatment from two causes. One would be disassociation of the coordinately linked chains from one another to uncover vulnerable existing terminal aldehyde groups and the other would be hydrolytic scission of glucosidic linkings giving short chains and consequently new aldehyde groups. Since chain shortening through hydrolysis causes a break in the continuity of the skeleton on which the coordinately linked groups function, it is probable that disassociation takes place more readily in fragments than in the original longer chains. Incipient hydrolysis ought to cause a very rapid production of reducing groups from the two directions already mentioned. An increase in reducing properties within certain limits may come, however, entirely through disassociation of one chain from another during some treatment and and not by hydrolytic scission. It is probable that the great increase in alkali-labile value in making soluble starch by dry grinding or by the Lintner acid process a t the outset comes from vulnerable terminal aldehyde groups which have become exposed through disassociation of the chains to the ready attack of the hot aqueous alkali. Later, however, in the disassociative chains the slower hydrolytic breakdown of glucosidic linkings becomes the principal producer of smaller molecular weight reducing sugars, This is probably what causes the relations that give the shape to Figure 1 and the heterogeneity shown in the &amylose samples from starches that had a long pretreatment.
APRIL, 1936
INDUSTRIAL AND ENGINEERING CHEMISTRY
Experimental Procedure
505
Benzylation
GRINDING AND ELECTROPHORESIS. Batches of air-dried commercial cornstarch were ground in a 4-gallon ball mill using 600 grams of starch to 8 kg. of pebbles and rotated at 45 to 55 r. p. m. for different lengths of time from 168 to 1848 hours. A portion of each batch was dispersed in water as follows: 150 grams of the ground sample were suspended in about 300 cc. of methanol to avoid lump formation, and the mixture was cautiously poured into 4 liters of boiling distilled water with vigorous stirring. This limpid dispersion was cooled then to room temperature and put in an electrophoretic cell, toluene was added, and 220 volts were applied (7,9 ) . When the supernatant liquor was clear and the sediment was tightly packed on the lower membrane, the liquid was siphoned off and the solids in it were determined by drying an aliquot at 100" to 105" C. The sediment was removed and L. McMASTER AND W. M. BRUNER again poured into 4 liters of boiling distilled water. When cool the process was repeated as before. A third dispersion into Washington University, St. Louis,Mo. boiling water was made, followed by electrophoresis. After the third portion of wash water was removed, the a-amylose was put into a tared dish and dried in the oven at 70" C. and weighed. DETERMINATION OF AMYLOSE FREEFROM RETROGRADED HE production of modern disinfectants (6). A suspension of the ground starch in 50 cc. of MATERIAL callsfor chemical compounds of high phenol methanol was poured cautiously with stirring into 300 cc. of boiling water. The boiling was continued for a few minutes to insure coefficients. It is therefore desirable to the breaking up of any lumps. The dispersion was then cooled prepare a t a reasonable cost such compounds as 0- and to room temperature and enough 12 per cent caustic solution p-benzylphenols which, when added to disinfectants, will was added to bring the alkali concentration to 2.5 per cent. increase their phenol coefficients. An improved method of The solution was cooled and concentrated hydrochloric acid was added in excess with constant stirring and cooling. The a-amypreparing these benzylphenols from benzyl chloride and an lose readily flocked out, and, after standing a short time, the excess of phenol has been developed. No use is made of precipitate was centrifuged off and washed repeatedly until the catalysts, organic solvents,l or reagents to separate the isowashings were neutral to methyl orange. The material was mers. There is also obtained a minimum yield of dibenzylthen dried in an oven at 70" C. and weighed. FRACTIONATION OF ,&AMYLOSE. The p-amylose fractions were phenols. The method is to melt the phenol, heat it to the obtained as the supernatant liquid from the electrophoretic desired temperature of benzylation, and stir vigorously with cells and were concentrated i n vacuo until they contained 5 to a mechanical stirrer while benzyl chloride is dropped slowly 6 per cent solids, and two volumes of methanol were added. into the reaction mixture. This procedure helps to minimize This mixture was allowed to stand 2 to 3 days, after which the supernatant liquid was removed and the precipitate was ground the formation of dibenzylphenols by preventing a local excess in methanol to a fine powder and then dried i n vacuo at 50" C. of benzyl chloride. Also, the principle of mass action is further The liquor which contained material not recipitated by the used by starting with an excess of phenol. methanol was concentrated i n vacuo at 40" to one-fifteenth of In the above reaction benzyl phenyl ether is first formed, its volume, and three volumes of methanol were added. The precipitate was allowed to settle and after several days was which, according to the well-known Claissen reaction, is removed and ground with methanol and dried i n vacuo at 50" C. rearranged to the benzylphenols under the conditions as stated. Ethyl alcohol as a precipitant will interfere with the alcoholNo benzyl phenyl ether could be identified in the undesired labile determination. A trace of electrolyte is necessary for by-products which are essentially the dibenzylphenols and a precipitation. ANALYSISOF CY-AMYLOSE FOR COMBINED FATTY ACIDS. The slight amount of tar. a-fractions were freed from extraneous fatty acids by triturating Benzylation was investigated at 125" C. using mole ratios under ethyl ether and dried in an oven at 60" to 70" C., and the (moles of phenol per mole of benzyl chloride) of 2 to 1, 4 to 1, amount of combined fatty acid was determined by the Rohrig etc., up to 10 to 1. Keeping the mole ratio constant at tube extraction method ( 1 ) after acid hydrolysis. This method was found to be more useful than the Taylor and Nelson technic 10 to 1, benzylation was tried a t 150' and 175" C. After for the small samples available here. Duplicate determinations benzylation was complete, the mixture was subjected to fracchecked within 1 per cent. tional distillation under diminished pressure and the benzylated products were thus separated without the use of solvents or reagents. This general method was followed in all cases. Literature Cited The yields of the monobenzylphenols when the tempera(1) Assoc. Official Agr. Chem., Methods of Analysis, p. 166 (1930). ture was kept a t 125" C. varied within wide limits. At the (2) Gore, IND. ENG.CREM.,20, 865 (1928). 10 to 1 mole ratio 89.5 per cent of the theoretical yield, based (3) Haworth, "Constitution of Sugars," London, Edward Arnold & Co., 1929; Hirst, Plant, and Wilkinson, J. Chem. SOC., 1932, on the weight of benzyl chloride used, was obtained, and 50 2375. per cent a t the 2 to 1 mole ratio. The yields of monobenzyl(4) Lintner, J.prakt. Chem. [N. S.],34,378 (1886). phenols when the mole ratio was kept a t 10 to 1 varied only (5) Meyer and Mark, Ber., 61,593 (1928); "Aufbau der hochpolyslightly with the temperature, the maximum yield being meren organ. Naturstoff," Leipzig, 1930. (6) Sidgwick, "Electron Theory of Valency," pp. 73, 134, London, obtained when the temperature of benzylation was 150' C. Oxford University Press, 1929; "Some Physical Properties of In all cases the yields of o-benzylphenol were greater than the Covalent Link in Chemistry," p. 27, Cornel1 University those of p-benzylphenol. Likewise, the quantity of residue in Press, 1933. the dibenzylphenol fraction was very small. (7) Taylor and Beckmann, J. Am. Chem. SOC.,51, 294 (1929). (8) Taylor, Fletcher, and Adams, IXD.ENG.CHEM.,Anal. Ed., 7, The excess phenol, the o-benzylphenol, the p-benzylphenol, 321 (1935). and the dibenzylphenols could be efficiently separated by (9) Taylor and Iddles, IND. EKG.CHEM.,18,713 (1926). fractionation under diminished pressure, using a 40-inch (101.6(10) Taylor and Lehrman, J. Am. Chem. SOC.,48, 1739 (1926). cm.) heated Vigreux column and a reflux ratio of about 5 (11) Taylor and Morris, I h i d . , 57, 1070 (1935). (12) Taylor and Salzmann, I b i d . , 55, 264 (1933). or 6 to 1. The o-benzylphenol (recorded melting point, (13) Taylor and Sherman, I h i d . , 55, 258 (1933). 21' C. for this labile form) and the p-benzylphenol (recorded melting point 84" C.) were obtained nearly colorless and of
of
PHENOL
6
RECEIVED September 27, 1935. Part of the material for this report is taken from a dissertation submitted by J. C. Keresztesy t a the Pure Science Faculty, Columbia University, in partial fulfillment of the requirement f o r the degree of doctor of philosophy.
1 Kalle and Company [German Patent 346,384 (Deo. 31, 1921)l and Courtot [Compl. rend., 187, 661 (1928)] mention the benzylation of phenol without the presence of a solvent. No details are given.
