Determination of the Carboxyl Content of Oxidized Starches hZ. F. MATTISSON’
AND
K. A. LEGENDRE, Clinton Foods Znc., Clinton, Zowa
The determination of the carboxyl content of commercial oxidized starches has long been used to estimate the degree of oxidation. Existing methods are based on similar procedures for cellulose. The usual method requires the starch to be leached with hydrochloric acid to remove any existing cations, followed by exhaustive washing to remove the hydrochloric acid. The starch is then treated with calcium acetate solution, which unites calcium to carboxyl, freeing acetic acid which is determined by titration. A new method is proposed wherein the starch is pasted after the acid leaching and washing and the liberated carboxyl groups are titrated directly. This procedure eliminates the involved calcium acetate treatment of the older method, and is simple, shorter, and more precise.
C
ORIlIERCML oxidized cornstarches are widely used in
papermaking, where their specific properties are used t o close the pores of the paper t o lay fuzz on the surface, to increase tensile fold and bursting strength, and to give a “feel” and rattle to the paper. The degree of oxidation is relatively small when compared to the periodate, chromic anhydride, and nitrogen dioxide starches frequently described in scientific research ( 3 ) . Oxidized starches resemble the parent starch in that they retain essentially the original granule structure, preserve the typical polarization crosses, and show the typical blue staining with iodine. However, oxidized starches differ from the parent starch in that they require a shorter cooking time t o produce a paste, and give a higher fluidity, incieased adhesiveness, lower rate of congealing, and greater translucency of the pastes. Commercial oxidized starches are usually produced by the treatment of an 18’ BB. starch slurry with a solution of sodium hypochlorite (7.5 to 9.0% chlorine). The degree of oxidation is, in general, controlled by fluidity determinations. After the desired degree of oxidation has been reached, the residual hypochlorite is eliminated by the addition of sodium bisulfite. The slurry is then acidified to a p H of 6.5, washed, filtered, and dried. The resulting products are less colored than the parent starch or the acid-modified starches. Apparently, the free caustic of the sodium hypochlorite has neutralized most carboxyl groups formed during the oxidizing process; however, the subsequent acidification t o pH 6.5 produces a small variable amount of acidity in the dry starch, and the washing introduces calcium cations, thereby transforming some sodium salts of carboxyl groups into the calcium salts. The acidity of the finished oxidized starches may result from one or more of the following causes: 1. Residual mineral acid or sulfur dioxide retained in the starch granules. 2. Lactic or amino acids retained by the starch from thc steeping process. 3. Fatty acids contained in the extraneous fat adsorbed on the starch. 4. Carboxyl groups attached to anhydroglucose units of the starch molecule and freed from their cation content by the acidification of the starch slurry to p H 6.5.
The deteimination of this acidity is essential. Since most of the carboxyl groups are not liberated from their salts by the acidification of the slurry t o pH 6.5, all method. pro-
’ Present address, Commercial Solvents Corn , Terre Haute, Ind.
posed for the determination of the carboxyl content of oxidized starch require a preliminary de-ashing treatment,-Le., the starch is treated with diluted hydrochloric acid with subsequent removal of the excess hydrochloric acid with distilled water. The copious washing required removes not only cations, but also some of the sulfur dioxide, some of the organic acids and, undoubtedly, some of the water-soluble starch fractions of low degree of polymerization and therewith some carboxyl groups, for the solubility of oxidized starches increases with increasing degree of oxidation. The acidimetric value of the cation-freed starch as determined by various procedures has been used as a measure of the carboyyl content. This value should be corrected for the acidity of the original starch which has not been subjected t o the de-ashing pretreatment but only washed with distilled water to remove the water-soluble acidic substances. This coriection is deemed advisable, especially in the case of the lower oxidized starches, for here the correction is of considerable magnitude and may influence the characterization of these starches. These considerations have prompted an investigation of possible simplification and improvement of existing methods for carboxyl analysis, because a rapid and precise method for the determination of the carboxyl content of oxidized starches would aid starch chemists in identification and manufacturing control of these products. SELECTION OF METHODS
The extensive literature on the determination of the carboxyl content of polysaccharides pertains almost exclusive’y to the carboxyl content of celluloses and oxidized celluloses. Of the various methods proposed, the calcium acetate method appeared to be the best for oxidized starches because it seemed the simplest and least time-consuming procedure. Ludtke ( 4 ) was the originator of this method of cation exchange of carboxylic acid groups with calcium acetate and titration of the liberated acetic acid. He used 0.5% hydrochloric acid and carbon dioxide-free distilled water in the de-ashing pretreatment and 0.01 A: sodium hydroxide for titrating a t room temperature to the phenolphthalein end point. Yackel and Kenyon (9) proposed as standard conditions for the method: 0.5 S calcium acetate, 2 hours’ treatment time a t 25 “C., and titration with 0.1 -V sodium hydroxide. Other investigators found that concentrated solutions of calcium acetate had a strong buffering effect on the liberated acetic acid, resulting in unsatisfactory end points. For this reason, Heymann and Rabinov (8) critically reviewed previous research and investigated the normality of the calcium acetate and other salts for the liberation of the carboxylic acid. They found that 1.0 AV calcium acetate solutions gave most unsatisfactory liberations and that 0.1 N calcium acetate was preferable t o 0.5 -V calcium acetate, as the liberation end point was sharper. Meesook and Purves ( 5 ) found that carboxvl values tend to be too low when the equilibrium pH of mixtures of oxidized polysaccharides and calcium acetate solutions falls below 6.3. Davidson and Neve11 ( 1 ) specified an equilibrium pH of 6.5 to 6.7, the use of freshly prepared 0.1 S calcium acetate, slow agitation during a reaction time of 17 hours, and a mixed indicator to finish titration with 0.01 N sodium hydroxide at pH 8.4 t o 8.6. They recommended the use of ammonia-free distilled viater for the preparation of the solutions and for mashing purposes. Kenyon and coworkers ( 7 ) proposed the use of electrometric titrations for determining the acetic acid liberated in the Yackel and Kenyon modification of the calcium acetate method.
1942
V O L U M E 2 4 , NO. 1 2 , D E C E M B E R 1 9 5 2 Finally, Wilson (8)investigated several methods and concluded that in the base exchange by the calcium ion for the hydrogen ion, the pH range must be within the limits of 7.0 to 8.6, for above pH 8.6 reactions other than acid-base equilibriums become significant. She showed that 0.1 .V calcium acetate met the pH range specified, but that the slight slope of the neutralization curve resulted in a lack of precision. Various suggestions advanced by the above authors have been investigated and incorporated into a new modification of the calcium acetate method, which is discussed below and is compared to another procedure which the authors developed and call the “paste titration method.” It is based on a procedure which antedates the calcium acetate method and now serves frequently a. a routine method for the analysis of starch samples or for manufacturing control-Le., the starch sample is pasted and the pListeis titrated hot with standard sodium hydroyide employing phenolphthalein as the indicator. EXPERIMENTAL
Calcium acetate of 0.1 normality was chosen instead of 0.5 S calcium acetate because experiments confirmed the opinion of Ifeymann and Rabinov ( 2 ) and Wilson (8)that the calcium acetate-acetic acid system exerts less buffering capacity at the lower normality (see Figure 1, in which B refers to a sample of a low oxidized starch). All data and calculations are based on dry substance starch.
1943 water which has been used in the above procedure and are then submitted to the calcium acetate treatment. The acidity is determined as described above, B. The corrected acidity is
( A )- ( B ) .
CALCULATIONS.( A - B , A; x 100 = milliequivalents of weinht acidity/100 grams of staGh. Milliequivalents of acidity/100 grams of starch X 0.045 = apparent % ’ carboxyl. Paste Titration Method : ACIDITY OF DE-ASHEDSTARCH. Samples, 5.000 or 0.1600 gram, the latter for the more highly oxidized starches known as gums, are transferred to a 150-ml. beaker, 25 ml. of 0.1 N hydrochloric acid are added, and the mixture is allowed to remain for 0.5 hour with occasional stirring. The slurry is filtered through a fritted-glass crucible (medium porosity) and washed with distilled water until the wash water is free from chlorides. The de-ashed starch is transferred to a 600-ml. beaker, slurried in 300 ml. of distilled water, and heated to boiling over a gas burner or in a steam cooker. Sufficient time (about 5 to 7 minutes) should be allowed to ensure thorough gelatinization. The pasted starch is titrated hot with 0.1 AT sodium hydroxide to the phenolphthalein end point, C. BLAKK, ACIDITYOF ORIGINAL STARCH.The weighed samples are transferred to a 600-ml. beaker, pasted, and titrated hot as described above, B. The de-ashing pretreatment is omitted as well as the washing with distilled water, since comparative tests with aashed samples did not show any significant difference. The acidity is determined as described above, D. The corrected acidity is (C) - (D). CALCCLATIOSS. (‘ - D, x AV x 100 = milliequivalents of weight acidity/100 grams of starch. Milliequivalents of acidity/100 grams of starch X 0.045 = apparent % carboxyl. ~
STARCH SAMPLES
A series of seven commercial oxidized starches was obtained, A to G, inclusive, representing increasing degrees of hypochlorite oxidation. A sample of common pearl starch was included for the first member of this series. The moisture was obtained, and all data are calculated to dry basis starch.
9 50
9.00 8.30 8.00 PH
750 Table I.
700 6.50
1
Modified Calcium Acetate %lethod
(Acidity as milliequivalents per 100 grams of starch) d B A - B Starch De-ashed Water-Washed Corrected
6.00 0
50
1.00 1.50 2.00 ml. ,025-N NaOH
2.50
Figure 1. Titration of Calcium Acetate 1. 0.1 N calcium acetate 2. 0.5 Ncalcium acetate 3. 0.1 N calcium acetate plus 1 gram of starch B 4. 0.5 N calcium acetate plus 1 gram of starch B
Table 11. Paste Titration 3Iethod
Modified Calcium Acetate Method : ACIDITYOF DE-ASHED STARCH. Water free of carbon dioxide and ammonia is used throughout for the preparation of solutions and de-ashing. Samples, 5.000 to 0.500 gram, of starch are used, the amount decreasing with the degree of oxidation, and are so chosen that the equilibrium pH of the calcium acetate solution a t the end of this treatment falls within the range of 6.5 to 6.7. The sample is transferred to a 150-ml. beaker, slurried with 25 ml. of 0.1 N hydrochloric acid, and occasionally stirred. After a reaction time of 30 minutes, the slurry is filtered through a fritted crucible (medium poropity) and the starch is washed with ammoniafree distilled water until free from chlorides. The starch is transferred to a 100-ml. flask, 10 ml. of a 1 S calcium acetate solution or its equivalent are added, and the solution is made to volume with ammonia-free water, so that the final concentration of the calcium acetate is 0.1 S. The flasks are shaken occasionally during a reaction period of 30 minutes, the slurries are filtered into a dry suction flask, and a 50-ml. aliquot is titrated potentiometrically to pH 8.3 with 0.25 to 0.01 N sodium hydroxide. rl blank titration is made on the calcium acetate solution used. The net de-ashed acidity is obtained by subtraction, A . BLANK,ACIDITYOF STARCHAFTER WASHINGWITH WATER. The procedure is the same as described above, but for the omission of the de-ashing pretreatment with 0.1 N hydrochloric acid. The samples are washed with the same volume of ammonia-free
(Acidity as milliequivalents per 100 grams of starch) C - D C 4 Starch De-ashed Oriqinal Corrected Pearl 2 3 2 5 n
Methanol extraction was omitted beeawe the results of tests made on samples B, D, and F, which were extracted with 85% methanol for 1 hour either once or five times according t o Schoch ( 6 ) , were not significant (Table IV). EXPERIMENTS
The acidity determinations obtained by the modified calcium acetate method are shown in Table I, the corresponding data for the paste titration method in Table 11. Duplicate determinations were run in each test. The deviations from the mean of
1944
ANALYTICAL CHEMISTRY
the corrected acidities were calculated from the deviations from the mean of the uncorrected and the original acidities. Table I11 contains the results of the corrected acidity calculated as per cent carboxyl and moles of carboxyl per 100 anhydroglucose units (A.G.U.).
Table 111.
% COOH
CONCLUSIONS
The data given in Tables I to IV indicate that the difference between the two methods is small and in most cases well mithin the limits of experimental error. The precision of the paste titration method is better because the buffering capacity of the pasted starch system is greater than that of the calcium acetateacetic acid system. I t is more rapid and is simpler than the corresponding calcium acetate method, and may even enable less experienced analysts to obtain reproducible data. The paste titration method is, therefore, recommended for routine control and analysis. Both methods show that starches which were oxidized to a varying degree had essentially the same original acidity, which was approximately the same as the original acidity of the unoxidized starch. As the unoxidized starch contains only a small amount of carboxylic groups attached to anhydroglucose unitsif any at all-it seems improbable that such carboxyl groups were the main cause for the original acidity of the oxidized starches. This acidity might be rather attributed t o the presence of “acidic impurities,” such as, for instance, fatty acids. Therefore, deducting the original acidity from the acidity of the de-ashed samples is recommended. This correction makes it also unnecessary to remove free fatty acids by solvent extraction before determining the acidity. The results of Table IV show that one methanol extraction which removes the greater part of the free fatty acids causes only a comparatively small decrease of the corrected acidity. Base exchange methods of analysis or the paste titration method measure the corrected or uncorrected acidity of only that portion of a sample which is insoluble under the conditions of the deashing pretreatment. Therefore, the exact content of carboxylic groups attached to anhydroglucose units in the original starch samples has not been determined. Except in the case of very highly oxidized starches, the “apparent carbovyl content” of
Corrected Acidity Expressed a s Apparent Carboxyl
Oxidized Starch
Calcium acetate method
A B
0.16
0.077
C
0.26 0.39 0.48 0.58
D E
F G
0.77
Table IV. Oxidized Starch
a
Paste titration method 0.081 0.16 0.30 0.42 0.53 0.60 0.83
Moles of COOH/ 100 A.G.U. 0 . 2 t o 0.3 0 . 95 t o 01..61
1.3 t o 1 . 6 1 . 6 t o 1.9 2.0 to 2 . 2 2.7 t o 3.0
Per Cent Decrease on lMethanol Extraction
No. of F a t by Extractions Hydrolysis
De-Ashed .4ciditya
Ori inal Aciiitya
Corrected Aciditya
Paste titration method for acidity.
de-ashed oxidized starches based on the corrected acidity is believed to be a better approximation to the “real carboxyl content” than calculations based on the uncorrected acidity. LITERATURE CITED
(1) Davidson, G F., and Nevell, T. P., Shirley Inst. Afem., 21, 85100 1947) (2) Heymann E., and Rabinov, G . , J . Phys. Chenz., 45, 1152-66 (1941). (3) Kerr R. IT., “Chemistry & Industry of Starch,” 2nd ed., Chap. 111. XI. XXIV, New York, Academic Press, 1950. (4) Ludtke, hl., Anuew. Chem., 48, 650-1 (1935). (5) hleesook, € 3 . and Purves, C. B., Paper Trade J., 123, KO.18, 3 5 4 2 (1946); Terh. Assoc. Papers Ser., 29, 508-15 (1946) (6) Schoch. T. H., J . Am. Chem. Soc., 64, 2954-6 (1942). (7) Unruh, C. C., McGee. P . -4., Fowler, W. F., Jr., and Kenyon, W 0 , J. Chem. SOC..69, 347-9 (1947). (8) Kilson. B., Saensk Paperstidn., 51, 45-9 (1948). (9) Yackel E C , and Kenyon, IT, O., J . Am. Chem. Soc., 64, 121-7 (1942).
RECEIVED for review June 18. 1952. Accepted September 8, 1952
Composition of a Typical Grape Brandy Fusel Oli A. DINSMOOR WEBB, RICHARD E. KEPNER, AND ROBERT M. IKEDA Department of Viticulture and Department of Chemistry, Uniaersity of California, Davis, C a l g .
A
LOSG-term program is under xvay in these laboratories, the primary objective of which is the isolation and identification of the substances contributing to the aromas and flavors of fresh grapes and grape fermentation products. The first phase of this project has been concerned with the investigation of the composition of a typical grape brandy fusel oil obtained from a large California distillery. Early work in the field has not resulted in a detailed knowledge of the composition of grape fusel oils (2, 6, 8, 10, 15) and in the more recent reports there have been errors in designating the isomers of amyl alcohol which are actually present. While the identity of the grape5 used in the production of the fusel oil investigated was not recorded, it is very probable that they were all varieties of Vitis vinzfera L., as this genus predominates in California. The composition of this sample of fusel oil is reported on a xater- and ethyl alcohol-free basis, as the amount of these substances in a fusel oil depends upon the extent ot the washing process used after the fusel oil has been removed from the rectifying column. Dehydration of the fusel oil sample prior to fractiona-
tion is necessary to avoid obtaining complex water-containing azeotropes. It was found, by titrations with the Karl Fischer reagent, that repeated dryings over magnesium sulfate, freshly dehydrated a t 400” for 3 hours immediately before use, left about 0.2% water in the fusel oil. Complete dehydration was accomplished by adding a calculated excess of anhydrous ethyl alcohol and removing the water as the ethyl alcohol-water azeotrope a t the beginning of the fractional distillation of the sample. The dried fusel oil was found to contain about 0.02 meq. of acid and 0.15 meq. of ester per gram. The free acids were removed from a separate sample of the fusel oil by use of ion exchange resins. The acids present were identified by chromatographing the mixed p-phenylphenacyl derivatives according to the procedure of Kirchner, Prater, and Haagen-Smit (7). The principal free arids were found to be acetic and butyric, although there were traces of several other acids present in amounts too small to identify. Reaction of an aliquot part of the fusel oil with 2,Cdinitrophenylhydrwine demonstrated that there were trace amounts of
