Rapid Estimation of Dialdehyde Content of Periodate Oxystarch through Quantitative Alkali Consumption B.
T.
HOFREITER, B. H. ALEXANDER], and I. A. WOLFF
Northern Utilization Research Branch, Agricultural Research Service,
A rapid and reliable method was required for determination of the extent of oxidation of numerous samples of periodate oxidized starches prepared in the course of a research program on these materials. Such a method, described here, is based on the discovery of reaction conditions under which a mole of alkali is consumed per mole of dialdehyde unit in oxystarch. There was good agreement between results obtained by this method and by other independent procedures over a wide range of dialdehyde contents. The simplicity of this method permits a single determination to be made in 10 minutes.
J
ACKSOS and Hudson (6) have established that oxidation of starch by periodate results in cleavage of the anhydroglucose units between carbon atoms 2 and 3, and have assigned the structure shown in Figure 1 to the repeating unit of the modified starch. In connection n-ith a research program on periodate oxidized starches now under way a t this laboratory, a rapid and reliable analytical method was required for determining, on a routine basis, the extent of conversion of anhydroglucose residues in starch to dialdehyde units. Although a number of procedures for carbonyl determination using hydroxylamine (S), sodium chlorite ( I ) , cyanide ( I ) , bisulfite (I), hypoiodite ( I ) , sodium borohydride ( 7 ) , or hydrolysis techniques ( 4 ) aere considered, and some were tested experimentally, none had the rapidity and simplicity of the method described here, which allon-s analysis of an oxystarch sample in less than 10 minutes.
Heating Time, Seconds 15 30 45 60 120
I n the course of investigating the reaction of alkali with periodate oxidized starch for analytical purposes two reaction temperatures were employed, each having been tested for varying Agricultural
Temperature ( " C.) of Reaction Mixture When Heated On steam bath I n 70' C. bath 47 45 49 60 67 55 72 58 79 63
Table I. Per Cent Oxidation of Periodate Oxystarch Determined by Three Independent Methods Per Cent Oxidationn Periodate consumed in preparationb 1.0 5.0 10
20 40 48 59 73 76 78 89 96 97 98
Hydroxylaminee 2.2 6.0 11 21
Alkali consumption 1.4 6.0
11 22
e a Number of dialdehyde units in 100 repeating units. b Corrected for additional periodate consumed a t nonreducing end groups, as estimated by formic acid production during the oxidation. Determined by elight modification of t h e method employed b y Gladding and Purves (3). d Periodate oxidized cellulose. Periodate oxidized dextran.
Table 11. Comparison of Dialdehyde Contents of Selected Oxystarch Samples Measured by Alkali Consumption (AC)
RESULTS
Present address, Beltsville, Md.
111.
It can be seen in Figure 2 that the reaction of periodate oxidized starch with alkali does not cease upon consumption of 1 mole of alkali per dialdehyde unit. Yet there is apparently a definite primary reaction n-hich occurs involving this stoichiometric ratio and which is manifest in an inflection point of the curves (steam bath) or in the markedly lowered rate of reaction (70' C.) beyond that point. The rate of secondary reaction is considerably more pronounced a t the higher temperature. Heating times might be selected to form the basis of an analytical method to be carried out at 70" C. At this temperature it is obvious that higher precision might be expected, since slight deviation from the time selected would have less influence on the alkali consumption. However, for convenience, and because the method was required in these studies primarily as a guide, most
From 0.1500 to 0.2000 gram of oxystarch of known moisture content is weighed into a 125-ml. Erlenmeyer flask. To the flask is added, via pipet, 10 ml. of standardized, carbonate-free 0.25N sodium hydroxide, and the flask is gently swirled and immediately placed on a steam bath having a circular opening 5.5 em. in diameter for exactly 1 minute. There should be rapid flow of steam during the heating peiiod. The flask is then cooled immediately under running tap water with rapid swirling for 1 minute, and 15 ml. of standardized 0 . 2 5 5 sulfuric acid is added by pipet. To the contents of the flask are added 50 ml. of water and 1 ml. of neutral 0.2% phenolphthalein. Titration of the acid solution is carried out using 0 . 2 5 S sodium hydroxide delivered from a 10-ml. microburet, graduated to 0.02 ml. Alternatively, the titration may be carried out potentiometrically if desired. The percentage of dialdehyde units in the oxystarch is given by the equation:
1
Peoria,
time periods and on products oxidized to different dialdehyde contents. The results are summarized in Figure 2. For the reactions which are indicated as having been carried out a t 70" C., the flasks were immersed to a depth of 1 cm. in a water bath held a t that temperature. The temperatures attained by the liquid reaction mixtures under conditions used were as follows.
EXPERIMENTAL
161 where 161 is the average molecular weight of the repeating unit in starch, 50% of which has been converted to dialdehyde units.
U. S. Department o f Agriculture,
Research
Center,
(Under two reaction conditions with values obtained by other methods) Sodium Periodate Sample AC, Steam AC, 70' c . , Borohydride HydroxylConsumed ( ~ i ~ ~Bath, ~ 1 ~ 311n. 2 ) 2 hiin. (7) amine in Preparation 99 97 99 85 .. 63 60 56 60 59 C 11 11 12 12 10
;
1930
V O L U M E 27, NO. 12, D E C E M B E R 1 9 5 5
1931
of these analyses were carried out using the steam bath temperature as described in the experimental section. That the consumption of alkali in the initial reaction phase is actually a measure of dialdehyde content is confirmed by the good agreement in most cases between analytical values obtained bv the alkali consumption method and those from three independent procedures for assaying the dicarbonyl content of oxypolysaccharides. Comparative data are shown in Tables I and 11.
radation, production of acidic groups, and formation of colored products. Standardized conditions have been developed under which 1 mole of alkali is consumed for each dialdehyde group in oxystarches having widely varying degrees of oxidation. Indications are that the conditions are valid also for periodate oxidized dextran and cellulose. Although the mechanism of action and reaction products resulting from this alkaline reaction are unknown a t this time, it is probable that the predominating reaction is a dismutation of the Cannizzaro type.
Table 111. Precision of Analyses by Alkali Consumption Method Approximate Dialdehyde Content, %
No. of Samples in Set
Av. Dev.
Std. Dev.
99 99 99 99
9 10 10 9
1.53 1.74 0.88 1.58
2.14 2.12 1.25 1.99
60
10 9
0.71 0.97
0.92 1.34
42
Reaction Conditions Steam b a t h , 1 min. Steam bath, 1 min. Steambath,lmin. Steam b a t h , 1 min. potentiometric to p H 7.1 70°C.,80sec. S t e a m b a t h , 1 min.
Operator 1 1 2
1 1
1
Values obtained by the alkali consumption procedure might be expected to be somewhat high, since the secondary reaction with alkali is undoubtedly proceeding during the initial heating period, This is shown by the trend in Table I1 for values obtained a t steam bath temperature to be higher than those when reaction was a t 70" C. However, values were not consistently higher than those obtained from the independent methods used. TIME,SECONDS
-7
Figure 2. Effect of reaction time on consumption of alkali at different temperatures and dialdehyde contents
----
CH
HC
Figure 1. Repeating unit of periodate oxystarch
The precision to be expected of the alkali consumption method is exemplified by the data of Table 111, which include analyses run on different samples, different days, by different operators, by use of indicator or potentiometer, and a t 70' C. versus steam bath temperature. The average standard deviation of the analyses for essentially completely oxidized samples was 1.88.
Analyses carried out on s t e a m b a t h Analyses carried out i n 70' C. w a t e r bath
The alkali-consumption procedure is currently in routine use a t this laboratory for assaying periodate oxidized starches in research directed toward improved procedures for preparing these materials. Future work on the underlying chemical changes involved in this alkali reaction is contemplated. ACKNOWLEDGMENT
The authors are indebted to R. L. Mellies and J. C. Rankin for the determinations of dialdehyde content of oxystarches by means other than the alkali consumption method, and to P. R. Watson for his assistance in evaluating the precision of the present method. LITERATURE C I T E D
DISCUSSIO;\ (1)
Literature reports (2, 6 ) have indicated the ocrurrence of an internal Cannizzaro reaction when certain polyaldehydes produced by the periodate oxidation of carbohydrates were treated with alkali. In one case ( 2 ) stoichiometric consumption of 1 mole of alkali per mole of the simple dialdehyde, L'-methoxyL-methyldiglvcolaldehyde (prepared by periodate oxidation of a-methyl-L-rhamnopyranoside), was demonstrated. Acid hydrolysis of the product from the alkali treatment, and identification of the fragments, confirmed the occurrence of a Cannizzaro reaction. Holvever, treatment of starch in a similar manner resulted in consumption of only 0.7 mole of alkali per dialdehyde repeating unit. In general, periodate oxidized starch, cellulose, and structurally related polysaccharides are characterized by instability in even mildy alkaline solution, manifested by deg-
Ellington, A. C., and Purves, C. B., Can. J . Chem., 31,
801-13
(1953).
E.'hI., Wilson, E. J., Jr., and Hudson, C. S.,J . Am. Chem. SOC.,6 4 , 8 7 2 - 3 (1942). (3) Gladding, E. K., and Purves, C. B., Paper Trade J . . 116. No. (2) Fry,
14, 26-31 (1943). (4)
Grangaard, D. H., Gladding, E. K., and Purves, C. B., Tech. Assoc. Papers, 25, 385-90 (1942); Paper Trade J., 115, KO.7 , 41-8 (1942).
(5) Head, F. S. H., J . Textile Inst., 38, T 3 8 9 - T 4 0 7 (1947). (6) Jackson, E. L., and Hudson, C. S., J Am. Chem. Soc., 6 0 , 98991 (1938). (7) Rankin, J. C., and AIehltretter, C. L., Korthern Utilization Research Branch, unpublished results, RECEIVED for review May 23, 195.5. Accepted August 22, 1955. Presented in part before the Carbohydrate Division, 127th Meeting, ACS, Cincinnati, Ohio, March-April, 1955.
