Determination of Dialdehyde Units in Periodate-Oxidized Cornstarches J. C. RANKIN and C. L. MEHLTRETTER Northern Utilization Research Branch, Peoria,
111.
Two methods are presented for quantitatively determining the extent of oxidation of cornstarch by periodate. Reduction of carbonyl groups in oxystarch by the sodium borohydride method has given percentages of dialdehyde units in close agreement with those obtained by an indirect procedure. In the latter method the unchanged anhydroglucose residues of periodate oxystarches are oxidized by additional sodium metaperiodate and the percentage of oxidized units in the oxystarch is obtained by difference. Comparison of these results with those calculated from periodate analysis in the preparation of the oxystarches has indicated the reliability of the methods for determining dialdehyde content of oxystarch.
I
N A study of periodate-oxidized cornstarches of widely varying degrees of oxidation several methods of analysis were developed at this laboratory for estimation of the extent of conversion of starch to oxystarch. One of these, a rapid quantitative alkali consumption procedure, has been described by Hofreiter, Alexander, and Rolff (6). I n this paper are reported two methods which were developed concurrently 11 ith the alkali procedure and appear to be accurate and precise for determination of dialdehyde units in periodate oxystarch.
Table I. Initial Mole Ratio, Periodate t o 4GUL" 2.00 0.95 0.85 0.80 0.65
I n the second procetiuw nsetl, instead of direct meaaurenie~itof aldehyde groups in the oxidized units, the oxystarrh n-as subjected to complete oxidation with sodium metaperiodate iindcr conditions in which overoxidation was not observed. Dialdehyde content of t,he oxystarch was calculated from the differeiicnr I)+ h e e n periodate consumcd on renewed oxidation and t,hat rrquired for complete Oxidation of a cornstarch control. Correction vias made for periodate used in oxidizing the nonretlucing end groups present. I-alidity of the results of both methods \vas substantiated by coniparison with dialdehyde values computed from periodate analysis in the initial preparation of the oxystarches. EXPERIMENTAL
Preparation of Periodate Oxystarches. Some of the osystarch samples used for analysis were prepared as described belov; others were produced under different oxidation conditions by other workers a t this laboratory and are so indicated in the tables.
Commercial cornstarch defatted by methanol extraction \vas rised; it contained 0.0i% ash, 0.05% nitrogen, and 0.03% fat (hv acid hydrolysis). Three- and 5-gram samples in 1% suspen51011 in carbon dioxide-free distilled water n-ere oxidized with various concentrations of sodium metaperiodate. The rcactiori flasks were shaken continuously in the dark a t 25" C. Samples (10 ml.) of clear liquor were withdrawn by pipet a t intervals and diluted to 100 ml. Bliquots of 5 ml. were then taken for tictermination of periodate by the method of Fleury and Lange (.$, 6).
Preparation of Oxystarches at 2.5' C. A t Isolation Moles periodate Moles HCOOII consumed per produced per mole AGU mole AGU 1.00 0.04 0.94 0.03 0 84 0.02 0.80 0.02 0.63 0.01
Dialdeiij-de Units. scb
90 91 83 78 62
Measurement of the aldehyde groups in ouystarch R B S achieved in one procedure by quantitative reduction with sodium borohydride. The relatively rapid method used by Lindberg and Misiorny (8) for estimation of reducing monosaccharides was found to be convenient and applicable to the analysis of oxystarch samples. The procedure is based on the hydrogen consumed in the conversion of carbonyl to alcohol groups. Difference between hydrogen evolved on hydrolysis of a sodium borohydride blank containing no oxystarch and of a mixture after reaction of excess reagent with the oxystarch sample is the amount of hydrogen required for reduction of the carbonyl groups in the dialdehyde units of oxystarch. While this work was in progress, Lindberg and Theander ( 9 ) reported the use of sodium borohydride for the determination of carbonyl groups in periodate oxycellulose. Although the results obtained by those investigations averaged 4% lower than the values calculated from periodate consumed in the preparation of the oxycellulose, corresponding values for periodate ouystarch were in closer agreement. Refinements in apparatus and in operating technique are presented in this manuscript, and these undoubtedly contribute to the more accurate analyses obtained with oxystar ch,
MAGNETIC STIRRING MOTOR
1
Figure 1. Apparatus for sodium borohydride method
Formic acid producsed in the oiidation of starch was estimated on a 10-ml. aliquot of the diluted solution as described by Rankin and Jeanes (IO). rlfter 24 hours these analyses showed reaction to be essentially complete and the oxystarch was isolated. The filtered product was washed free from periodate and iodate with water and finally with acetone. It was then equilibrated at room 1012
V O L U M E 2 8 , NO. 6, J U N E 1 9 5 6 humidity. Data for the oxystarch prepaiations are given in Table I. Sodium Borohydride Method. Figure 1 illustrates the asacmhlrd apparatus used. A 500-ml. leveling bulb was connected 11) an appropriate length of rubber tubing to a 100-ml. gas measuring buret graduated t o 0.1 ml. and provided with a threen a y c,apillary stopcock. One end of the stopcock was connected with the capillary side arm of the specially designed flask cap n i t h rubber tubing. The cap had a female ground-glass joint :ind :i scaled-in, 20-ml. dropping funnel with capillary stopcock.
1013
The volume of hydrogen involved during sample and blank runs must be corrected for vapor pressure of water a t 25" C., and to standard conditions of temperature and pressure. Periodate Oxidation. Samples of 200 mg. of periodate oxystarch and defatted cornstarch were weighed into 250-ml. glassstoppered Erlenmeyer flasks. T n o hundred milliliters of a 0.007M solution of sodium metaperiodate (1.10 moles per mole of anhydroglucose unit, an excess of theory for the complete oxidation of starch) was then pipetted into each flask. The reartion mixtures were allon-ed to stand in the dark a t 25" C Flasks were shaken periodically, usually after the aliquots, free of oxystarch particles, \?-ere removed for analysis. Measurement of periodate reduced by oxidation of the samples was performed a t time intervals of 24 hours for a period of 10 days. An average value from duplicate runs \vas obtained for each time interval of oxidation DISCUSSION O F PROCEDURES
Borohydride Procedure. A new blank was determined each day, becaause sodium borohydride in 0.1N sodium hydroxide was not completely stable. It was less stable in the reaction mixture 1Yhic.h was buffered a t p H 9. As a result, hydrogen was continuously evolved during reduction of oxystarch. The amount of borohydride solution specified in the procedure is large enough t o 0 permit this loss of reagent and &ill be in excess of that required co \H C to reduce 100 mg. of lOO7c osystarch. ,/IO G N 1,indbei-g and Rlisiorny ( 8 )H observed thatA 2 hours n-asI sufficient oc time to reduce monosaccharides completely with sodium borohydride. A 2-hour reaction period also vias adequate for com(C) (d) plete reduction of periodate oxystarch. An oxystarch sample of high carbonyl content gave essentially the same analysis on reFigure 2. Structures resulting from oxidation duction nith borohydride for a period of i hours as was obtained of starch b y periodate after 2 hours. Periodate oxystarch of masimum carbonyl content contains repeating units with a molecular weight of 160; this value \$-as s hooks on the cap and the 50-nil. 1,eaction flask provided a used in calculations of the percentage of dicarbonyl units in osyis of ,securing these parts with springs. A magnetic stirrer aas starch. Taking into consideration the nonreducing end groups used for agitation of the mixture a t the end of the reaction. Iteagents required for this determination \yere as follow: in st'arch, which comprise 3 to 470 of the anhydroglucose residues, sodium borohydride, 0.26121, 500 mg. in 50 ml. of 0 . l K sodium t,he average molecular weight for the oxidized nnhyclroglucose h!droxide; boric acid, 0.ldl: sulfuric acid, 2X. units of oxystarch is 159. Tcrminal glucosidic units in starch are A 100-mg. sample of periodate oxystarch was weighed into the oxidized by periodate to dinldrhyde units having one carbon 50-ml. reaction flask. The stirring magnet was added, follon-ed by 4.0 ml. of 0.1X boric acid. After the apparatus was assembled atom less than the repeating unit of oxystarch. Correction was (Figure I ) , the leveling bulb was filled with 300 ml. of water not made for such units, however, because it would not, apprcsaturated with hydrogen. Air was expelled from the system l)y ciablq- affect the values obt:iined. Dialdehyde content of oxymoving the leveling bulb to the top of the buret with the threestarches determined by horohydride reduction are given in n-:iy stopcock open to the atmosphere. The stopcock was closed \rhen the buret was completely filled with n-ater. The leveling Table I1 and are the average of a t least two analyses. bull) was then lowered and the stopcock opened to the reaction flask. This operation was repeated until water filled the capillarjportion of the buret with the three-way stopcock open to the reartion chamber. Three milliliters of 0.2611f sodium boroTahle 11. Analysis of Periodate Oxystarches b>-Different hydride solution was then pipetted into the dropping funnel. %lethods The borohydride solution was added to the oxystarch-boric acid niisture and the stopcsock closed when the solution level reached the capillary portion of the funnel. All the borohydride was borohydride washed into the reaction flask with 3 ml. (pipet) of water by the oxystarcli reduction same procedure. The addition of a constant volume of solution 98 97 97 was assured by maintaining the same level of liquid in the capil91 89 89 .. 90 90 1:iry throughout t,he reaction. Reduction of oxystarch was com. . i 8 75 plete within 2 hours. The nlixture was then acidified by s l o n 78 i8 77 addition of 3 ml. of 2 S snlfuric acid t o liberate hydrogen from 78 77 75 ci:4 .. 01 unreactetl sodium borohydride. This addition was made as (io 00 above, leaving the capillarv tube filled with acid. The reaction $) 46 41 40 mixture was then stirred for 5 minutes to dissipate hydrogen 10 20 19 20 10 11 12 hiibbles in the solution. Buret readings vere recorded at, the Id 100 end of I5 and 30 niinntes liy aligning the water meniscus b 0 " of the leveling bulb with that of the buret. The readings n-ere a l a t i o n a l Bureau of Standards .-lucose xveraged, and temperature and atmospheric pressure were noted. b Corn9tarch control sample. The blank determination was made in the same manner, but h?;drolysie of sodium borohydride wis effected immediately after addition of the solution5 t o the flask. The per cent dialdehyde iinit3 in 0x3-starch was calculated from Standard deviation of the method for 19 determinations on a the equation: sample of oxystarch of 950/, dialdehyde content was 1.0%; for a 1 2y0 oxystarch, based on six trials, the standard deviation was % dinldehyde units = 1.1%. (ml. HPfrom blank run - ml. Hz from sample run) Periodate Oxidation Method. Oxidation of repeating anhydrodry sample wt. (gram) x 2 x 22,400 glucose units (Figure 2, a) of starch by periodate required 1ti0
,P-
(.-
ANALYTICAL CHEMISTRY
1014 1 mole for cleavage of the bond between carbon atoms 2 and 3 with formation of a dialdehyde structure (Figure 2, b) ( 7 ) . Periodate consumed in the oxidation of the nonreducing and reducing end groups of starch can be determined by the amount of formic acid produced. Although insignificant amounts are required for reducing end groups ( 2 ) ,it was s h o m experimentally that, in agreement mith expectations, an additional 0.03 to 0.04 mole of periodate was consumed in eplitting the bond between carbon atoms 3 and 4 of the nonreducing end groups (Figure 2, c ) to yield the structure shown in Figure 2, d, and formic acid. Thus, starch completely oxidized t o oxystarch was found to reduce I .03 to 1.04 moles of periodate per anhydroglucose unit. A4nderson, Greenwood, and Hirst (1) considered the uptake of periodate per anhydroglucose unit of various starches to be 1.03 to 1.05 moles. Figure 3 illustrates the course of periodate oxidation of a number of representative periodate oxystarches 11-hich had different dialdehyde contents. Only slight or no further consumption of periodate by oxystarches m s found after the seventh day of oxidation. The more highly oxidized starches required less time to reach maximum reduction of periodate. Listed in Table I11 are periodate data for all isolated oxystarches R-hich were analyzed by the oxidation procedure. Average values are reported in column 2 for periodate reduced during the seventh, eighth, ninth, and tenth days of oxidation of the oxystarch samples. Because it was found that the starch control consumed 1.03 moles of periodate per anhydroglucoseunit for complete oxidation to oxystarch, subtraction from this value of the periodate consumed by the oxystarch sample (column 3) gave the moles of periodate corresponding to the number of oxidized units in the sample. A more exact calculation of dialdehyde content of the oxystarch (column 5 ) m-as obtained from this value by correcting for periodate used in the production of formic acid during oxidation of the nonreducing end groups. This correction, listed in column 4,was obtained in the preparation of oxystarches. Beloiv the 50% oxidation level, periodate used for formic acid production ivas not significant and no correction was required.
Table 111. Sample SO.
Ib 2b 3b 4b 5b 6C
7c U C
Gc
1oc 11c
Periodate Oxidation of Oxystarches at 23” C. Mole Periodate Consumed per Mole of AGU 1.03a For HCOOH 4v. 417. production 0.02 1.01 0.04 0.11 0.92 0.03 0.21 0.82 0.02 0.24 0.79 0.02 0.40 0.63 0.01 0.02 1.01 0 04 0.03 1.00 0.04 n 23 n 80 0 02 0.62 0.ii 0.00 0.19 0.00 0.84 0.00 0.92 0.11
Dialdehyde Units,
%
97 89 80 77 62 97 96 78
41 19 11
0.03 90 12d 0.10 0.93 13d 0.17 0.86 0.02 84 0 . 0 2 78 14d 0.23 0.80 15d 0.39 0.64 0.01 63 16d 0.42 0.61 0.01 60 a T h e cornstarch control sample consumed 1.03 moles of periodate per mole of AGU for complete oxidation t o oxystarch (Figure 3). b Periodate oxystarches from Table I . c Periodate oxystarches prepared by Sloan and others ( 1 1 ) : d Periodate oxystarches prepared electrolytically b y a modified procedure of Dvonch and RIehltretter (SI.
Standard deviation of the method for four trials on a sample of oxystarch of 62% dialdehyde content was 0.8%. RESULTS
I n Table I1 comparison is made of the diddehyde content of various oxystarches, computed from periodate consumed during preparation with dialdehyde contents found by sodium borohydride reduction and by reoxidation with periodate. .-iverage
difference in per cent of dialdehyde units found for various oxystarches by the different methods is about &I%. The good agreement of results by the three methods indicates that borohydride reduction probably is restricted to aldehyde groups and that p H 9, under the conditions used in the analysis, does not significantly affect the oxystarch.
I
+
$
0
I
2
3
4
5
6
7
8
g 5 9 9
1
0
TIME, DAYS
Figure 3.
Rate of oxidation of oxystarch at 25’ C.
Dialdehyde units measured b y periodate analysis during preparation
ilpparently borohydride reduction was not affected by heterogeneity of the reaction mixture because insoluble and soluble oxystarch samples gave equally good values in comparison with the other procedures. Both the reduction method and the periodate reoxidation procedure were found to be reliable and quantitative for determination of the dialdehyde content of electrolytic periodate oxystarches. Because of the relative rapidity of the borohydride reduction procedure, it is preferred for routine analosis of periodate oxyqtarches. ACKNOWLEDGMENT
The authors n i s h to thank J. \T7. Sloan of the Cereal Crops Section of the Sorthern Utilization Research Branch for furnishing them xith a number of the samples of periodate oxystarch used. LITER4TURE CITED (1) Anderson, D. 11. IT.,Greenwood, C. T., Hirst, E. L., J . Chem. SOC.1955, 225. (2) Brown, F., Dunstan, R., Halsall, T. G., Hirst, E. L., Jones. J. K. N., S u t u r e 156, 785 (1945). (3) Dvonch, W.,Nehltretter, C. L., J . Ani. Chem. SOC.74, 5522 (1952). (4) Fleury, P., Lange, J., J . pharm. chim. 17, 107 (1933). (5) Hofreiter, B. T., Alexander, B. H., Tl’olff, I. d.,ANAL.CHEM. 27, 1930 (1955). (6) Jackson, E. L., “Organic Reactions,” vol. 11, p. 361, Wley, iiew York, 1944. ( 7 ) Jackson, E. L.. Hudson, C. S., J . Am. Chem. SOC.59, 2049 (1937). (8) Lindberg, B., Msiorny, d.,Szensk Papperatidn. 55, 13 (1952). (9) Lindberg, E., Theander, 0..Ibid., 57, 83 (1954). (10) Rankin, J. C . , Jeanes, .1., J . Am. Chem. Soc. 76, 4435 (1954). (11) Ploan, J. W., Hofreiter, B. T., JIellies, R. L., Wolff, I. A , , un-
published manuscript.
RECEIVED for review August 18. 191j.5. Accepted February 21, 1966. Division of Carbohydrate Chemistry, 128th Meeting, ACS, Minneapolis, RIinn., September 1955.
