SEPARATION O F ISOMERIC GLYCOSIDES PRODUCED BY TRANSGLYCOSYLATION
OF STARCH WITH ETHYLENE GLYCOL F. H. O T E Y , FLORENCE L. BENNETT, BONNIE L. ZAGOREN. AND C. L. M E H L T R E T T E R Northern Regional Research Laboratory, C. S.Department of Agriculture, Peoria, Ill.
An acid-catalyzed reaction of starch with ethylene glycol at 120" to 130" C. yields a mixture of isomeric glycosides that has potential for use as an industrial polyol. The major glycosides present were separated by carbon-Celite column adsorption followed by gradient elution. Course of the elution was determined by thin-layer chromatography. 2-Hydroxyethyl a-D-glucopyranoside was eluted with water and crystallized from the concentrated solution. The corresponding /3-glucoside was removed from the column with 3.7% ethanol. A third product, isolated after elution with 6y0 ethanol, was identified as ethylene bis(a-Dglucopyranoside). Quantitative gas chromatographic analysis showed that the crude glycoside mixture contained 45 to 48% 2-hydroxyethyl a-D-glucopyranoside, 2 1 to 24% /%anomer, and at least 1 170 ethylene bis(a-D-glucopyranoside).
A
preparation of glycol glycosides by transglycosylation of starch with ethylene glycol has been developed in this laboratory (8,9 ) . The crude mixture obtained has potential use as a polyol ra\z material for surfactant (7) and rigid urethane foam manufacture ( 9 ) . Further development of this mixture for industrial applications \vould be facilitated by a knowledge of the
types and amounts of glycol glycosides present. This paper describes the isolation and quantitation of the major glycoside components in the mixture and their identification as 2hydroxyethyl a-D-glucopyranoside (hereafter referred to as glycol a-D-glucoside) ; 2-hydroxyethyl P-D-glucopyranoside (glycol P-D-glucoside) ; and ethylene bis(a-D-glucopyranoside) (glycol diglucoside).
FACILE
t
h
bH 2 -Hydroxethyl a m D glucopyranoside "Glycol CY D glucoside"
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OH 2 .Hydroxyethyl /3. D glucopyranoside "Glycol @. D glucoside"
H OH Ethylene bis[a-o.glucopyranoside) "Glycol diglucoside" 228
I & E C P R O D U C T R E S E A R C H A N D DEVELOPMENT
h
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A
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Excellent yields of the three major components were obtained by separation of the crude glycoside mixture on a carbon column (76). Preferential displacement from the carbon was achieved with alcohol-water adjusted to p H 3 with formic acid (73). .4 rapid means of following the course of the desorption is provided by thin-layer chromatography (TLC). Isolation of the pure glycosides allowed quantitation of the crude mixture with gas-liquid chromatography IGLC). I n this study, the trimethylsilyl derivatives (73) of the glycosides were separated on a column packed with diatomaceous earth (Diatoport-S) and silicone (SE-52). T h e only previous report of crystalline glycol a-D-glucoside is by Bourquelot and Bride1 (2). They obtained an SYGyield of the glucoside by enzymatic synthesis from glucose and ethylene glycol after 93 days of reaction. Glycol 8-D-glucoside has also been obtained by biochemical synthesis ( 7 ) and by the action of ethylene glycol on acetobromoglucose ( 3 - 5 ) .
Separation of Glycosides on Carbon
Activated carbon (260 grams of Darco G-60) and diatomaceous earth (260 grams of N o . 535 Johns-Manville Celite) were mixed in a bottle on a ball-mill drive for 1 hour. T h e mixture was suspended in about 2 liters of 10A' hydrochloric acid and stirred intermittently for 12 to 18 hours. T h e solids were then separated by filtration and washed with distilled \.vater until a water suspension of the solids had a p H of a t least 4, after standing 2 to 3 hours. A water suspension of the solids was poured into a glass tube (60- X 6-cm. diameter) plugged with glass \vool. This gave a column 52 cm. long. T\yenty grams of the crude glycoside mixture, obtained by in 100 ml. of water reaction of ethylene glycol with starch (8), was adsorbed on the carbon and desorption was carried out by gradient elution mith aqueous ethanol adjusted to p H 3 with formic acid. Different concentrations of aqueous ethanol were siphoned into a n intermediate container, so that fractions were calculated to contain the follokving concentrations : fractions 2 to 8!kvater; 13 to 29, 0 to 3.77, ethanol; 37 to 42, 5.6 to 6,3y0 ethanol; and 43 to 54, 6.3 to 9.0% ethanol. T h e eluate \vas collected in 400-ml. fractions on a n automatic
fraction collector a t the rate of 5 ml. per minute. Course of the desorption process was followed by T L C . T L C plates were prepared and sprayed as described by Weill and Hanke (75) and developed with benzene-methanol (1 to 1). T h e reported Ro values are the rate of travel of the glucoside per rate of travel of glucose.
Glycol a-D-Glucoside. Water (fractidns 2 to 8) desorbed the a-monomer ( R , 0.90), which yielded 7.8 grams of sirup upon concentration to dryness. The sirup crystallized as needles after standing at room temperature for 2 weeks { [CY]: +133' (H20, c, 2) Recrystallization from a mixture of acetone and methanol yielded pure glycol a-D-glucoside { m.p. 101-02@C., [a]: +137.5' (HzO, c, 2 ) ) . A melting point for this compound was not found in the literature, although Bourquelot (2) reported the follo\ving optical rotation: [ a ] : $135.5' ( H 2 0 ,c, 3). Hydrolysis of this glycoside with 0.5.Y sulfuric acid for 3 hours a t loo@ C. yielded the calculated amount of glucose and glycol (6: 7 7). Glycol @-D-Glucoside. The @-monomer ( R , 1.1) was desorbed next with dilute ethanol (fractions 13 to 29). Concentration of the eluate to dryness yielded 3.5 grams of sirup which did not crptallize upon standing. The pure glycoside was finally crystallized from a mixture of ethanol and ethyl acetate after seeding { m . p . 137-38' C.? [a]: -30.4' ( H 2 0 . c: 1.7); lit. ( 3 ) : m.p. 137-38' C., [a]: -30.2' ( H 2 0 ) } . Glycol Diglucoside. X dimer (X, 0.49) was desorbed by increasing the ethanol concentration (fractions 37 to 42) and yielded 2.2 grams of white solid upon concentration to dryness { [a]: + l l O @ (H90, c: 1.3)}. The product did not crystallize, but a small amount was precipitated from a mixture of ethanol and ethyl acetate after standing in the cold for 1 Lveek (melting range, 90' to 96@ C.: [CY]: +148@ (H?O, c. 1.8) Purity of the product \vas questionable because of the melting range; however, T L C indicated only one compound. The product was nonreducing to Fehling's solution, and upon hydrolysis in 1 S sulfuric acid for 4 hours a t 100' C.: only glucose and ethylene glycol were detected by paper chromatography.
1.
1.
1
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lal 4
10
Figure 1. sor bito I
Time, min. 15
25
30
35
Gas chromatogram of crude glycol glycoside mixture and
Column operated isothermally at 230' C. for 18 minutes and then at 300' C. for remaining time 1 . Sorbitol (internal standard) 2. Glycol a-D-glucoside 3. Glycol P-D-glucoside 4. Glycol diglucoside
VOL. 4
NO. 4
DECEMBER 1 9 6 5
229
Analysis of the hydrolyzate (6, 77) showed the presence of 2 moles of glucose per mole of glycol. T h e most probable structure for the compound is that of glycol maltoside, glycol isomaltoside, or glycol diglucoside. T L C of hydrolyzate samples during the course of hydrolysis showed the presence of only glucose, glycol a-b-glucoside, and the original dimer. This study indicated a glycol diglucoside structure. T h e dimer consumed 3.83 moles of periodate and liberated 1.74 moles of formic acid during oxidation ( 7 0 ) ,which also supports a diglycoside structure. Probably the a-anomer predominated, because glycol di-P-n-glucoside has a n [CY]: -35.2' (H20)
(4. Additional mixed dimers (2.0 grams) were obtained from the concentration of fractions 43 to 54. This mixture had the following properties: Ro values of 0.63 to 0.49 on T L C ; [CY]: +86' (H20, c, 1.5); m.p. 83-93' C. Acid hydrolysis produced 2 moles of glucose per mole of glycol. The mixture consumed 3.4 moles of periodate and liberated 1.5 moles of formic acid during 48 hours of oxidation. At least three other products were desorbed from the carbon column, as indicated by different Ro values with TLC, but none were sufficiently resolved for absolute identification. Because of their lower RGvalues, it is assumed that the compounds were mixed glycol oligosaccharides. Quantitative Analysis of Glycoside Mixture
GLC proved useful for analysis of the crude glycoside mixture. Analyses were made with a n F & M Scientific Model 810 with a 4-foot copper column packed with Diatoport-S containing 57, SE-52. Samples were prepared by mixing 10 mg. of cxbohydrate with 1 ml. of anhydrous pyridine, 0.2 ml. of hexaniethyldisilazane, and 0.1 ml. of trimethylchlorosilane ( 7 3 ) . Figure 1 is a typical chromatogram. Peaks were identified from chromatograms made with pure samples of the glycosides obtained from the carbon column. Quantitative analysis by GLC of the crude glycoside was made as described by Sparagana, Keutmann, and Mason (72). T h e three glucosides, isolated on the carbon column, were separately mixed with a known amount of internal standard (sorbitol) and then each mixture was chromatographed. T h e area ratio of the internal standard to the compound in question divided by the respective weight ratio gave the desired calibration constant. A known mixture of sorbitol and crude glycoside was then chromatographed and percentages of the three glucosides were determined by using the calibration constant. These analyses showed that the crude glycoside mixture contained 45 to 48Y0 glycol ol-n-glucoside, 21 to 24% glycol p-D-glucoside, and 11 to 12y0glycol diglucoside. T h e presence of a higher percentage of dimers was indicated from other responses near the diglucoside peak and from the carbon column separation. Free ethylene glycol was detected by injecting the crude glycoside derivative a t a column temperature of 66' C. with propylene glycol as the internal standard. Quantitative analysis showed the glycoside mixture to contain 1 to 1.5% unreacted ethylene glycol. In earlier work (7)
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I & E C P R O D U C T RESEARCH A N D D E V E L O P M E N T
with quantitative paper chromatography and chemical analysis, a glycol glycoside mixture prepared from starch contained about 60% glycol monoglucosides, 20% glycol diglucosides, and 1 to 2% unreacted glycol. Paper chromatography, however, did not separate the a- and p-anomers as did carbon, TLC, and GLC. Discussion and Conclusions
Separation and identification of the major components produced during the transglycosylation of starch with ethylene glycol provide information that may help in further development of the product for industrial applications. Since no reducing groups are present, the product is sufficiently heatstable to withstand alkoxylation-a reaction necessary for most surfactant and foam applications. Chromatographic studies indicate that there is no appreciable buildup of sugar anhydrides. A combined evaluation of both the carbon column and chromatographic results indicates that the product mixture contains about 45% glycol a-D-glucoside, 21y0 glycol p-Dglucoside, 20% mixed dimers of glucose, 13% mixed glycol oligosaccharides, and 1yo free ethylene glycol. Acknowledgment
The authors thank J. H. Sloneker and J. S. Sawardeker of this laboratory for their assistance with the gas chromatographic analysis. Literature Cited
(1) Bourquelot, E., Bridel, M., Compt. Rend. 158,898 (1914). (2) Ibid., p. 1219. (3) Fischer, E., Fischer, H., Ber. 43, 2521 (1910). (4) Helferich, B., Hiltmann, R., Ann. Chem. 531, 160 (1937). f51 Kariala. S.. Link. K. P.. J . A m . Chem. SOC. 62. 917 (19401. (65 LaGbert, M.,Nekh, A.'C., Can. J . Res. 28B,'83 (1950). ' (7) Otey, F. H., Mehltretter, C. L., Rist, C. E., J . Am. Oil Chemists, Soc. 40, 76 (1963). (8) Otey, F. H., Zagoren, B. L., Bennett, F. L., Mehltretter, C. L., IND.ENG.CHEM.PROD.RES.DEVELOP. 4,224 (1965). (9) Otey, F. H., Z a g x m , B. L., Mehltretter, C. L., Ibid., 2, 256
(1963). (10) Rankin, J. C., Jeanes, A., J . Am. Chem. SOC.76, 4435 (1954). (11) Somogyi, M., J . Bid. Chem. 160, 61 (1945). (12) Sparagana, M., Keutmann, E. H., Mason, W. B., Anal. Chem. 35, 1231 (1963). (13) Sweeley, C. C., Bentley, R., Makita, M., Wells, W. W., J . A m . Chem. SOC. 85, 2497 (1963). (14) Taylor, P. M., Whelan, W. J., Chem. Ind. (London) 1962,44. (15) Weill, C. E., Hanke, P., Anal. Chem. 34, 1736 (1962). (16) Whistler, R. L., Durso, D. F., J . A m . Chem. Soc. 72, 677
(1950). RECEIVED for review April 30, 1965 ACCEPTED July 26, 1965 Division of Carbohydrate Chemistry, 149th Meeting, ACS, Detroit, Mich., April 1965. The Northern Laboratory is part of the Northern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture. Mention of firm names or commercial products does not constitute an endorsement by the U. S. Department of Agriculture.
