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Anal. Chem. 1981, 53, 736-737
Determination of Molecular Size Distributions of Modified Corn Starch by Size Exclusion Chromatography Robert G. Stone and Joseph A. Krasowskl” Westvaco Corporation, Laurel Research Laboratory, 11 10 1 Johns Hopkins Road, Laurel, Maryland 208 10
The molecular weight of starch must be modified from its native state to control the rheological and binding properties of industrial coatings prepared with the starch. The extent of starch conversion is routinely determined from viscosity measurements, which reflect average molecular weight. Characterization of starch molecular weight distributions by size exclusion chromatography (SEC) provides more useful information for process control and evaluation of coating properties. Low-pressure SEC has been used for the determination of molecular weight distributions of starches, as well as the separation of low molecular weight oligosaccharides (1-9). High-pressure liquid chromatography (HPLC) has been used to separate low weight oligosaccharides and substituted carbohydrates (10-13). Recently, the molecular weight distributions of modified cellulosics (14) and pectins (15)have been determined by HPLC using a Synchropak column which consists of a glycerylpropylsilane layer bonded to 10-pm macroporous spherical silica (16). Dimethyl sulfoxide (Me2SO) has been used in the lowpressure SEC separation of amylose fractions using columns packed with porous glass (3). While MezSO is known to be a “better” solvent for starch than water, the use of a polar column packing material, porous glass, has been shown to be undersirable because of adsorption effects (3). The addition of salts to a Me2SO-water solvent system in order to affect the molecular size of starches has not been reported. The ionic strength of the mobile phase can affect the hydrodynamic volume of a macromolecule, especially polyelectrolytes, and non-size-exclusion mechanisms affecting the elution behavior can be eliminated by increasing the ionic strength (17,18) of the mobile phase. This report describes the high-pressure SEC method that has been used in this laboratory to determine the molecular size distributions of starches that are of interest in the paper industry. Columns packed with a 10-ym macroporous spherical silica with a glycerylpropylsilane bonded layer were used. The effect of solvent composition, particularly ionic strength, on the molecular size distribution is discussed with the intention of using a single solvent for both ionized and neutral starches and being able to compare the distributions of both types. Potassium phosphate was used to increase the ionic strength because it has been shown that it does not form complexes with polysaccharides (19). The method is applied to hydroxyethylated and oxidized corn starches.
EXPERIMENTAL SECTION Apparatus. A Waters Associates (Milford, MA) Model 244 liquid chromatograph was used with the differential refractometer as the detector. The column system was composed of three 250 X 4.1 mm columns packed with SynChropak GPC, which is a 10-pm macroporous spherical silica with a carbohydrate bonded layer, available from Synchrom, Inc. (Linden, IN). The quoted exclusion limits were 500,500, and 100 A, and the molecular weight separation range was 5 X lo6 to 5 X lo3. The flow rate was 0.3 mL/min. The injection volume was 100 WLand the starch concentration was 0.125%. All samples were filtered throqh a 0.5-pm fiiter type FH using a Type AP prefilter (Millipore Corp., Bedford, MA) prior to injection. Samples that did not pass through the filter were not injected. The mobile phase was a mixture of dimethyl sulfoxide (Me2SO)and water which was varied between pure MezSO and 1:3 Me2SO:H20with 0.01 M potassium phos-
phate, pH 7.0. The 0.01 M potassium phosphate, pH 7.0, solution was prepared by adding the calculated amounts of the mono- and dibasic salts and adding phosphoric acid or potassium hydroxide until the pH equaled seven. A single 500-A column was used with the pure Me2S0 mobile phase. Glass-distilled solvents from Burdick &Jackson Laboratories, Inc. (Muskegon, MI), an MCB ManufacturingChemists, Inc. (Cincinnati,OH), were used without further purification except for filtering through a 0.5-pm Type FH filter (Millipore Corp.). Sample Preparation. Starch solutions were prepared by two methods. A 0.500% solution of the starch in Me2S0was heated to 100 “C for 30 min, diluted to 0.125% with the appropriate solvent to match the mobile phase, and filtered through a 0.5-pm Type FH filter. The second approach was to heat a 10% starch-water slurry in a jet cooker, in which the starch slurry is pumped through a cooking valve (Hydro-Thermal Corp., Milwaukee, WI) and mixed with steam so that the heat gelatinizes the starch, and the mechanical action of the expanding steam disrupts the swollen granules (21). The resultant starch paste was then diluted to 0.500% with MezSO and treated as above. Standard Preparation. Dextran standards (Pharmacia Fine Chemicals, Piscataway, NJ) were solubilized by heating in MezSO at 100 O C for 15-30 min and diluted t o 0.125%.
RESULTS AND DISCUSSION The SEC column system was calibrated by using dextran standards of known weight average molecular weight (ATw). The resulting calibration curve of log Mwvs. elution volume is shown in Figure 1 for the three columns: 500 A, 500 A and 100 A. The maximum of each dextran peak was taken to be MW.This assumption results in a small error (less than 270) in the calibration plot because the polydispersity is -2 (20) (the true Mw is just before the peak maximum). Elution volumes at the peak maximum for the dextran standards were reproducible to f0.05 mL. Exclusion limits for the 500 and 100 A columns overlap, and the individual column calibration plots have different slopes. This results in an inflection point at a Mwof about 190000. The dextran standards were used as secondary standards as the branched amylopectin and linear amylase components of the starches are not identical with dextran. Injection volume, sample concentration, and mobile phase flow rate were optimized for separation efficiency. An injection volume of 100 pL, a sample concentration of 0.125%, and a flow rate of 0.3 mL/min produced rapidly eluting dextran standards without peak broadening. As stated above, the polydispersity of the dextran standards is -2, resulting in very broad peaks that are not greatly affected by changes in the operating parameters. The measured signal to noise ratio for a 0.125-mg sample was 50. Varying the mobile phase from MezSO to a 3:l mixture of water and Me2S0 had no observable effect on the molecular size distributions of the dextran standards. This study was performed by using a single 500-A column, as pressure exceeding the column manufacturer’s recommended pressure limit would be obtained with the three column system and MezSO as the mobile phase. The effect that is produced by adding potassium phosphate (0.01 M, pH 7.0) to the mobile phase is shown in Figures 2 and 3, which are oxidized and hydroxyethylated starches, respectively. As can be seen in these figures, the salt addition shifted the chromatograms to higher elution volumes which indicates that the salt reduced the effective molecular volumes
0003-2700/81/0353-0736$01.25/00 1981 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 53, NO. 4, APRIL 1981
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7 r
24
,
42
46
54
60
66
Figure 4. Size exclusion chromatographs of two commerical hydroxyethylated corn starches: (A) high-viscosity starch; (B) low-viscosity starch (three columns).
*
4
5
6
7
Elution Volume (mL)
Figure 1. Calibration of SEC column system with dextran standards (three columns).
0.5
36
Elution Volume (mL)
3t 3
30
1.0
1.5
2.0
2.5
Elution Volume (mL) Figure 2. Effect of potassium phosphate on SEC of oxidized corn starch: (A) no potassium phosphate; (B) 0.01 M potassium phosphate (single column).
strength increases alone, as potassium phosphate is known to undergo no complex formation with polysaccharides (12). Increasing the phosphate concentration above 0.01 M did not produce any further changes in the molecular size distributions of the oxidized starch. The molecular size distribution of the hydroxyethylated starch presented in Figure 3 is less dependent upon the solvent ionic strength as would be expected for a neutral starch. At solvent ionic strength, sufficient to minimize intramolecular interactions of ionic groups, the molecular size distributions of both oxidized and hydroxyethylated corn starches will reflect the extent of conversion (A&). The molecular size distribution of the dextran standards shifted by small amounts that were within experimental error. Material balance determinations were not conducted with the starches. These experiments should be conducted to assure that irreversible absorption is not affecting the measured molecular size distributions. The molecular size distributions of two commerically available hydroxyethylated starches are shown in Figure 4. The difference in these distributions is manifested in large differences in Brookfield viscosity in concentrated aqueous dispersions (7000 Pa s for starch A and 0.40 P a s for starch B, 30% solids, 43 "C). Starch A exhibits a rather narrow size distribution over the range of 105-10e daltons, while starch B exhibits a much broader distribution with a peak maximum at about lo5 daltons and an appreciable amount of starch over the range 105-103 daltons.
LITERATURE CITED
0.5
1.0
1.5
2.0
2.5
Elution Volume (mL) Figure 3. Effect of potassium phosphate on SEC of hydroxyethylated corn starch: (A) no potassium phosphate: (B) 0.01 M potassium phosphate (single column). of the starches. (These data were also obtained with the single 500-Acolumn.) The solvent ionic strength has a more pronounced effect on the molecular size distribution of the oxidized starch than on the hydroxyethylated starch. The oxidation of starch is known to introduce ionizable carboxyl groups on the starch molecule, which should interact internally to form an extended molecule in the absence of salt. The addition of the salt shifted the distribution to smaller molecular sizes indicating that the intramolecular interaction of the carboxyl groups is lessened by the presence of the salt. It is believed that these changes were produced by ionic
(1) Sabbagh, N. K.; Fagerson, I. S. J . Chromatogr. 1073, 86, 164-169. (2) John, M.; Dellweg, H. Sep. Purlf. Mefbods 1073, 2(2), 231-257. {3) Dintzls, F. R.; Tobin, R. J . Chromatogr. 1074, 88, 77-85. (4) Ebermann, R.; Praznlk, W. Sfarke 1075, 27, 329-333. (5) Ebermann, R.; Praznlk, W. Sfarke 1075, 27, 361-363. ( 8 ) Sabbagh, N. K.; Fagerson, I. S. J. Chromatogr. 1078, 720, 55-63. (7) Bruun, H.; Henriksnas, H. Stark8 1077, 29, 122-126. (6) Praznik, V. M.; Ebermann, R. Sfarke 1970, 37,288-293. (9) Bao, Y. T.; Bose, A.; Ladlsch, M. R.; Tsao, G. T. J . Appl. Polym. Scl.
1080, 25, 283-275. (10) Gum, E. K., Jr.; Brown, R. D., Jr. Anal. Biochem. 1077, 82, 372-375. (11) McGinnis, G. D.; Fang, P. J . Chromatogr. 1078, 753, 107-114. (12) Ladisch, M. R.; Huebner, A. L.; Tsao, 0. T. J. Chromafogr. 1078, 747, 165-193. (13) LadlSCh, M. R.; TmO, 0. T. J. ChrO/7WbQr.1076, 766, 65-100. (14) Barth, H. G.; Regnler, F. E. J . Chromafogr. 1080, 792, 275-293. (15) Barth, H. G. J. Liq. Cbromafogr. 1980, 3, 1461-1496. (16) Regnier. F. E.; Noel, R. J . Cbromafogr. Sci. 1078, 74, 316-320. (17) Vollmert, B. "Pobmer Chemistry"; Springer-Verlag: New York, 1973; Chapter 4. (18) Barth, H. G. J . Chromatogr. Scl. 1980, 78, 409-429. (19) Foster, A. B. Adv. Carbohydr. Chem. 1057, 72, 81-115. (20) Dextran Information Booklet, Pharmacia Fine Chemicals: Piscataway, NJ.
(21) Whistler, R. L.; Paschall, E. F. "Starch: Chemistry and Technology"; Academic Press: New York, 1967; Voi. 2, p 129.
RECEIVED for review August 25,1980. Accepted January 16, 1981.
