Chapter 20
Size Exclusion Chromatography of Polysaccharides in Dimethylacetamide—Lithium Chloride 1
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André M . Striegel and Judy D. Timpa †
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Chemistry Department, University of New Orleans, New Orleans, LA 70148 Agricultural Research Service, U.S. Department of Agriculture, Southern Regional Center, P.O. Box 19687, New Orleans, LA 70179 2
Size exclusion chromatography has long been the method of choice for characterization of polymers and polysaccharides. A chief impediment to this process has been the lack of suitable solvents for the polysaccharides with more intricate secondary valence bond networks, such as cellulose, chitin, and starch. To this effect the solvent N,N-dimethyl acetamide with lithium chloride (DMAc/LiCl) has been utilized. With it we can accomplish dissolution of a wide variety of polysaccharides without the need for prior derivatization or extraction. The homogenization of solvent and chromatographic mobile phase greatly simplifies experimental conditions and variables. Here we address the choice of this solvent system and present results of universal calibration and light scattering studies of cellulose and pullulans dissolved in DMAc/LiCl. Although the importance of polysaccharides in a variety of fields has been widely recognized for a number of years, no single common theory has emerged to explain the remarkable diversity and complexity of these molecules (1). This has led to the conclusion that even for biological systems no single unifying function seems to exist (2). Optimizing functions and understanding the roles of polysaccharides are directly dependent upon knowledge of the composition, structure, and molecular weight distribution (MWD), and the extent to which these properties affect the molecules' behavior. Physical properties of the polysaccharides will be dictated by molecular structure. As such, when comparing branched and linear polysaccharides of equal molecular weight, the branched molecule, being more compact, will have a smaller radius of gyration, its hydrodynamic volume will be smaller, and its solution viscosity lower than those of the linear polymer. The technique of size exclusion chromatography, or SEC (also termed gel permeation chromatography, or GPC), allows the determination of molecular weight averages and, more importantly, molecular weight distributions. With it the calculation †Deceased This chapter not subject to U.S. copyright Published 1996 American Chemical Society
In Strategies in Size Exclusion Chromatography; Potschka, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
Downloaded by UNIV LAVAL on November 30, 2014 | http://pubs.acs.org Publication Date: May 30, 1996 | doi: 10.1021/bk-1996-0635.ch020
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SEC of Polysaccharides
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of a number of other characteristic solution properties is also possible. Of particular importance when performing studies of this type is the homogenization of experimental conditions, i.e. solvent, mobile phase, temperature, flow rate, method(s) of detection, adequate linear standards for performing branching calculations, proper calibration techniques, etc. The first of these conditions, the choice of solvent, has caused considerable difficulty when studying polymers, as techniques for polymer characterization usually depend on dissolution of the polymer (3). Polysaccharides such as cellulose, starch, and chitin, among many, are extremely difficult to dissolve. For cellulose, metal complex solutions, which often degrade the molecule through oxidation, are unstable, and many are colored. An alternative approach that has been extensively used with cellulose is to convert to a derivative soluble in an organic solvent which can then be conveniently characterized. The methods for obtaining solutions of cellulose involving derivatization can lead to hydrolytic and/or oxidative changes. In addition to the steps required for preparation of the derivative with strict attention to avoid degradation, a narrow range in degree of substitution is required to achieve dissolution. Agents such as sodium hydroxide or other alkaline solvents capable of selectively removing the non-cellulosic components seldom leave the cellulose unchanged; the remaining insoluble cellulose may be degraded, altered in crystallinity, and/or not absolutely pure (4). To characterize a cellulose polymer that is truly representative of the fiber matrix has proven to be a challenge. A n even greater challenge is to find a means of characterizing not only the cellulose polymer, but also other polymers and polysaccharides that may co-exist in cotton fibers or plants at different stages of development, or in biological samples. Previous studies by our group have demonstrated that #,#-dimethyl acetamide with lithium chloride (DMAc/LiCl) has the capability of dissolving a wide variety of representative polysaccharides differing in molecular weight, branching, linkage, and anomeric configuration, without the need for prior extraction, derivatization, or fractionation (5,6). We have determined D M A c / L i C l to be a thermodinamically good solvent that will break the secondary valence links of the molecule and buttress the macromolecular backbone. An additional advantage is that the solvent and the chromatographic mobile phase are thus identical. In this paper we will address our choice of this particular solvent, and give examples of results. Previously we have utilized SEC with dual detection: A concentration detector (refractive index or RI) preceded by a differential viscometer (DV). Use of these has permitted the application of the concept of universal calibration. Recently we have incorporated a multi-angle laser light scattering (LS) detector into our experimental set-up. With it absolute macromolecular characterization of molecules with radii of gyration (Rg) between approximately 10 nm and 50 nm is possible. In terms of molecular weight this translates approximately from 1,000 Da to over 1,000,000 Da for random coils, higher for spherical molecules and lower for rigid rods. Here we use the techniques of universal calibration and light scattering to characterize the solution properties of cellulose and pullulans dissolved in DMAc/LiCl. Average molecular weights and molecular weight distributions will be presented, and we will show differences in the detection ability of LS versus RI or DV.
In Strategies in Size Exclusion Chromatography; Potschka, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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STRATEGIES IN SIZE EXCLUSION CHROMATOGRAPHY
Experimental Materials. Samples included cellulose 5 (J. T. Baker Chemical C o , Phillipsburg, NJ, cat. no. 1528-1) and pullulan 1 (Pfanstiehl, Waukegan, IL, cat. no. 12474). Polystyrene standards were from Toyo Soda Manufacturing (Tokyo, Japan), types F-288, F-20, F80, F-10, F-128, F-4, F-40, F-2, A-5000, F - l , with nominal molecular weights (Da) of 2.89 xlO , 1.9 xlO , 7.1 xlO , 1.02 xlO , 1.26 xlO , 4.39 xlO , 3.55 xlO , 1.96 xlO , 6.2 xlO , 1.03 xlO , respectively. The solvent was Ν,Ν-dimemylacetamide (Burdick & Jackson, Muskegon, IL), dried with molecular sieves (Baker, activated type 3A). Lithium chloride (Baker) was oven-dried and stored in a desiccator. 6
Downloaded by UNIV LAVAL on November 30, 2014 | http://pubs.acs.org Publication Date: May 30, 1996 | doi: 10.1021/bk-1996-0635.ch020
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Sample Preparation. Sample preparation was performed following a simplified version of the protocol used with cotton (7). 30 mg of polysaccharide was added to 5 mL D M Ac in 10 mL ReactaVials with a conical magnetic stirrer in a heating block. The temperature was raised to 150°C and maintained with stirring for 1 hour. The mixture was allowed to cool to 100°C and 0.250 g dried LiCl was added. The vials were shaken by hand and returned to the heating block, where the mixture was maintained with stirring at 100°C for 1 hour. The temperature of the block was lowered to 50°C and samples were stirred at this temperature overnight. The solutions were quantitatively transferred to 50 mL volumetric flasks and diluted to volume with DMAc. They were then filtered through a solvent-resistant Teflon disposable filter. An extraction apparatus was employed with 10 cm glass syringes fitted onto filters with 4 mL glass vials held in the small volumetric holder. The final concentration of each polysaccharide was 0.6 mg/mL in DMAc/0.5% LiCl. This concentration of LiCl was chosen based on the Ekmanis procedure for cotton dissolution (7,
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