Kavita Mistry 1, 2 Ira Krull 1 Nelu Grinberg 2 1 Department of Chemistry, Northeastern University, Boston, MA, USA 2 Analytical Research Department, Merck & Co. Inc., Rahway, NJ, USA Size-exclusion capillary electrochromatographic separation of polysaccharides using polymeric stationary phases We report the successful size-based separations of large, neutral polysaccharides using capillary electrochromatography (CEC). As the polysaccharides possessed little chromophore for photometric detection, two separate approaches were taken. In the first approach, indirect detection was combined with size-exclusion chromatography using a sulfonated polystyrene/divinylbenzene stationary phase. The separations were performed using a 300 Å pore size stationary phase under aqueous conditions. Non- size based interactions were minimal using this material, resulting in an effective cali- bration range of molecular masses 180 to 112 000 g ? mol 21 for pullulans. In the second approach, the polysaccharides were derivatized with phenylisocyanate and were sub- sequently separated on columns made using a combination of high capacity ion- exchanger and a neutral polystyrene/divinylbenzene material of various pore sizes. The sulfonated ion-exchange phase provided the electroosmotic flow, while the mixed pore size material provided the extended calibration range. The linear range for this primarily nonaqueous system using tetrahydrofuran was determined to be from molec- ular masses 738 to 404 000 g ? mol 21 of the original, untagged pullulan. This approach overcame the limited solubility issue associated with analysis of some polysaccha- rides. Analysis of pullulan and amylose samples by CEC correlated well with results obtained by conventional high-performance liquid chromatography (HPLC). The size- exclusion electrochromatographic separations provide an alternative mode for deter- mining the relative molecular weights of polysaccharides with reduced sample and solvent consumption, as well as analysis times. Keywords: Capillary electrochromatography / Indirect detection / Nonaqueous / Polysaccharides / Size-exclusion DOI 10.1002/elps.200305381 1 Introduction Pullulans are highly water-soluble, linear, long-chain glu- cans produced extracellularly in a fermentation process by the yeast-like fungus Aureobasidium pullulans [1–4]. It is a homopolymer composed primarily of maltotriosyl units linked through a,1–4 and a,1–6 glycosidic linkages in the ratio of 2:1. Occasionally, maltotetraosyl residues replace some of the maltotriosyl units. This polysaccha- ride is of economic interest due to its vast applications in the food, pharmaceutical and cosmetics industries, which take advantage of its properties such as adhesive- ness, film formability and biodegradability [5]. In addition, pullulans are often used as standards for aqueous size- exclusion chromatography (SEC) analysis of polymers [6, 7]. Similarly, amylose is also a linear homopolymer con- sisting of D-glucose units linked via a,1–4 glycosidic bonds [8, 9]. However, its solubility in water is low [5, 10]. This plant-derived polysaccharide is a constituent of starch, along with amylopectin, a branched polysaccha- ride of glucose units. The amylose content of the starch granules varies with the source and variety of the starch [11]. The properties of these polysaccharides, which are often used as thickeners, stabilizers and texturizing agents, are dependent on the molecular weight distribu- tion (MWD) [3, 10]. Hence, analysis of the polysaccha- rides, with respect to heterogeneity of composition and molecular weight (MW), is of significant importance. Correspondence: Ira Krull, Department of Chemistry, North- eastern University, 360 Huntington Ave., Boston, MA, 02115 USA E-mail: iraSK@aol.com Fax: +617-373-8795 Nelu Grinberg, Analytical Research Department, Merck & Co., Inc., RY818-C215,P.O. Box 2000 Rahway, NJ, 07065 USA E-mail: Nelu_Grinberg@merck.com Fax: +732-594-3887 Abbreviations: DMPC, tris(3,5-dimethylphenylcarbamate); ELSD, evaporative light scattering detection; M n , number-aver- age molecular weight; M p , molecular weight based on peak max- imum; M w , weight-average molecular weight; MW, molecular weight; MWD, molecular weight distribution; PIC, phenylisocya- nate; SEC, size-exclusion chromatography; SEEC, size-exclu- sion electrochromatography Electrophoresis 2003, 24, 1753–1763 1753  2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0173-0835/03/1106–1753 $17.501.50/0 CE and CEC