Size-Exclusion Chromatography of Polysaccharides with Pulsed Amperometric Detection (PAD)

INTRODUCTION Size-exclusion chromatography is used in carbohydrate research to determine molecular weight distributions of polysaccharides. A variety of detection methods have been used, among them viscometry, lowangle laser light scattering (LALLS), refractive index, ultraviolet/visible absorbance, fluorescence, and precision differential refractometry coupled with ultraviolet detection.This application note focuses on pulsed amperometric detection, a method that provides sensitive and specific detection of carbohydrates. Size-exclusion columns separate biological macromol ecules by hydrodynamic size. The Zorbax size-exclusion columns used in this study contain highly stable spherical, silica-based packings with diol functional groups. The packings are stable over a pH range of 3.0 to 9.0. The Zorbax SE-250 separates globular proteins by molecular size over a range of 5000 to 300,000 daltons. The Zorbax SE-450 separates globular proteins by molecular size over a range of 15,000 to 1,000,000 daltons. The exclusion volume for these columns is about 6 mL, while the included volume is around 13 to 15 mL. (Some volumes on the chromatograms shown in this application note are different from those given by the column manufacturer. These differences may be due to the non-size-exclusion effects caused by the weak ionic strength of the eluent.) Carbohydrates are not globular but rather linear or branched, so their behavior differs greatly from that of proteins under native conditions on size-exclusion columns. Fractionated pullulan standards were used to determine the separation range of linear carbohydrates. Pullulan is a straight chain of maltotriose units. Carbohydrates can be classified by several categories based on their structure: linear or branched, type of linkage, and type of sugar residue. Table 1 on page 2 presents some common polysaccharides and their structural information.