CARBOHYDRATE FRACTIONATION FACILITY

The aim of this proposal is to establish a Carbohydrate Fractionation Facility that uses the latest HPLC techniques for rapidly separating and quantitating complex mixtures of monosaccharides, oligosaccharides and glycopeptides with maximal resolution and sensitivity. The facility will consist of three HPLC Systems designed to separate analytical or quantitative amounts of carbohydrates on the basis of charge, size, lectin- binding properties and anionic properties at high pH. A Rainin HPLC system presently available to the group will be used for separations based on charge and size. A Perkin Elmer HPLC system is requested to be dedicated for use in lectin-affinity HPLC which separates carbohydrates according to their abilities to bind different plant lectins. The third system in the facility will be the Dionex Carbohydrate Gradient HPLC system, a novel method developed recently, which allows rapid determination of as little as 0.1n mole complex carbohydrates and the separation of oligosaccharides with identical composition that differ only in a single glycosidic linkage. The Dionex system includes a pulsed amperometric detector (PAD) that is extremely sensitive and will detect 10-100 pmoles carbohydrate. A second PAD is requested for use with the Rainin HPLC system. This method of detection cannot be applied to monitoring lectin-affinity HPLC since high concentrations of sugars are used in the buffers. Therefore, a continuous flow Berthold radiation detector is requested for efficient monitoring of these columns. The combination of HPLC systems and detectors described represents "state-of-the-art" technology in rapid and sensitive analysis of complex mixtures of eukaryotic carbohydrates and would be enormously helpful to the research programs described herein.