Desalting and Freeze-Drying Techniques to Prepare Plant Derived Neutral Sugar Extracts for Quantitative HPLC

Abstract

Desalting and sample concentration are frequent prerequisites for the quantitative HPLC analysis of sugars and polyhydric alcohols in extracts of biological material. However, desalting using Dowex mixed-bed resins and sample concentration by conventional freeze-drying adversely affect the recovery of the carbohydrates being analysed, making quantitation almost impossible. In the present paper we report modified sample preparation procedures which overcome this technical problem. Efficient desalting was achieved by batchwise deionisation with an Amberlite mixed-bed deionisation resin, incorporating thymolphthalein to provide a sensitive indicator of resin exhaustion. Complete recovery of neutral sugars was possible provided the resin was subsequently washed with water. Concentration of dilute extracts with full recovery of neutral sugars was achieved by freeze-concentration using a Savant Speed-Vac centrifugal freeze-concentrator. The combination of these desalting and freeze-concentration procedures gave excellent recoveries of the neutral sugars together with dramatic improvements in HPLC column life and chromatographic resolution.