Analysis of sub-microgram quantities of cellodextrins by aqueous liquid chromatography using a differential refractometer

Abstract

The analysis of water-soluble cellodextrins using liquid chromatography is readily achieved with a variety of packings. Direct injection of enzyme incubation mixtures allows quantitation of 10 mM cellodextrins in hydrolysis mixtures, resulting in a method which is useful for kinetic studies. Reported here are operating procedures for a 4% cross-linked, styrene-divinyl benzene cation exchanger (Aminex 50W-X4 (Bio Rad Lab., Griffin, CA, USA), 20–30 μm particle size) in the Ca⁺⁺ form, packed in a column of dimensions 6 mm i.d. × 60 cm long. Using this column, resolution of the cellodextrins, celloheptaose through cellobiose and glucose was possible with 91 mM H₂SO₄ as the eluent. Requirements of the separation system included use of a pulsation free syringe pump to minimize baseline fluctuations, the use of Ca⁺⁺ as the counterion to give a column operational life of 500–1000 injections, and injection of sample volumes of up to 25 μL. cellodextrins were quantified at sub-microgram (nmole) levels using a differential refractometer as the detector. Examples of this technique for analysis of the acid hydrolysis of cellodextrins and enzymatic hydrolysis of cellodextrins and carboxymethylcellulose are described.