Separation of fructan isomers by high performance anion exchange chromatography

Abstract

Most cereal grasses accumulate substantial quantities of isomeric oligofructosylsucroses (fi-uctan) during vegetative and reproductive growthlb3. While fructan involvement in sugar translocation, partitioning, and utilization has not been completely elucidated, previous studies have indicated that patterns of accumulation fluctuate diurnally and seasonally lS4. To examine fructan metabolism in cereal grasses an efficient method for isomer separation is necessary. Our investigation of seasonal growth patterns among several species of winter cereals led to the development of a method for separating fructan isomers, using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), that will be useful in future metabolic studies. Hitherto, combinations of reverse and normal phase HPLC methods have been utilized for fructan separation, but these often require extended separation times ‘-’ HPAEC-PAD has been used to quantify three fructan . trisaccharides in Helianthus tuberow L., Aesculus hippocastanum L., Asparagus ofjkinalis L., and Allium cepa L. 9-11; however, this methodology has not been used to characterize fructan in Avena sativa L. (oat). We now describe a chromatographic method for routine analysis of cereal fructan in leaf and crown tissues containing neutral sugars and fructan up to dp 7. The method requires minimal sample cleanup, and analyses take less than twenty minutes to complete. Neutral sugars (glucose, fructose, and sucrose> and dp 3 and 4 fructans were chromatographed on a Carbo Pac-PAl analytical column (4 x 250 mm). A sodium hydroxide-sodium acetate gradient was used to facilitate the separation of monosaccharides, disaccharides, and oligosaccharides. Because plant tissue extracts typically contain much higher concentrations of glucose, fructose, and