High‐performance liquid chromatographic determination of oligosaccharides in leguminous seeds

Abstract

AbstractA simple and reliable high‐performance liquid chromatographic (h.p.l.c.) method has been developed for the determination of sucrose, raffinose and stachyose in leguminous seeds. A LiChrosorb NH2 column (Merck) and an acetonitrile+water solvent (65:35, by vol) at a flow rate of 1 ml min−1 were used for the separation. An interference type refractive index detector (Tecator) was used for the detection. With this detector the smallest amount of oligosaccharide detectable was 10 ng. With a traditional deflection type refractive index detector the smallest amount of oligosaccharide detectable was 240 μg. The oligosaccharides were extracted by placing the whole, dry seeds in boiling water for 30 min, blending the seeds and water, placing the whole suspension in a shaking bath at 60°C for 60 min, and removing the solid material by centrifuging. The extract was deproteinated by adding 65 parts of acetonitrile to 35 parts of extract (by vol), placing the mixture at 5°C for 60 min, and filtering off the resulting proteinaceous precipitate before injection into the chromatograph. The entire procedure has been successfully applied to soya beans, chick peas, garden peas and red kidney beans with recoveries of added raffinose in the range 97–102%.