Microdialysis clean-up and sampling in enzyme-based methods for the characterisation of starch

Microdialysis was used for sampling enzyme hydrolysis products of starch hydrolysed with beta -amylase, pullulanase, and/or isoamylase, to obtain information about the molecular structure of starch. Starches from waxy, normal, and high amylose maize, and from normal and genetically modified potato (amylose deficient) were used, and also commercial potato amyloses. The hydrolysis products were analysed using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Simultaneous sampling and sample clean-up were achieved with microdialysis, thus enabling on-line injection into the liquid chromatographic system. The molecular weight cut-off of the membrane allowed for diffusion of small molecules such as oligosaccharides through the membrane, but hindered large molecules, e.g. enzymes and large polysaccharides, from entering the chromatographic system. With microdialysis sampling, it was possible to investigate the short chain fractions of debranched starch in the presence of amylose without pre-fractionation. The microdialysis-HPAEC-PAD system was also used for determination of the A:B chain ratio and the P-limit value. After P-amylolysis, only liberated maltose diffused through the dialysis membrane, which resulted in on-line sample clean-up from branched P-limit dextrin as well as from the enzyme. The proposed method is fast and easy to handle since clean-up of the hydrolysate is achieved on-line with the chromatographic system. (C) 2001 Elsevier Science Ltd. All rights reserved. (Less)