Characterization of Electrophoretic Behavior of Sugar Isomers by Microchip Electrophoresis Coupled with Videomicroscopy

The electrophoretic behavior of oligosaccharide isomers was investigated by microchip electrophoresis ((mu)-CE) coupled with videomicroscopy using maltose, cellobiose, maltriose, and panose as oligosaccharide isomer models. The present study revealed for the first time that the formation of a carbohydrate-phosphate complex is a pH-independent rapid process, whereas the formation of a carbohydrate-borate complex is a highly pH-dependent slow process. As a result, phosphate buffer gave much better separation on oligosaccharide isomers than borate and borate-Tris buffers over a wide pH range in (mu)-CE. The imaging analysis of the complete process of sample loading and injection with field-amplified stacking (FAS) demonstrated that FAS could be used as an efficient method for manipulating the shape of injected sample plugs, and thus improving the performance of (mu)-CE in the absence of electroosmotic flow. However, once the ionic strength mismatch between sample and running buffer reached a critical threshold, a further increase in ionic strength mismatch deteriorated the effect of FAS, resulting in a surprising decrease in separation efficiency and peak distortion. Under optimal conditions, highresolution separation of some oligosaccharide isomers and a complex oligosaccharide mixture released from ribonuclease B was achieved using PMMA microchips with an effective separation channel of 30 mm.