Sampling, Dilution, and Loading Device-coupled High-Performance Liquid Chromatography Method for Successive On-line Analyses of Major Carbohydrate Products in Immobilized Cellulase Hydrolysate of Paper Cellulose
Cellulose is a polymer of glucose and is the main structural material of plants. The use of purified cellulase or the whole cell of cellulase containing microorganisms to hydrolyze the cellulose in waste agricultural products or paper products to produce useful glucose is a mandatory and important subject in the scientific world. 1 However, the analyses of monocarbohydrates such as glucose, galactose, mannose, xylose, and arabinose by traditional methods are very difficult due to the similarity
... e to the similarity in their structures. 2 Modern detection techniques used to determine carbohydrates fall in 2 categories: high-performance liquid chromatography (HPLC) 3-6 and enzyme electrode biosensors. 7-10 When these methods were carried out in an off-line mode they usually posed the problems of sample transferring loss and contamination; they were timeconsuming and were incapable of providing immediate information for chemical or biochemical process evaluation to enable timely action. In contrast, on-line monitoring can quickly and accurately indicate not only substrate and product variations but also formation of and changes in intermediates. 11-14 Accordingly, the on-line procedure displays a significant advantage in product quality control. In order to analyze multi-components over a wide concentration range efficiently with on-line HPLC method, one needs sample dilution for high concentration analytes that can adjust the concentration of analytes within the linear range of the calibration curve. Most of the on-line dilution schemes     described in the literature were complicated and used expensive equipment, while off-line dilution procedures often exhibited sample loss and tedious procedures. It would be wise to simplify the on-line dilution scheme in a more convenient and economical way. In this paper, a simple specific designed device was developed that can couple the HPLC analysis system to an immobilized cellulase bioreactor system. 19 The specific designed device was operated manually; however, it can easily handle the sampling, dilution, and loading steps on-line. Thus, the concentration of the major carbohydrate products in cellulase hydrolysate can be on-line analyzed simultaneously. By using a refractive index (RI) detector, 4 kinds of quantification methods were compared to elucidate the suitability of the internal standard calibration method 20 with on-line dilution procedure for our on-line HPLC analysis system.