Multi-Criteria Decision Making in High-Submicellar Liquid Chromatographic Separation of Reducing Sugars by using UV-Detection

Di- and mono-saccharides are compounds with similar property and high hydrophilicity. Therefore, the analysis of saccharides is challenging. The colorimetric methods can provide the total amount of carbohydrates but it is nonspecific. Because of the lack of effective chromophores or fluorophores in the structure of monosaccharides, the application of high performance liquid chromatography (HPLC) method is greatly limited. Specialized carbohydrate columns in combination with refractive index detector (RID) was often used in direct HPLC analysis of monosaccharides. However, the RID was not as common as ultraviolet (UV) or fluorescence (FL) detector for most of the researchers and the sensitivity was usually not satisfying [1]. Besides, since monosaccharides are non-volatile, direct gas chromategraphy (GC) method is also not applicable [2]. In this study, a simple and sensitive pre-column derivatization method for analysing reducing sugars is developed with labelling reagents of the containing chromophore moieties. The reagent 1-phenyl-3-methyl-5-pyrazolone (PMP) with strong UV absorbance at 245 nm is used as one of the popular labels that can react with reducing carbohydrates under mild conditions, requiring no acids catalyst and causing no desialylation and isomerisation. PMP derivatization increased the hydrophobicity of carbohydrates; therefore HPLC is quite suitable for analyzing PMP derivatives. Therefore the labelled di- and monosaccharides were separated by high-submicellar chromatographic (HSC) technique and detected by UV detector. In this technique, the surfactant monomers exist in the mobile phase, which are dissolved in the hydro-organic medium. The mobile phases were prepared with acetonitrile and an aqueous solution of sodium dodecyl sulphate (SDS) according to a face-centred cube central composite design. The considered factors in modelling were SDS concentration, mobile phase pH and volume percentage of acetonitrile. Multiple linear regression method was used for the construction of the best model based on experimental retention times. Pareto-optimal method was used to find suitable compatibility between separation quality and analysis time of sugar derivatives (manose, glucose, galactose, xylose and maltose) based on the prediction of the retention behaviour through empirical retention models. The optimum mobile phase composition for separation and determination of the saccharide analytes were [SDS]=0.03 molL-1, 30% v/v acetonitrile and mobile phase pH 5.