Date of Award
Master of Science (MS)
Food, Nutrition, and Culinary Science
Professor Feng Chen, Committee Chair
Dr. Lance Beecher
Dr. Xi Wang
Detection of carbohydrates has always been a big challenge in the world, which is still attracting numerous researchers to develop different methods to overcome various difficulties. Reducing sugars, a special group of carbohydrates containing a reducing end, have provided a possibility to combine one or more chromophores to facilitate the carbohydrate detection in spite of the lack of chromophoric group in original carbohydrates. After such kind of chemical derivatizations, the sugar derivatives can be analyzed by high performance liquid chromatography (HPLC) with ultraviolet detector (UV) and diode array detector (DAD), which have been the most common methods for the carbohydrate detection. In order to optimize the sugar detection via the HPLC-UV and/or DAD, this study applied the chemical derivatization to add an extra luminophore into carbohydrates molecules, for which 1-phenyl-3-methyl-5-pyrazolone (PMP) was used in this experiment. The optimal conditions for derivatizations of glucose and glucosamine with PMP were obtained through the response surface methodology (RSM) experimental design, which suggested the optimal conditions, under a fixed value at pH 13 of the buffer solution, for the glucose-PMP and glucosamine-PMP derivatizations at 71°C for 134 minutes and 73°C for 96 minutes, respectively. The delicate difference among the optimal conditions might result from the difference of the inner-structure and inner environmental pH values of the carbohydrates. Nevertheless, this method has been proven to be a feasible and practical method with high sensitivity to determine the most monosaccharides except fructose, and disaccharides such as lactose and maltose, as well as oligosaccharides which contain the reducing end. In addition to the effect of inner pH environment, multiple sugar rings and optical isomerism of carbohydrates might also play important roles in the yield of sugar-PMP derivatives. Furthermore, this research involved the study of the detective power in terms of the detective sensitivity, accuracy and linearity of two common detectors, i.e., DAD and evaporative light scattering detector (ELSD), on the sugar-PMP derivatives, and the efficiency in terms of the separation capability of two common HPLC columns, i.e., C18 column and amide column. Because of different principles of DAD and ELSD in chemical detection, both popular detectors have different sensitivities and selectivities for carbohydrates. DAD is able to analyze the sugar-PMP derivatives, while ELSD is good at detecting both the PMP free sugars, sugar PMP derivatives and other sugar derivatives such as sugar alcohols, etc. Moreover, the results have demonstrated that the amide column could efficiently separate the PMP free carbohydrates rather than the sugar-PMP derivatives, and on the contrary, the C18 column was able to separate the sugar-PMP derivatives rather than the sugar themselves.
Wang, Weizheng, "Optimization of HPLC Detection of PMP Derivatives of Carbohydrates" (2017). All Theses. 2682.