Development of a Method of Analysis by High Performance Liquid Chromatography for Products of the Nitric Acid Oxidation of D-Glucose

Abstract

This thesis explored the development of a faster and more efficient means of qualitative and quantitative analysis of the products of the nitric acid oxidation of D-glucose and other simple sugars, for the Shafizadeh Rocky Mountain Center for Wood and Carbohydrate Chemistry.

During the research, analysis was carried out based on previous work completed in a similar area using two Aminex HPX-87H+ cation-exchange columns at different temperatures, and plumbed in series. Standards were filtered and injected on to the columns, then eluted with 5 mM sulfuric acid. A total run time of 33 minutes enabled the elution of all products and by-products of the reaction. Retention times of standards and the use of spiking helped specify and quantify unknowns in samples from a series of oxidation reactions involving D-glucose and other aldoses.

The PrevailTM Organic acid (OA) column was said to provide 'unsurpassed resolution of organic acids'. It was therefore investigated, and a method was developed and refined in order to optimise conditions enabling the column's use for the required analyses. The optimised parameters were established as: ambient temperature with an eluent of 10 mM KH2PO4 adjusted to a pH of 2.1 with phosphoric acid. The sample size was 5 uL with a flow rate of 0.3 mL/min, giving a total run time of approximately 13 minutes.

The Aminex HPX-87H+ column method and the PrevailTM OA method were compared to determine the superior method for the analyses intended. While some improvements were made for detection in the PrevailTM OA method, results were not satisfactory. This was due in part to limits imposed on the PrevailTM OA column method, which prevented the use of gradient elution. The Aminex HPX-87H+ column method outlined herein provides superior resolution for the nitric acid oxidation of D-glucose to D-glucaric acid, and in conclusion it is suggested that the Aminex HPX-87H+ column method be used.