Quantitative 2D HSQC NMR Analysis of Mixed Wood Sugars in Hemicellulosic Hydrolysate Fermentation Broth

摘要

Due to the complex composition of lignocellulosic feedstocks accurate quantification of the fermentable sugars can be challenging. These types of samples generally contain a mixture of 5- and 6-carbon sugars and while high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy have been used to quantify concentrations of sugars in hemicellulosic extracts and hydrolysates both of these methods had problems. In the case of HPLC the carbohydrate column had a relatively short lifespan when running these types of samples. In the case of proton NMR (~1H-NMR) the close proximity of the various peaks corresponding to the anomeric protons for the different sugars caused complications. Even more problematic was when the samples were not completely hydrolyzed as the oligosaccharides generate peaks with very similar resonance frequencies to those of the monosaccharides making integration difficult. In order to quantify the sugar concentrations of samples taken during the fermentation of dilute acid hydrolyzed hemicellulosic extracts a two dimensional heteronuclear single quantum coherence (2D-HSQC) technique was used. The addition of the ~(13)C axis resulted in almost no overlapping of the peaks between the various carbohydrates. This makes integrating these peaks less problematic and improves the accuracy of the carbohydrate analysis in the fermentation samples. In addition to being able to quantify the sugars, the ethanol concentrations which were previously determined using a separate gas chromatography method can be also be determined from the same NMR sample which reduced the number of samples that needed to be prepared.