Methods for Determining Sugars and Hydroxymethylfurfural in Biomass

Common biomass sugars and hydroxymethylfurfural (HMF) are routinely analyzed during biofuel processing. Sugars are monitored for optimizing processes to maximize the yield of ethanol. HMF, a byproduct of processing, is quantified because it can have an inhibitory effect on ethanol yield. Additionally, there is a growing interest in HMF as a platform compound that can be used to synthesize several compounds (solvents, fuels, etc.) currently derived from crude oil. This presentation describes high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) methods for the determination of (i) sugars and (ii) HMF in acidhydrolyzed biomass using electrolytically generated hydroxide eluent, separation on strong anion-exchange columns, and electrochemical detection with disposable gold working electrodes. Biomass samples often have a sugar concentration over 100 g/L, and the common sugars are fucose, sucrose, arabinose, galactose, glucose, xylose, mannose, and fructose. The method for sugar analysis described here has a short run time (8 min) with resolution of all the sugars, linear range of 0.5–2 g/L, retention time (RT) precisions of <0.01–0.12%, peak area precisions of 1.7–2.7%, and acceptable recoveries (70–112%). This method is capable of handling the high concentration biomass samples with minimal sample preparation (i.e., the strong acid hydrolysate just needs to be diluted, and the column is not affected by the amount of sulfate in the diluted sample). The proposed method for HMF has a broad linear range (0.1–1000 μg/mL), low detection limit (0.04 μg/mL), high precision, and good recovery (112%). These methods require no eluent preparation, and due to short analysis time, good sensitivity, and consistent response, can be used for routine sugar and HMF analysis. IntroductIon Biomass Sugars The uncertainty associated with the supply of petroleum and the general shifts in public opinion toward environmentally-friendly lifestyle choices have increased interest in and research on feasible alternative fuels. Biofuels have emerged as an attractive alternative to fossil fuel. Biofuels have significantly less carbon output and contain fewer toxins, making them a safer and cleaner alternative. Currently, the largest producers of biofuel are Brazil, USA, France, and Germany. A common feedstock for bioethanol production is corn stover, which consists of the leaves and stalks of maize plants left after harvesting. Corn stover processing involves dilute acid treatment followed by enzymatic reactions to convert the sugars to ethanol. It is critical to analyze and quantitate the sugars during bioethanol production. Often the sugars in biomass samples are quantified by HPLC using refractive index detection.1 In the HPAE-PAD method, carbohydrates are detected by measuring the electrical current generated by their oxidation at the surface of a gold electrode. The products of this oxidation reaction also poison the surface of the electrode and must be cleaned between measurements. This occurs automatically, twice a second, during the analysis. HPAE-PAD is extremely selective and specific for carbohydrates because pulsed amperometry detects only those compounds that contain functional groups that are oxidizable at the detection voltage employed. The proposed HPAE-PAD method using a CarboPac® SA10 column has a short run time of 8 min and resolves all of the eight common biofuel sugars.2 In comparison, other columns (e.g., CarboPac PA1)3 can also elute biomass carbohydrates rapidly (in less than 10 min), resolving monosaccharides from disaccharides and trisaccharides. However, the analysis time for resolving the individual monosaccharides is typically long (>2 h). Methods for Determining Sugars and Hydroxymethylfurfural in Biomass Lipika Basumallick, Deanna Hurum, and Jeff Rohrer, Dionex Corporation, Sunnyvale, CA, USA No w s old un de r th e Th erm o S cie nti fic br an d