Improved analytical methods for carbohydrate analysis in biofuel research.

Essential for continued growth of the biofuels industry is the need for continued development of rapid, robust, and accurate carbohydrate quantitation methods. As new feedstocks are identified and developed, understanding how to optimize the total amount of energy obtainable will be of primary consideration for biorefineries. Conditions needed to obtain the optimal energy yield will require the testing and monitoring of different chemical and biological treatment technologies. Monitoring of carbohydrates, specifically monosaccharides and sucrose, is required to evaluate the effectiveness of the applied bioprocessing conditions. However, due to the increasing number of possible treatment technologies and potential combinations, the number of samples to be analyzed for optimization becomes the rate limiting step. Therefore rapid, accurate, and robust analytical methods for carbohydrate analysis are critical for researchers as they investigate potential feedstocks. Currently, high performance liquid chromatography with refractive index detection (HPLC-RI) and a ligand-exchange column is the standard method the biofuels industry utilizes for interrogation of carbohydrates in research samples. Overall, the HPLC-RI methods have proven to be both robust and easy to use, providing a large detection range and requiring little to no sample dilution. However, the caveats of this approach include long analysis times (45 - 60 minutes), limited resolution of select carbohydrates and an inability to separate sucrose from cellobiose. In addition, the use of a universal detector creates the possibility of false positives due to interferences caused by co-eluting compounds. An alternative method is the application of high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) for carbohydrate analysis. The primary advantage of the HPAE-PAD approach is the selectivity of PAD for carbohydrates, while a reduction of analysis time is also feasible. A series of experiments was conducted to improve the resolution of monosaccharides and sucrose while reducing analysis time. Initial experiments utilized the addition of carbonate to a commercially available column to improve analyte resolution and reduce overall analysis time to ~5 min and employed for the analysis of several sorghum types and process streams. Additionally, a commercially produced column, inspired by the carbonate-modified column was tested in an interlaboratory collaboration involving government, industry, and academic labs.