Does size matter? Separations on guard columns for fast sample analysis applied to bioenergy research

Stefan Bauer, Ana B. Ibáñez

Increasing sample throughput is needed when large numbers of samples have to be processed. In chromatography, one strategy is to reduce column length for decreased analysis time. Therefore, the feasibility of analyzing samples simply on a guard column was explored using refractive index and ultraviolet detection. Results from the guard columns were compared to the analyses using the standard 300 mm Aminex HPX-87H column which is widely applied to the analysis of samples from many biotechnology- and bioenergy-related experiments such as biomass conversions or fermentations. The 50 mm Rezex RFQ Fast Acid H(+) guard column was able to separate the most common fermentation products (ethanol, acetone, iso- and n-butanol) and promising precursors (furfural and 5-hydroxymethylfurfural) of biofuels and value-added chemicals. Compound profiles in fermentation samples were analyzed with similar accuracy compared to results using the 300 mm column. However, separation of glucose and xylose was not achieved. Nevertheless, it was possible to monitor the consumption of one of the two sugars during fermentation if the other one was absent or remained constant over the course of the experiment. If correct peak integration and interference subtraction was applied, concentration profiles from enzymatic digestibility experiments and even more complex samples (e.g. acetone-butanol-ethanol (ABE) fermentation) were reliably obtained. With the 50 mm guard column, samples were analyzed up to ten-times faster compared to the 300 mm column. A further decrease in analysis time was achieved by using the 30 mm Micro Guard Cation H guard column. This column is especially suitable for the rapid analysis of compounds with long elution times on the standard 300 mm column, such as biofuel-related alcohols (e.g., n-butanol, n-hexanol) and furan- and tetrahydrofuran-type molecules. Applied to a suitable set of samples, separations on a guard column can give rapid and sufficiently accurate information on compound changes over the course of an experiment. Therefore, it is an inexpensive and ideal tool for processing a large amount of samples, such as in screening or discovery experiments, where detecting relative changes is often sufficient to identify promising candidates for further analysis.