Attempted fractionation of LB Lennox medium via reversed phase high performance liquid chromatography

Abstract

Mass balance analysis is a highly useful tool for chemical engineering analysis of biological processes. Specifically, composition and amounts of inputs to a cell could be correlated with measurement of metabolic byproducts and outputs for inferring metabolic fluxes flowing through specific cellular metabolic pathways. However, the composition of many common microbiological growth medium remains ill-defined with batch-to-batch variation. Thus, a need exists for developing methods for effective fractionation and separation of common growth medium. Using 5 g/L yeast extract, 10 g/L tryptone, and LB Lennox medium as model systems, this work attempted the fractionation of the three complex growth mixtures using C-18 reversed phase high performance liquid chromatography (RP-HPLC). Results revealed no effective fractionation of the three mixtures. More importantly, experiment results indicated that appropriate choice of detection wavelength for visualizing the chromatogram made a huge difference to understanding the effectiveness of fractionation achieved. Specifically, in the case of yeast extract, tryptone and LB Lennox medium, 194 nm may be a more appropriate detection wavelength compared to 280 nm. Collectively, C-18 RP-HPLC was not effective in separating 5 g/L yeast extract, 10 g/L tryptone and LB Lennox medium with hydrophilic mobile phases (ethanol/water mixture). Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY Short description: Visualization of the reversed phase HPLC chromatogram at 194 nm revealed broad peaks and lack of distinct peaks indicative of fractionation of LB Lennox medium by the chromatography method. More importantly, complex ensemble of different components in the growth medium present a significant separation challenge that precludes separation or fractionation by modern liquid chromatography methods. Overall, combinatorial use of both hydrophobic and hydrophilic mobile phase with a C-18 reversed phase column could reveal the presence of a couple of main fractions in a mixture otherwise unable to be separated into distinct components. C_FIG_DISPLAY