Abstract
Despite the rise of ‘omics techniques for the study of biological systems, the quantitative description of phenotypes still rests to a large extent on quantitative data produced on chromatography platforms. Here, we describe an improved liquid chromatography method for the determination of sugars, carboxylates, alcohols and aldehydes in microbial fermentation samples and cell extracts. Specific emphasis is given to substrates and products currently pursued in industrial microbiology. The present method allows quantification of 21 compounds in a single run with limits of quantification between 10−7 and 10−10 mol and limits of detection between 10−9 and 10−11 mol.
Highlights
High performance liquid chromatography (HPLC) has been widely used for quantification of compounds in biological samples [1]
0.22 μm PES syringe filter (Millipore: Cork, Ireland) and pre-diluted microbial fermentation samples) we have been able to make more than 200 injections per batch of analysis without change in column performance
A series of preliminary experiments were conducted to monitor the interaction of the retention times (RT) of compounds from various classes with column temperature (30, 50 and 65 ̋ C), mobile phase concentration (2, 4, 6, 8, 10, 12 and 14 mM) and flow rate (0.4, 0.5 and 0.6 mL/min) and found that a column temperature of 40 ̋ C, aqueous solution of
Summary
High performance liquid chromatography (HPLC) has been widely used for quantification of compounds in biological samples [1] It is precise, quantitative and highly reproducible, but, depending on the analysis, HPLC can be slow and the analysis of different compound classes are best performed with dedicated columns and methods [2]. While still based on cation-exchange, the method provides optimized operation temperature and mobile phase composition for a recently commercialized column. It has been optimized for simultaneous quantification of at least 21 compounds, including carbohydrates to varied alcohol products via central metabolism and has been applied to three very different samples
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