Journal des techniques de chromatographie et de séparation
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Determination of Impurities in Bioproduced Succinic Acid

Rousova J, Ondrusova K, Karlova P and Kubatova A

At present, significant research resources are directed towards development of renewable products for replacing petrochemicals such as succinic acid. The critical component of this research is the identification of impurities which have a detrimental impact on further processing of succinic acid. We have adapted derivatization with gas chromatography - mass spectrometry to identify and quantify more than 120 impurities in several succinic acid samples. This study focused on petroleum based succinic acid as well as bio-based samples that use a modified E. coli strain for fermentation. To enable an accurate quantification of both the target product and common impurities, we evaluated the acetonitrile extraction efficiency as an alternative to direct derivatization, and then compared several derivatization agents for trimethylsilylation. A prior acetonitrile extraction was shown to be essential to detect impurities in trace concentrations. N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) was most efficient for derivatization of saccharides and low molecular weight monocarboxylic acids. However, the presence of pyridine was necessary for derivatization of saccharides and polyalcohols with BSTFA, whereas low molecular weight acids had to be quantified without pyridine. Fourteen representative bioproduced succinic acid samples differing in production stage, and cultivation method were characterized. The screening of initial process (1st stage of synthesis) samples showed monocarboxylic acids as most abundant and suggested occurrence of saccharides. Thus we have developed method allowing for quantification of carboxylic acids and saccharides with limits of detection between 0.02-0.3 ng. In initial process bacterial samples and also petrochemical sample, formic, acetic, lactic, oxalic, benzoic, citric and malic acids as well as glycerol, butanediol, and glucose were found in a range of 0.02-1160 μg/g. In final processed samples, formic and acetic acid, and glucose were found in concentration lower than 0.001% demonstrating effectiveness of process as well as applicability of the method as quality control of the process.