Evaporative light scattering detection based reversed-phase ultra-high-performance liquid chromatography method to quantify intermediates and end products of biodiesel production.

A fast method based on reversed-phase ultra-high-performance liquid chromatography using evaporative light scattering detection (RP-UHPLC-ELSD) was developed for monitoring the intermediates and end products of biodiesel production. Gradient elution of water, acetonitrile, and a mixture of acetonitrile:2-propanol:n-hexane was used. With a minimal and easy sample preparation, fatty acid methyl esters (FAME), fatty acid ethyl esters (FAEE), free fatty acids (FFA), monoacylglycerols (MAG), diacylglycerols (DAG), and triacylglycerols (TAG) were successfully separated. The developed method was used to monitor an ethylic enzymatic transesterification of soybean oil and to characterize the ester content of methyl and ethyl biodiesel. The ester content obtained was compared with the reference method by gas chromatography and flame ionization detector (GC-FID), with similar results for both methyl and ethyl biodiesel. The presented method is a simple and fast alternative, a 17 min run, to monitor the transesterification process, simultaneously quantifying all the analytes produced in the reaction: biodiesel and its intermediates. Limits of detection (LOD, between 29 and 307 ng) and quantification (LOQ, between 48 and 614 ng), linearity (R2>0.99), precision (between 0.30 and 6.58%), and accuracy (between 81.6 and 119.9%) were determined for the twenty-one compounds.

Development and validation of HILIC-UHPLC-ELSD methods for determination of sugar alcohols stereoisomers and its application for bioconversion processes of crude glycerin.

The recent increase in the production of crude glycerin through the manufacture of biodiesel has imputed a commercial issue, the excess of this raw material in the market and its constant devaluation, which resulted in the need for new technologies for its use. Crude glycerin can be used in biotechnological processes for the production of high value-added compounds. This study presents novel, simple and fast methods based on ultra-high performance liquid chromatography (UHPLC) using evaporative light scattering detection (ELSD) for simultaneous analysis of ten sugar alcohols with a hydrophilic interaction chromatography (HILIC) column. The selected compounds and their possible stereoisomers have major commercial importance and they can be obtained by biotechnological routes. Under optimized conditions, threitol, erythritol, adonitol, xylitol, arabitol, iditol, sorbitol, mannitol, dulcitol and volemitol can be analyzed simultaneously within 15.0 min. The use of different column temperatures was a key parameter to reach the selectivity during the separation of some stereoisomers. Regression equations revealed a good linear relationship (R > 0.995) over the range from 50.0 to 800.0 ng. Limits of detection (LOD) and quantification (LOQ) ranged from 30.0 to 45.0 ng and 50.0-75.0 ng, respectively. The HILIC-UHPLC-ELSD methods showed good precision with low coefficient of variation (CV%) for the intra- and inter-assays experiments (≤ 5.1%) and high repeatability in terms of retention times for each analyte (≤ 0.5%). The accuracy was confirmed with an average recovery ranging from 92.3 to 107.3%. The developed methods employ an analytical technique more accessible and suitable for routine analyzes and have shown to be suitable for simultaneous analysis of sugar alcohols present in crude bioconverted glycerin samples using different classes of microorganisms.