HPLC Analysis of Monoesters and Byproducts

HPLC analysis was performed according to Hollenbach et al., using a Kinetex EVO C18 (2.6 µm, 250 × 4.6 mm) from Phenomenex (Aschaffenburg, Germany) with an accompanying guard column (4 x 3.0 mm ID) of the same phase using an Agilent (Waldbronn, Germany) 1260 series liquid chromatograph equipped with a quaternary pump, an autosampler and a column oven [49 (link)]. For detection, an evaporative light scattering detector from BÜCHI Labortechnik (Essen, Germany) was used. Mobile phase was a gradient of acetonitrile (A) and water (B) with a total flow rate of 1mL/min. This method reliably separates monoesters from substrates and by-products such as diesters [49 (link)]. Only products with a purity of at least 95% determined by the area % of the HPLC chromatograms were used for further investigations.