Studies on the Effects of Process Conditions on Separation of B1, B2 and B3 Vitamin Mixture Using HILIC and RPLC Chromatography

In this work, a series of experiments on the retention of B1 (riboflavin), B2 (thiamine) and B3 (nicotinic acid) vitamins in the HILIC and RPLC conditions have been performed involving the effects of organic modifier type and content, pH of the eluent and buffering salt (ammonium acetate) concentration in the mobile phase as well as temperature of the system. For the needs of this study, three columns of different features have been chosen: Acclaim™ Mixed–Mode HILIC–1 (Dionex, Sunnyvale, CA, USA), Eurospher II 100–5 HILIC (Knauer, Berlin, Germany) and the RPLC column Nucleodur® C18 Gravity-SB (Macherey-Nagel, Duren, Germany). The influence of acetonitrile and methanol content in the eluent and process temperature have been tested and, based on that, the most promising systems have been selected regarding the possible separation of the vitamin mixture. Both the pH and buffering salt concentrations in the eluent have been adjusted in order to indicate the most effective system, which turned out to be the one involving the Nucleodur column and the eluent with 90% methanol, at pH 6 and Cbuff = 20 mmol/dm3, which enables separation of the mixture within a time as short as 2.5 min at a 1.0 mL/s flowrate in isocratic conditions.