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

Abstract

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), Eurospher II 100−5 HILIC (Knauer) and the RPLC column Nucleodur® C18 Gravity-SB (Macherey-Nagel). 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.

Research progress on the detection of water-soluble vitamins in food using liquid chromatography

“…Chromatographic conditions include chromatographic columns, column temperature, mobile phase, flow rate, and elution gradient. Liquid chromatography methods mainly include high-performance liquid chromatography (HPLC) [28][29][30][31][32][33][34][35][36][37] and liquid chromatography-tandem mass spectrometry (LC-MS/MS) [38][39][40][41][42][43][44][45][46][47]. HPLC has been widely used for the determination of WSVs due to its high sensitivity and low cost [40].…”

“…Most WSVs are highly polar with varying degrees of hydrophilicity, making them more easily separated and eluted first using RP-HPLC mode, which also offers higher selectivity [39] and is more advantageous in terms of time and cost compared to normal phase mode. Due to its simplicity and efficiency [36], most researchers utilize RP-HPLC for the separation and determination of WSVs [1,[28][29][30][31][32][33][34][35]37].…”

“…Swen Langer and John K. Lodge [19] first used HILIC to simultaneously detect multiple WSVs extracted from complex food matrices, employing a gradient elution of 10 mM aqueous ammonium acetate (pH 5.0): acetonitrile (95:5), enabling the simultaneous detection of six B-group vitamins within 18 minutes, with intra-day and inter-day repeatability of 1.6% -3.6% and 1.8% -11.1%, respectively. Due to the excellent solubility of bioactive compounds in the HILIC environment, the application of HILIC in the separation of WSV mixtures has been a strong trend in recent years [36]. Huiliang Geng et al [53] manipulated the carbon load of the stationary phase by preparing a novel HILIC polar stationary phase, HPG-Sil, through surface-initiated polymerization of hyperbranched polyglycerol with silica.…”