Simultaneous Separation of Water- and Fat-Soluble Vitamins by Selective Comprehensive HILIC × RPLC (High-Resolution Sampling) and Active Solvent Modulation

Abstract

The simultaneous liquid chromatographic analysis of water- and fat-soluble vitamins is challenging because of their wide polarity range. Typically, water-soluble vitamins are separated and analyzed by hydrophilic interaction chromatography (HILIC) while fat-soluble vitamins are analyzed by reversed-phase liquid chromatography (RPLC). The combination of these two retention principles in a column coupling or multidimensional liquid chromatography approach seems to be a logical consequence to solve the problem. In this work, a selective comprehensive HILIC × RPLC 2D-LC approach is investigated. In this method, the polar water-soluble vitamins are resolved in the first dimension (1D) on a 2-pyridylurea mixed-mode phase operated by a HILIC gradient and the coeluted fat-soluble vitamins in the early part of the chromatogram are comprehensively transferred in ten 40-µL fractions into a second dimension (2D) separation by RPLC on a C8 core–shell column. This mode of separation is also known as high-resolution sampling. The separations in 1D and 2D were optimized systematically and the retention mechanism on the mixed-mode column interpreted by support of these chromatographic data. The solvent incompatibility of 1D HILIC and 2D RPLC conditions due to sampling of acetonitrile-rich fractions from 1D into 2D RPLC led to severe peak broadening when a direct fraction transfer was carried out. An isocratic refocusing step could partly improve the situation for the stronger retained fat-soluble vitamins. Active solvent modulation with a specifically designed valve which allows a bypass of the weak eluent from the 2D pump to the column head and dilution of the fractionated sample from the sampling loop completely solved the problem and provided perfect peak shapes and chromatographic efficiencies.