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

Bäurer, Stefanie, Guo, Wenkai, Polnick, Stefan, Lämmerhofer, Michael
Chromatographia 2019 v.82 no.1 pp. 167-180
fat soluble vitamins, fractionation, hydrophilic interaction chromatography, reversed-phase liquid chromatography, solvents, water-soluble vitamins
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.