Simultaneous Separation of Water- and Fat-Soluble Vitamins by Selective Comprehensive HILIC × RPLC (High-Resolution Sampling) and Active Solvent Modulation
- 374 Downloads
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.
KeywordsReversed-phase HILIC Mixed-mode chromatography Active solvent modulation High-resolution sampling Two-dimensional LC Water-soluble vitamins Fat-soluble vitamins
ML is grateful to Agilent Technologies for financial support through an Agilent Research Award. Stephan Buckenmaier (Agilent Technologies, Waldbronn, Germany) is gratefully acknowledged for technical support.
This study was funded by an Agilent Technologies Research Award (Grant Number Agilent Research Gift #4068).
Compliance with ethical standards
Conflict of interest
Author ML has received a research grant from Agilent Technologies. Other authors declare no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- 1.Papadoyannis IN, Tsioni GK, Samanidou VF (1997) Simultaneous determination of nine water- and fat-soluble vitamins after SPE separation and RP-HPLC analysis in pharmaceutical preparations and biological fluids. J Liq Chromatogr Relat Technol 20(19):3203–3231. https://doi.org/10.1080/10826079708000485 CrossRefGoogle Scholar
- 5.Phinney KW, Rimmer CA, Thomas JB, Sander LC, Sharpless KE, Wise SA (2011) Isotope dilution liquid chromatography-mass spectrometry methods for fat- and water-soluble vitamins in nutritional formulations. Anal Chem (Washington, DC, U S) 83(1):92–98. https://doi.org/10.1021/ac101950r CrossRefGoogle Scholar
- 7.Klejdus B, Petrlová J, Potěšil D, Adam V, Mikelová R, Vacek J, Kizek R, Kubáň V (2004) Simultaneous determination of water- and fat-soluble vitamins in pharmaceutical preparations by high-performance liquid chromatography coupled with diode array detection. Anal Chim Acta 520(1):57–67. https://doi.org/10.1016/j.aca.2004.02.027 CrossRefGoogle Scholar
- 9.Gentili A, Caretti F(2013) Analysis of vitamins by liquid chromatography. In Elsevier Inc., pp 477–517. https://doi.org/10.1016/B978-0-12-415806-1.00018-8
- 13.Tayade AB, Dhar P, Kumar J, Sharma M, Chaurasia OP, Srivastava RB (2013) Sequential determination of fat- and water-soluble vitamins in Rhodiola imbricata root from trans-Himalaya with rapid resolution liquid chromatography/tandem mass spectrometry. Anal Chim Acta 789:65–73. https://doi.org/10.1016/j.aca.2013.05.062 CrossRefGoogle Scholar
- 14.Taguchi K, Fukusaki E, Bamba T (2014) Simultaneous analysis for water- and fat-soluble vitamins by a novel single chromatography technique unifying supercritical fluid chromatography and liquid chromatography. J Chromatogr A 1362:270–277. https://doi.org/10.1016/j.chroma.2014.08.003 CrossRefGoogle Scholar
- 20.Stoll DR, Harmes DC, Staples GO, Potter OG, Dammann CT, Guillarme D, Beck A (2018) Development of comprehensive online two-dimensional liquid chromatography/mass spectrometry using hydrophilic interaction and reversed-phase separations for rapid and deep profiling of therapeutic antibodies. Anal Chem 90(9):5923–5929. https://doi.org/10.1021/acs.analchem.8b00776 CrossRefGoogle Scholar
- 21.Lämmerhofer M, Richter M, Wu J, Nogueira R, Bicker W, Lindner W (2008) Mixed-mode ion-exchangers and their comparative chromatographic characterization in reversed-phase and hydrophilic interaction chromatography elution modes. J Sep Sci 31(14):2572–2588. https://doi.org/10.1002/jssc.200800178 doiCrossRefGoogle Scholar
- 22.Bäurer S, Polnick S, Sánchez-Muñoz OL, Kramer M, Lämmerhofer M (2018) N-Propyl-N′-2-pyridylurea-modified silica as mixed-mode stationary phase with moderate weak anion exchange capacity and pH-dependent surface charge reversal. J Chromatogr A 1560:45–54. https://doi.org/10.1016/j.chroma.2018.05.012 CrossRefGoogle Scholar
- 23.Jandera P, Hájek T, Česla P (2011) Effects of the gradient profile, sample volume and solvent on the separation in very fast gradients, with special attention to the second-dimension gradient in comprehensive two-dimensional liquid chromatography. J Chromatogr A 1218(15):1995–2006. https://doi.org/10.1016/j.chroma.2010.10.095 CrossRefGoogle Scholar
- 24.Stoll DR, O’Neill K, Harmes DC (2015) Effects of pH mismatch between the two dimensions of reversed-phase × reversed-phase two-dimensional separations on second dimension separation quality for ionogenic compounds—I. Carboxylic acids. J Chromatogr A 1383:25–34. https://doi.org/10.1016/j.chroma.2014.12.054 CrossRefGoogle Scholar
- 26.Stoll DR, Sajulga RW, Voigt BN, Larson EJ, Jeong LN, Rutan SC (2017) Simulation of elution profiles in liquid chromatography—II: Investigation of injection volume overload under gradient elution conditions applied to second dimension separations in two-dimensional liquid chromatography. J Chromatogr A 1523:162–172. https://doi.org/10.1016/j.chroma.2017.07.041 CrossRefGoogle Scholar