Hydrophilic-interaction chromatography

Hydrophilic interaction chromatography (HILIC) is particularly used for strongly polar compounds, that is, if these have a low retention at RP chromatography or 

The HILIC functional principle can be described as follows. On polar phases, polar molecules have retention when solvents that are more nonpolar than the stationary phase and that can be mixed with water are used. A layer of water develops on the phases surface. The polar sample spreads between this layer of water and the mobile phase. 

There is a great selection of stationary phases for HILIC silica, derivatized silica with diol, nitrile, amino or amide groups, silica with a hydrophilic polymeric coating, and various polymers. They all were especially developed for HILIC whereas 

HILIC is suited for very polar analytes such as glycosylated molecules which are not retained on reversed phases and which cannot be separated on classical adsorbents because they are not eluted by the nonpolar mobile phase. 

Two retention indices are then measured, log kgs (log k obtained with a mobile phase containing 95% of acetonitrile and 5% of buffer solution) and log ko (retention factor obtained with a pure aqueous mobile phase). The difference between these two values (log ko 9s) is correlated to the 1-octanol/water partition coefficient of the neutral form of tested compounds (Equation 5.13)  

As electrostatic interactions exist between the HILIC zwitterionic stationary phase and charged solutes, particular attention has to be paid to the rigorous control of experimental conditions such as pH, buffer type and ionic strength. 

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Hydrophilic interaction chromatography on Asahipak NH2P or Excel-pak CHA-P44 with pulsed amperometric detection has been used to fractionate malto-oligosaccharides.266 The Asahipak NH2P is a polyvinyl alcohol support with a polyamine bonded phase, and the Excelpak is a sulfonated polystyrene in the Zn+2 form. Amine adsorption of sialic acid-containing oligosaccharides was performed on a Micropak AX-5 column (Varian) using acetonitrile-water-acetic acid-triethylamine.267... 

Soga, T., Inoue, Y., and Yamaguchi, K., Determination of carbohydrates by hydrophilic interaction chromatography with pulsed amperometric detection using postcolumn pH adjustment, /. Chromatogr., 625, 151, 1992. 

Alpert, A.J. (1990). Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds. J. Chromatogr. A 499, 177-196. 

Hydrophilic interaction chromatography (HB IC) or aqueous normal-phase chromatography (ANP) refers to the use of polar stationary phases (e.g., bare silica, silica, or polymeric phases with bonded zwitterionic ligands, diol phases) in combination with a mobile phase rich in organic solvent but containing a significant amount of water (typically at least 3%). Bell [97] summarized the advantages of this technique as follows ... 

Additional modes of HPTC include normal phase, where the stationary phase is relatively polar and the mobile phase is relatively nonpolar. Silica, diol, cyano, or amino bonded phases are typically used as the stationary phase and hexane (weak solvent) in combination with ethyl acetate, propanol, or butanol (strong solvent) as the mobile phase. The retention and separation of solutes are achieved through adsorp-tion/desorption. Normal phase systems usually show better selectivity for positional isomers and can provide orthogonal selectivity compared with classical RPLC. Hydrophilic interaction chromatography (HILIC), first reported by Alpert in 1990, is potentially another viable approach for developing separations that are orthogonal to RPLC. In the HILIC mode, an aqueous-organic mobile phase is used with a polar stationary phase to provide normal phase retention behavior. Typical stationary phases include silica, diol, or amino phases. Diluted acid or a buffer usually is needed in the mobile phase to control the pH and ensure the reproducibility of retention times. The use of HILIC is currently limited to the separation of very polar small molecules. Examples of applications... 

Bard, B., Carrupt, P.A. and Martel, S. (2009) Determination of partition coefficients of basic drugs using hydrophilic interaction chromatography. Journal of Medicinal Chemistry, 52, 3416-3419. 

Determination of alkane/water partition coefficients of polar compounds using hydrophilic interaction chromatography. Journal of Chromatography A, submitted. 

Recently, Tai, and Gohda [546] have reported a method using a novel technique named hydrophilic interaction chromatography, which use a polar stationary phase with aqueous-organic mobile phase. It enables to quantify acciffately AA and related compounds, to run at high flow rate thanks to the low back pressiffe, and to be easily coupled with an MS. 

Figure 25-15 Stationary phases for hydrophobic and hydrophilic interaction chromatography...

E. S. Grumbach, D. M. Wagrowski-Diehl, J. R. Mazzeo, B. Alden, and P. C. Iraneta. Hydrophilic Interaction Chromatography Using Silica Columns for the Retention of Polar Analytes and Enhanced ESI-MS Sensitivity, LCGC 2004,... 

Hsieh, Y., and Chen, J. (2005). Simultaneous determination of nicotinic acid and its metabolites using hydrophilic interaction chromatography with tandem mass spectrometry. Rapid Commun. Mass Spectrom. 19 3031-3036. 

Song and Naidong [129] analyzed omeprazole and 5-hydroxyomepra-zole in human plasma using hydrophilic interaction chromatography with tandem mass spectrometry. Omeprazole and its metabolite 5-hydroxy omeprazole and the internal standard desoxyomeprazole were extracted from 0.05 ml of human plasma using 0.5 ml of ethyl acetate in a 96-well plate. A portion (0.1 ml) of the ethyl acetate extract was diluted with 0.4 ml of acetonitrile and 10 /il was injected onto a Betasil silica column (5 cm x 3 mm, 5 /rm) and detected by atmospheric pressure ionization 3000 and 4000 with positive electrospray ionization. Mobile phase with linear gradient elution consists of acetonitrile, water, and formic acid (from 95 5 0.1 to 73.5 26.5 0.1 in 2 min). The flow-rate was 1.5 ml/min with total rim time of 2.75 min. The method was validated for a low limit of quantitation at 2.5 ng/ml for both analytes. The method was also validated for specificity, reproducibility, stability, and recovery. 

Jandera, P. 2008. Stationary phases for hydrophilic interaction chromatography, their characterization and implementation into multidimensional chromatography concepts. J. Sep. Sci. 31 1421-1437. 

Nguyen HP, Yang SH, Wigginton JG, Simpkins JW, Schug KA (2010) Retention behavior of estrogen metabolites on hydrophilic interaction chromatography stationary phases. J Sep Sci 33 793-802... 

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