Hydrophilic interaction liquid chromatography (HILIC)--a powerful separation technique

Abstract

Hydrophilic interaction liquid chromatography (HILIC) provides an alternative approach to effectively separate small polar compounds on polar stationary phases. The purpose of this work was to review the options for the characterization of HILIC stationary phases and their applications for separations of polar compounds in complex matrices. The characteristics of the hydrophilic stationary phase may affect and in some cases limit the choices of mobile phase composition, ion strength or buffer pH value available, since mechanisms other than hydrophilic partitioning could potentially occur. Enhancing our understanding of retention behavior in HILIC increases the scope of possible applications of liquid chromatography. One interesting option may also be to use HILIC in orthogonal and/or two-dimensional separations. Bioapplications of HILIC systems are also presented.

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Fig. 1

SciFinder Scholar search results documenting the continuously growing research area of HILIC, based on the numbers of publications per year

Fig. 2

HILIC combines the characteristics of the three major methods in liquid chromatography

Fig. 3

Schema of the separation mechanism in a HILIC system

Fig. 4

Surface excesses of acetonitrile and water on the HILIC adsorbent (a). Total adsorbed amounts of acetonitrile and water per unit surface area of HILIC adsorbent as a function of the volume fraction of acetonitrile in the binary eluent (b). Based on data from [79]

Fig. 5

Model of the electric double layer on the stationary bonded-phase surface. Based on data from [81]

Fig. 6

Effect of the mobile-phase aqueous buffer concentration on the HILIC retentions of the test compounds obtained using HILIC monolithic columns. Adapted from the data in [83]

Fig. 7

Comparison of the selectivities obtained for polar pharmaceuticals using a reversed-phase ion pairing (LiChrospher RP-Select B column) and b HILIC (Zorbax NH2) separation modes. Compound numbering is the same for both chromatograms. Adapted from the data in [10]

Fig. 8

Comparison of columns through the cluster analysis of retention data for peptide separation. Adapted from data in [106]

Fig. 9

Scheme of the LC × LC sample transfer interface with two sampling loops or two X-Terra trapping columns. Adapted from the data in [111]

Fig. 10

Theoretical representation of orthogonality in comprehensive LC: a NP-LC × RP-LC separation of lemon oil, b RP-LC × RP-LC separation of steroids. Adapted from the data in [113]

Fig. 11

Schematic diagram of the ACN concentration profile in a sequence of alternating HILIC and RP gradients on a single column. Adapted from the data in [99]

Fig. 12

Three-dimensional chromatogram for the comprehensive LC × LC separation of phenolic acid and flavone test standards with an optimized two-dimensional parallel gradient. Adapted from the data in [116]