6
May/June 2013
Hydrophilic Interaction Liquid Chromatography: an Investigation into the Solvent and Column Selectivity
by Monica Dolci, Thermo Fisher Scientific, Runcorn, United Kingdom
This article summarises the results of an investigation into hydrophilic interaction liquid chromatography (HILIC) focusing on the retention mechanisms of ten stationary phases. Additionally to a column characterisation study, the effect on retention of changing acetonitrile content, column temperature, mobile phase pH and buffer concentration was investigated. The understanding and characterisation of HILIC retention mechanisms coupled with a better understanding of how common experimental parameters affect the separation mechanism will allow not only the judicious selection of the column, but also the appropriate HILIC conditions when developing separations.
Introduction
The ability to retain and separate polar and hydrophilic molecules can be very challenging during method development. If using conventional reverse phase liquid chromatography (RPLC), ion pair reagents, mobile phase pH modification, concentrated buffers or highly aqueous mobile phases have to be employed. Such options have potential detrimental effect upon column stability, mass spectrometric detection and sample solubility, and often still offer poor retention. If using normal phase liquid chromatography, poor reproducibility and difficulty in interfacing with mass spectrometry can be expected.
Hydrophilic interaction liquid chromatography (HILIC) is a viable alternative for the analysis of polar compounds. HILIC has been described as ‘reversed reversed phase liquid chromatography’ [1], where the stationary phase is either polar or charged (for example, unmodified silica, amino, cyano, amide, diol or ion exchange bonded phases). The mobile phase is highly organic (>70% solvent, typically acetonitrile) containing also a small percentage of aqueous solvent/buffer. The water/buffer solvent forms an aqueous-rich sub-layer adsorbed to the polar surface of the stationary phase into which polar analytes preferentially partition. The resulting retention order is approximately the opposite of the order analytes elute from a reversed phase chromatographic system [2].
McCalley and Neue demonstrated the existence of the water-rich layer on the silica surface under the typical HILIC conditions [3];
Figure 1: Chromatograms for α (CH2) test. Analyte: 1) toluene; 2) 5-methyluridine; 3) uridine. Column Name Syncronis HILIC (5 µm)*
Hypersil GOLD HILIC (5 µm)* Hypersil GOLD Silica (5 µm)* Accucore HILIC (2.6 µm)* Acclaim HILIC-10 (3 µm)
Phase type Zwitterion
Polyethyleneimine Unbonded Silica Unbonded Silica Proprietary^
Acclaim Mixed Mode HILIC-1 (5 µm) Mixed Mode Diol
Hypersil GOLD Silica (1.9 µm) Syncronis Silica (5 µm)
Experimental HILIC (3 µm) Acclaim Trinity P1 (3 µm)
Unbonded Silica Unbonded Silica Polyacrylamide NSH**
Column dimension (mm)
100 x 4.6 100 x 4.6 100 x 4.6 100 x 4.6 150 x 4.6
150 x 4.6
100 x 2.1 100 x 4.6 150 x 3.0 150 x 3.0
Surface area (m2
320 220 220 130 300
300
320 220 100 100
/g)
Pore size(Å)
100 175 175 80
120 120
100 90 90
300
Table 1: Specifications of the HILIC columns used. ** NSH: Nanopolymer Silica Hybrid. Columns marked with the asterisk were used in the chromatographic parameters investigation
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