Hydrophilic interaction chromatography

[2] He described the chromatographic mechanism for it as liquid-liquid partition chromatography where analytes elute in order of increasing polarity, a conclusion supported by a review and re-evaluation of published data.

However, HILIC is more than just simple partitioning and includes hydrogen donor interactions between neutral polar species as well as weak electrostatic mechanisms under the high organic solvent conditions used for retention.

[13] For the detection of polar compounds with the use of electrospray-ionization mass spectrometry as a chromatographic detector, HILIC can offer a ten fold increase in sensitivity over reversed-phase chromatography[11] because the organic solvent is much more volatile.

His subsequent publication further described orientation effects[15] which others have also called ion-pair normal phase[16] or e-HILIC, reflecting retention mechanisms sensitive to a particular ionic portion of the analyte, either attractive or repulsive.

ERLIC (eHILIC) separations need not be isocratic, but the net effect is the reduction of the attraction of a particularly strong polar group, which then requires less strong elution conditions, and the enhanced interaction of the remaining polar (opposite charged ionic, or non-ionic) functional groups of the analyte(s).Based on the ERLIC column invented by Andrew Alpert, a new peptide mapping methodology was developed with unique properties of separation of asparagine deamidation and isomerization.

For strongly ionic functional groups of the surface (i.e. sulfates or phosphates) one could instead use a lower amount of buffer so the residual charge is not completely ion paired.

An example of this would be the use of a 12.5mM (rather than the recommended >20mM buffer), pH 9.2 mobile phase on a polymeric, zwitterionic, betaine-sulfonate surface to separate phosphonyl antibiotic mixtures (each containing a phosphate group).

This enhances the influence of the column's sulfonic acid functional groups of its surface chemistry over its, slightly diminished (by pH), quaternary amine.

Commensurate with this, these analytes will show a reduced retention on the column eluting earlier, and in higher amounts of organic solvent, than if a neutral polar HILIC surface were used.