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Hydrophilic Interaction (HILIC) Columns for Alternate Selectivity and Greater Retention of Polar Compounds
by Scott Anderson, Alltech Associates, Inc.
Introduction
Separation of highly polar compounds can be difficult by traditional reversed phase, as highly polar analytes often lack the hydrophobicity needed for adequate retention on reversed phase packings. Moreover, many reversed phase packings are not capable of operating under highly aqueous conditions often needed for retention of polar compounds. Over the past few years many improvements have been made to reversed phase column packings for greater retention of polar analytes. Modifications such as polar endcapping, polar embedded groups, and reduced phase bondings have all greatly increased polar compound retention under reversed phase conditions. However, these separation mechanisms utilize highly aqueous conditions with mostly non-polar packings, conditions that are often discouraged for use with LC/MS and ELSD applications since detector sensitivity is greatly reduced.
Hydrophilic Interaction Chromatography (HILIC) columns separate polar compounds that have limited retention on traditional reversed phase packings, and supply a completely different selectivity than reversed phase packings. Unlike reversed phase, HILIC columns retain highly polar compounds with only small amounts of water in the mobile phase. These more volatile mobile phases increase sensitivity with LC/MS and ELSD applications. HILIC packings consist of highly polar surfaces, making selectivity with HILIC columns complementary or alternate to that obtained with reversed phase packings.
Here we demonstrate the alternate selectivity of the Alltech® Alltima™ HP HILIC column compared to reversed phase for two highly polar drug compounds. Albuterol and Bamethan (Figure 1) are two drug compounds whose retention by reversed phase requires large amounts of water in the mobile phase. HILIC provides an excellent means by which to retain these two compounds under more ideal LC/MS conditions and with different selectivity than reversed phase.
Experimental
Equipment: Hitachi L701 pump HPLC System, Linear 205 variable UV detector, Alltech 570 Autosampler, 10 μL sample loop, Alltech Allchrom Data System.
Sample Preparation: Standards of Albuterol and Bamethan were obtained from Sigma-Aldrich (St. Louis, MO). Two vials, each containing a mix of both compounds, were prepared by weighing and diluting each analyte to a final concentration of 0.5 mg/mL in the respective mobile phases.
Chromatographic Conditions: See Figure 2.
Results and Discussion
As shown in Figure 2, Alltima HP HILIC provides excellent retention and alternate selectivity of these two compounds under highly organic LC/MS compatible mobile phases. Utilizing the unique selectivity advantages of HILIC allows separation of polar analytes not well separated by traditional reverse phases and without the need for highly aqueous mobile phases. This leads to increased sensitivity in LC/MS and ELSD applications. The use of HILIC for separations of polar compounds gives the chromatographer another tool for solving challenging separations and improving sensitivity.
Figure 1: Chemical structures of Albuterol and Bamethan.
Figure 2: Alltima HP HILIC Columns provide alternate selectivity compared to reverse phase.
Alltech Associates, Inc.
2051 Waukegan Road
Deerfield, IL 60015
Grace Davison Discovery Sciences
2051 Waukegan Road
Deerfield, IL, 60015
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