HPLC HINTS & TIPS for Chromatographers

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Tip # 116: Hydrophilic Interaction Chromatography (HILIC)

Perhaps you have a polar sample which shows poor or no retention under reverse phase conditions. HILIC may provide you with an alternative method for retention and separation.

Sometimes referred to as aqueous normal phase chromatography, this hybrid technique utilizes a stationary phase which is very polar (e.g. silica, amino or a diol column) and a mobile phase which is made up mostly of organic phase with some water added. The retention mechanism is based on the idea that adding a polar phase (water in this case) to a polar surface will result in a water layer forming. Typically this water layer results when as little as 2 or 3% water is added to the mobile phase. The polar charged analyte(s) will partition into and out-of this adsorbed water layer (a cation exchange process takes place, but their also can be a purely electrostatic mechanism going on as well. pH also plays a role in the solution chemistry so be sure and experiment with it too). Unlike conventional reverse phase chromatography, in HILIC increasing the organic content of the mobile phase increases the retention! Put another way, increasing the water content of the mobile phase and decreasing the organic portion (as in an HILIC gradient method) results in retention and then elution of very polar analytes.

> Bill Letter, 02/21/12

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