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. HILIC is a unique mode of chromatography which uses numerous retention mechanisms. The most important mechanisms involve surface layer liquid-liquid partitioning, adsorption and various types of ionic interactions.
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 low percentage of polar phase (water in this case) to a polar surface will result in a
water layer forming. Typically this hydrophilic layer results when as little as 2 or
3% water is added to the mobile phase. The remainder of the mobile phase is an organic solvent (ACN is the most popular, but many others can be used). The polar charged analyte(s) will
partition into and out of this adsorbed water layer (often, a cation exchange process
takes place, but their may be a purely electrostatic mechanism going on as
well). 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.
With the HILIC mode, sample elution (retention) decreases as you increase the polarity of the organic solvent. Based on this information, good HILIC column wash solutions usually use alcohols in place of ACN (IPA, Ethanol and Methanol; with Methanol being a stronger eluter). For best results, consider incorporating an alcohol wash after each analysis. Allow plenty of time for the column to equilibrate too.
As with other modes of chromatography, the use of additives, buffers and pH can all play a role in retention and separation plus improve reproducibility. When developing methods, be sure and evaluate their role. Because of the low water content of most methods, buffers must be chosen carefully to insure full solubility. Ammonium formate and acetate are popular as are acids such as formic acid. Regarding pH, the low aqueous portion will mean that the actual pH of the final solution will be much closer to neutral.
With the HILIC mode, sample elution (retention) decreases as you increase the polarity of the organic solvent. Based on this information, good HILIC column wash solutions usually use alcohols in place of ACN (IPA, Ethanol and Methanol; with Methanol being a stronger eluter). For best results, consider incorporating an alcohol wash after each analysis. Allow plenty of time for the column to equilibrate too.
As with other modes of chromatography, the use of additives, buffers and pH can all play a role in retention and separation plus improve reproducibility. When developing methods, be sure and evaluate their role. Because of the low water content of most methods, buffers must be chosen carefully to insure full solubility. Ammonium formate and acetate are popular as are acids such as formic acid. Regarding pH, the low aqueous portion will mean that the actual pH of the final solution will be much closer to neutral.
- Caution: Sales and marketing people sometimes stick an HILIC label on an existing silica column to create a new product. No "special" HILIC columns are needed to develop an HILIC method. Since HILIC is a mode of chromatography, not a support type, most any high quality, NP silica column can be used.
As some HILIC methods may be hard to reproduce (very sensitive to changes in composition and long equil times) they are best used by more experienced cinematographers, only after conventional methods have been unsuccessful.
