Here’s to HILIC: tailor-made for the MS analysis of polar analytes and highly water-soluble species

There is much discussion these days about Hydrophilic Interaction Chromatography (HILIC) and the options it provides today’s analysts. And there should be.  It’s powerful for analysts working with polar metabolites or highly water-soluble compounds, namely when using MS/MS techniques. Since phase chemistry is Restek’s specialty, and we have designed our Raptor SPP LC column line for use with MS/MS, we’d like to illustrate how HILIC can help you.

If you look back to the beginning, the founder of chromatography, Mikhail Tsvet, set the stage for it all. In 1906, Mikhail established the first separation using what is now considered normal phase. It’s not the “norm”, but it was first. This was used for the separation of fat-soluble compounds using a polar stationary phase.  The elution order, as a result, was non-polar to polar.  Once scientists figured out how to attach a phase, like a C18, they then made something, well, opposite.  A non-polar stationary phase used in conjunction with a polar mobile phase yields the “reverse”, polar to non-polar elution; hence “reversed phase.” This marked the true beginning of liquid chromatography.   

Aqueous normal phase and HILIC fall between these two classic modes. They utilize a polar stationary phase and a less polar mobile phase. So, we get a mix of the two; the retention profile of normal phase with the use of aqueous mobile phases. For the mass spectrometrist, this is big. We can now retain polar compounds without getting too funky with the mobile phase. While all HILIC columns are either highly polar or more polar than a C18, the mobile phase does the work. More specifically, the mobile phase’s interaction with the silica does much of the work – without traditional additives and reagents that kill MS sensitivity. And bonus – stationary phase selectivity enhances HILIC-mode-friendly columns’ versatility and adds to the mechanisms available for complex retention needs.

How it works: mobile phase choice and gradient manipulation create the formation of an aqueous layer with the polar silica or phase support, and this allows the use of high organic solvent for a secondary partitioning mechanism to occur. It’s this magic that allows polar compound analysis using MS-friendly mobile phases.

Adding stationary phase into the equation widens the selectivity profile. The new Raptor FluoroPhenyl phase exhibits this versatility. While you can run in HILIC mode for compounds such as 4-MEI Restek - Analysis of 4-Methylimidazole on Raptor FluoroPhenyl by LC-MS/MS, it also resolves difficult to retain Vitamin D metabolites or basic drugs like taxanes Restek - Analysis of Taxanes on Raptor FluoroPhenyl by LC-MS/MS.

We view HILIC as a mode, rather than a technique. Modes are normal phase, reversed phase, ion exchange, HILIC, etc.; techniques are workflow- and instrumentation-based, and take into account more than the column and mobile phase. But neither of these is as important to consider when discussing HILIC as the retention mechanism. To consider retention mechanism, look at the analytes of interest for direction. Retention via cation exchange and polarizability mechanisms respond well to HILIC mode analysis. Nitrogen-containing analytes and Lewis bases are candidates. 

We believe the HILIC mode offers impressive versatility for the MS/MS analyst. It opens the door for polar analytes, water-soluble bases and species, and otherwise non-C18-amenable compounds – without extensive method development and additives. Be on the lookout for more mixed-mode and HILIC applications and products from Restek.