What is HILIC Separation? What Does It Mean For You?

By Scott Krepich, Food and Environmental Application Scientist Hydrophilic Interaction Chromatography (HILIC) is a variant of normal-phase liquid chromatography that partly overlaps with other chromatographic applications such as ion-chromatography and reversed-phase liquid chromatography. However, not all researchers know when the best time to use HILIC separation is. So, I am going to break down all the information and help answer the question we are all asking: When should I use HILIC? Answer:  While this seems like it should be an easy answer, HILIC is a little more complicated than that. While an advocate of the HILIC separation mode, I can attest to the myriad of challenges, making it less ideal for many practical scenarios. HILIC can be a powerful and effective technique for the most challenging cases if you can navigate a couple of common pitfalls and target the most appropriate scenarios. The most common applications where HILIC can be used is with LC-MS/MS.

1. Polar analytes: This is most difficult in reversed phase, as their weak retention limits the scope of selectivity and method optimization parameters.
2. Trace amounts: For solubility reasons in acetonitrile, which is typically a necessary injection solvent.
3. Mass Spec Detection: Other, more familiar solutions for polar analytes are not mass spec compatible, whereas HILIC elution compositions are ideal with mass spec

All three of these points are necessary to make a HILIC separation mode among the primary method development routes, with bioanalytical and single residue pesticide methods topping the list. Common solutions for polar analytes are ion-exchange (typically non-volatile buffers not compatible with mass-spec), high aqueous reversed-phase on polar functionalized or phenyl-phases (poor retention and poor de-solvation within an electrospray ion-source), ion-pairing agents (not mass spec compatible or memory effect concerns), and basic mobile phase pH’s to neutralize weak base analytes (less columns are stable at basic pH’s and theoretically may suppress positive ion-mode sensitivity). Considering these common solutions have their own downsides, you might be wondering why HILIC isn’t among the top solutions for a broader range of polar analyte applications, so here are some of the common technical issues that arise:

1. Inject related solvent breakthrough—Acetonitrile, or acetone-rich injection solvents, are typically a requirement for the HILIC separation mode and notoriously more finicky towards the injection solvent than reversed phase.  This includes more injector maintenance and check valve nuances, that seem to be exacerbated in a HILIC separation.
2. Ionic interactions—These can be more difficult to control and are influenced by pH, buffer concentration, and temperature.  Often HILIC will rely on these interactions, and to be robustly reproducible. Small methodology adjustments may be necessary.
3. Non-traditional ion-suppression regions—Since we’re utilizing polar interaction for retention and selectivity, not all ion-suppression components will elute un-retained at the void. Resulting with the results coming out with analytes of interest in the heart of a gradient.  The potential unpredictability of this, if not characterized through thorough infusion studies, may be the biggest technical concern of a HILIC separation mode.
4. Depletion of the aqueous-enriched layer—HILIC may be distinctly different than normal phase, not just in its use of reversed phase solvents, but also mechanistically.  While the relatively less polar acetonitrile is the weak solvent (encouraging retention), with aqueous buffer being the strong solvent (encouraging elution), it still needs to be aqueous in the mobile phase—to keep the polar stationary phase saturated with moisture.  This is to facilitate the partitioning (retention) in and out of the stationary phase through the aqueous enriched layer.

Ionic and dipole interactions in the stationary phase are still significant for selectivity, but the primary retention mechanism is thought to revolve around this equilibrium partitioning.  Conceptually, it may be the reason why acetonitrile is the only solvent that works as the primary weak solvent, as it is more prone to facilitate such a partition.  Consider when mixing acetonitrile and water mobile phase pre-mixes, the solution gets cold (endothermic), and takes mechanical energy to get them to combine even though they are miscible.  Acetone is the only other solvent in a true HILIC separation, with others like methanol disrupting the partition (and relying on ionic interactions if successfully retaining polar compounds). These challenges can be overcome, and so I wanted to add one subtle benefit to HILIC for polar analytes over reversed-phase, and that is related to the sample matrix and mobile phase eluotropic strength.  In reversed phase, if we’re retaining polar analytes with a low eluotropic strength (high aqueous), anything mid-polar and non-polar in the sample will be retained on the column stationary phase.  These will either elute unpredictably in subsequent injections, saturate the column and limit lifetime, require a longer gradient and re-equilibration to elute, and/or lead to irreproducibility.

Questions about HILIC and if it is right for you? Chat with our Technical Experts nearly 24/7. They are happy to help!