Why HILIC is what your polar compounds need for purification

Sometimes, we tend to get very familiar with certain methods and try to adapt them to fit to all our applications. But just because you can cut with a knife well, does not mean it is the best way to saw wood. Let me introduce you to a chainsaw instead. Prepare to meet a new separation method for the blog, HILIC, and to see what it can do for the purification of your polar compounds!

Last Saturday I went to a Chinese restaurant with a few study mates of mine. As I am accustomed to, I ate my noodle dish with chopsticks, as did my Chinese friend, whereas my Caucasian and Indian friends opted for forks. This got us started on a lively discussion on different cutlery used by different cultures. I already knew that using chopsticks can be beneficial: you eat slower, take smaller bites, practice some coordination skills. My Indian friend jumped in and said eating with hands also has advantages. Again, you eat slower, end up doing some unintentional hand exercises, form a strong connection to your food and may expose your gut to some friendly bacteria.

Later, when giving our discussion a second thought, I had to admit to myself that the forks of this blog are normal and reversed-phase chromatography. But even if you are comfortable with your fork, that does not mean you should never try chopsticks or your hands once in a while.

Let me take this opportunity to introduce you to then to an alternative method for purification, mainly hydrophilic interaction chromatography (HILIC).

Just like you can’t eat broth with chopsticks, HILIC is also not suitable for just any purification application you can think of. But just like chopsticks are ideal for eating noodles and sticky rice,

HILIC is ideal for separation of highly polar compounds.

Highly-polar compounds cannot be usually separated with our typical forks, namely normal phase (NP) or reversed phase (RP) chromatography. In normal phase conditions, the compounds are too sticky to the polar stationary phase, so elution takes too long. Polar compounds are characterized by good solubility in aqueous mobile phases and are not compatible with typical NP solvents. With RP, the highly polar compounds do not interact at all with the non-polar stationary phase and end up eluting with the solvent front. This prevents separation from occurring at all.

So let me introduce you to some chopsticks, HILIC! This type of separation takes place on a polar stationary phase, but under reversed-phase solvent conditions using water. In this situation, the mobile phase forms a water-rich layer on the surface of the polar stationary phase compared to the water-deficient mobile phase. The gradient elution starts with a low-polarity organic solvent and elutes polar analytes by increasing the polar water content

HILIC; chromatography

In HILIC, analytes are polar as in ion chromatography, the stationary phase is highly polar as in normal phase chromatography and the mobile phase consists of a water mixture as in reversed phase chromatography. However, the gradient elution begins at high organic solvent concentration and water is the strongest eluent used in your chromatography system.

Although the mixed-mode mechanism of HILIC is still under study today, the main retention mechanism is believed to be portioning of the analyte between the organic-rich mobile phase and the water-enriched later. Other interactions, such as hydrogen bonds, electrostatic interactions and dipole-dipole interactions contribute to HILIC separations:

HILIC; chromatography

If you want to try HILIC yourself, I recommend acetonitrile due to its good miscibility with water, good HILIC retention and low viscosity. However, many other polar organic solvents can be used as well. Relative solvent strengths in HILIC are approximately:

acetone < propanol < acetonitrile < ethanol < dioxane < DMF ~ methanol < water

In terms of your chromatography surface, any polar phase can be used for HILIC separations. For example:

Type of phaseExample of phaseComposition of phaseSuitable for separation ofRetention mechanism
NeutralDiol, amidePolar functional groups, such as amides, aspartamide, diol, cross-linked diol, cyano, and cyclodextrinHydrophilic compounds and complex mixtures of neutral, anionic and cationic analytesHydrophilic interactions; no electrostatic interactions
ChargedPlain silica; aminopropyl phasesAnionic or cationic functional groupsNeutral polar compounds

The primary amino group of aminopropyl phases is positively charged; therefore, it exhibits high affinity for anionic acid compounds.

Silica surfaces contain acidic surface silanol groups that have a pKa of 3.5, this means that above pH values equal to the pKa these groups will become ionized, allowing the silica stationary phase to work as a cation exchanger that can interact with and retain positively charged basic analytes
Strong electrostatic interactions from anionic or cationic functional groups
ZwitterionicAmino acids, Sulfamic acidFunctional groups that are permanently positively charged (ammonium) and negatively charged (sulfonic acid)Due to their hydrophilic nature and weak ion-exchange properties, these phases are suitable for separation of neutral, acidic, and basic analytes, polar and hydrophilic compounds, and inorganic ionsWeak electrostatic interactions

A good rule for selecting the ideal HILIC phase is to keep in mind that neutral analytes typically less hydrophilic than charges ones and high hydrophilic phases are requited for their retention (such as zwitterionic and amide phases). On the other hand, charged compounds are retained too strongly on charged columns due to electrostatic attractions, so neutral and zwitterionic phases offer better results.

I just want to acknowledge the following sites where I drew in lots of information for this post: ACE HILIC Method Development Kit | ACE (ace-hplc.com); Making HILIC Work for You—Column Selection (chromatographyonline.com) and Separation Science: HILIC Stationary Phases (sepscience.com).

With this, I’m wrapping up another post on celebrating diversity in the lab! We’ve touched on this topic many times, from the various methods available for protein concentration, to matching vapor ducts to rotary evaporators. This time, I hope I’ve helped you take a good bite out of the hassle of purifying polar compounds. By the way, I’ve got a great example of using HILIC for purification of vitamins in a webinar! Have you ever tried it yourself? Let me know for what applications you have used HILIC in the comments below!

Till next time,

The Signature of Bart Denoulet at Bart's Blog