![[Readers Insight] Hydrophilic Interaction Chromatography (HILIC): A Nemesis to Polar Compounds](http://www.welch-us.com/cdn/shop/articles/1080_92fb8a9d-1864-4b07-aa94-a5aa5ffc7a06_750x.jpg?v=1766974581)
[Readers Insight] Hydrophilic Interaction Chromatography (HILIC): A Nemesis to Polar Compounds
Author: Sepuxianyun
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Introduction
In liquid chromatography practices, have you ever faced analytes that are difficult to retain on C18 columns? In previous articles, we discussed about various solutions that have limitations: adding chaotropic agents to increase retention, which only works with certain analytes; adding ion-pair reagents, which may irreversibly modify the stationary phase; and using mixed-mode chromatography, yet it is often plagued by the reproducibility issues.
If you have ever been troubled by such analytes, hydrophilic interaction chromatography (HILIC) can be a good choice for you.
What is HILIC?
Hydrophilic interaction chromatography is a chromatographic mode orthogonal to both reversed-phase and normal-phase chromatography. The stationary phase in HILIC is polar, while the mobile phase uses what is used in reversed-phase chromatography.
Retention in HILIC mode does not arise from a single dominant force (e.g., hydrophobic interactions in reversed-phase chromatography), but a combination of mechanisms: liquid-liquid partitioning, adsorption, ion exchange, and hydrophilic interactions. Therefore, both retention and peak shape for polar compounds are dependent on mobile phase composition (pH, organic solvent, etc.) and the chemistry of stationary phase.
Similar to normal-phase chromatography, in HILIC mode, acetonitrile functions as a weak eluent and water as a strong eluent; to emphasize the distinction, HILIC is sometimes described as "reversed-reversed phase".
During separation, a layer of polar solvent from the mobile phase forms on the surface of the polar stationary phase. Polar analytes tend to partition into this polar layer and are thereby retained. As a consequence, the proportion of polar solvent in a HILIC mobile phase typically ranges from 3% to 40% (sometimes up to 50%). If below 3%, a stable polar layer cannot form on the stationary phase surface; if above 40%, the equilibrium would be disrupted and become a conventional reversed-phase mode.
How to Develop Methods on HILIC Mode
In this section, we introduce the method development strategies on HILIC mode.
Classification of HILIC Stationary Phases
Bare silica, diol phases, etc.: These stationary phases can acquire a net negative charge depending on pH and thus exhibit cation-exchange character, making them suitable for retaining basic compounds. Conversely, they tend to repel anions, which can shorten retention of acidic species.
Amino and amide phases, etc.: In contrast to the foregoing, these phases can be positively charged by pH adjustment and thus exhibit anion-exchange character (note that amide groups are weakly basic and may not ionize fully, but amide-bonded phases typically have longer lifetimes than amino phases). Therefore, these phases are well suited for retaining acidic compounds. Because complete end-capping of silanols is rarely achievable, residual silanol ionization can still impart negative character to the surface, making the overall interaction mechanisms more complex.
Zwitterionic phases (e.g., sulfobetaine-based phases): These phases incorporate both anionic and cationic functional groups. When together with residual silanol effects, they create even more complex mechanisms and greater selectivity diversity.
Mobile Phase Selection
1. Elution strength of solvents:
The relative elution strength in HILIC follows the order: Acetone < Acetonitrile < Isopropanol < Ethanol < Methanol < Water. Acetone and acetonitrile are considered weak eluents, while alcohols and water are considered strong.
Acetone is seldom used due to its high UV absorption cutoff and toxicity. Acetonitrile-water system remains the most commonly employed.
In HILIC, the starting proportion of acetonitrile is generally greater than 70%, with the aqueous proportion increasing progressively (opposite to what is used in reversed-phase systems). If the analyte's retention remains insufficient, add a small amount of alcohol to the aqueous phase to modulate retention.
2. Choice of buffers and additives:
Given the high initial acetonitrile content, volatile ammonium salts such as ammonium acetate and ammonium formate are preferred due to their compatibility with organic-rich mobile phases. When phosphate buffers are used, care must be taken to avoid precipitation. Addition of counter-ions can improve both retention and peak shape.
3. Concentration of additives:
At low buffer concentrations, ion-exchange interactions dominate. As buffer concentration increases, ion-exchange effects are progressively suppressed and liquid–liquid partitioning becomes the primary retention mechanism eventually.
For basic analytes, pairing with anions can increase apparent hydrophobicity and thereby reduce retention. Conversely, for acidic analytes, increasing salt concentration can suppress silanol ionization and its associated electrostatic repulsion, resulting in increased retention.
4. pH selection:
pH should be judged by the acid–base properties of the analytes. Generally, a pH condition that promotes an analyte in its ionized state tends to favor retention in HILIC.
For example, on an amide column, raising the pH causes acidic compounds to become negatively charged while simultaneously ionize surface silanols. This makes the polarity of the stationary phase to increase, attracting the polar solvents in the mobile phase to adsorb on the surface of the stationary phase, thickening the polar layer, and making it easier for analytes to partition into that layer, thereby increasing retention. However, the concurrent increase in silanol ionization also introduces anionic repulsion. This example highlights the intricate nature of HILIC selectivity.
Further Practical Considerations
As with other chromatography modes, HILIC entails certain practical limitations. Below are two technical challenges that must be managed:
- Equilibration time: HILIC systems generally require longer equilibration times than reversed-phase systems. This requirement is particularly evident in gradient elution, where post-run re-equilibration should be extended to ensure column stability.
- Solvent effects: HILIC is most often used to retain highly polar compounds, which are notably water-soluble. If samples are prepared in pure water as the injection solvent, significant solvent effects can arise because the mobile phase is typically organic-rich. Therefore, careful selection of the sample diluent is important. If dissolution in pure water is unavoidable, keeping sample concentration high and injection volume low will help minimize solvent effects.