HILIC is a chromatographic technique for the retention and separation of problematic polar compounds. In HILIC mode, water acts as the strongest solvent to effectively elute analytes off the column, and the organic solvent acts as the weak solvent. This is opposite to reversed phase chromatography.
HILIC phases retain polar, hydrophilic compounds that are not retained on standard reversed phase columns. For mass spectrometry (MS) applications with very polar compounds, HILIC (using an organic-rich mobile phase) provides a ten to twenty-fold sensitivity improvement. HILIC mode also eliminates the need for evaporation and reconstitution of samples dissolved in a highly organic solvent, allowing sample analysis throughput to be greatly increased.
Our HILIC columns are ideal for analyzing highly polar molecules such as urea, biurea, choline and butyrobetaine, tromethamine, ascorbic acid and related compounds, folic acid and its metabolites, nicotine and its metabolites, saponins, aminoglycoside antibiotics, glucosinolates, ionic liquids, organophosphonate nerve agent metabolites, etc.
This graph shows the relative retention of acids, neutrals, and bases for different Thermo Scientific HILIC (U)HPLC columns.
For basic compounds:
For neutral or slightly acidic compounds:
For acidic compounds:
* Hypersil GOLD Amino, Cyano, and Silica columns are all shipped in normal phase solvents. A solvent switch must be performed in order to use these columns in HILIC mode.
HILIC is a chromatographic technique that requires good method development in order to successfully separate difficult samples. When a HILIC protocol is set up properly, separations are highly effective and consistent. There are several important factors to consider when developing your HILIC method:
The mobile phase for a HILIC separation should consist of a low percentage of water and a high percentage of an organic solvent such as acetonitrile. It is essential that the mobile phase always contains at least 5% water as that water merges with the stationary phase and increases the stationary phase’s affinity to the analyte.
When running HILIC separations, it is important to use an organic solvent without alcohol functional groups because alcohol functional groups will disrupt the water layer required around the stationary phase, causing inconsistent results. Even small volumes of alcohols such as Methanol used during sample preparation or in the injection solvent can affect the chromatography. Acetonitrile is the most commonly used organic solvent without an alcohol functional group, making it ideal for use in HILIC.
HILIC is sensitive to minor pH changes. When setting up a HILIC method, preparing fresh reagents daily and routinely checking the pH of your samples can be imperative to your success.
HILIC applications require longer time for re-equilibration between samples due to the high sensitivity of the HILIC mobile phase system. Shorter equilibration times may cause irreproducible data or challenges with the robustness.
To learn more about how to set up a robust HILIC method, read our comprehensive guide to HILIC separations.
If you find it challenging to work with a HILIC method, know that there are a number of reversed phase stationary phases that are also capable of retaining relatively polar analytes. Before starting the development of a new HILIC method, explore whether one of these reversed phase columns might be an option for your application. Reversed phase columns are easier to maintain, and these columns can be run in 100% water.
Use Hypersil GOLD fully porous columns for simple samples, Accucore solid core columns for high-resolution separations, and Acclaim fully porous columns for complicated samples.
Learn more about reversed phase column options ›
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