Hydrophilic interaction chromatography (HILIC) can be considered as normal phase chromatography using an aqueous-organic mobile phase, so it is sometimes referred to as “aqueous normal phase chromatography”. The hydrophilic interaction column is more polar than the reversed-phase column, and the highly polar water is used as the B solvent. Increasing the proportion of water reduces sample retention (as opposed to reversed-phase chromatography). More polar and ionized solutes are more retained in HIC. In most HILIC separations, the proportion of water in the mobile phase is between 3% and 40%. When the proportion of water is greater than 40%, retention is generally weak.

Stationary Phase

The stationary phases routinely used in HILIC chromatography include: pure silica column, amino column, amide column, zwitterion column, etc. Pure silica columns have the advantage of less stationary phase loss and are most popular when using CAD, ELSD and LC-MS detectors. Amino columns, used in HILIC chromatography, are particularly suitable for carbohydrate (saccharide) separation. The zwitterionic column no longer needs to use ion-pairing reagents, has strong hydrophilicity, and can effectively retain weakly or not retained strong polar ionic compounds and basic drug molecules in RP-LC.

The same is Silica, NH2, different from the column used in normal phase chromatography, the column designed for HILIC chromatography, can be used in water-organic mobile phase, in other words, the water resistance of HILIC chromatography to the stationary phase The requirements are higher, otherwise there will be problems such as high baseline noise and short column life due to the hydrolysis of the stationary phase. Therefore, columns used in normal phase chromatography may not necessarily be used in HILIC chromatography. Columns suitable for HILIC chromatography can be used in normal phase chromatography.

Advantages of HILIC

Polar solutes are preferentially retained in HIC, which means that many substances that are poorly retained in reversed-phase chromatography can be easily separated using HIC.

Hydrophilic interaction chromatography has several potential advantages:

Good peak shape for basic solutes

Helps to improve the sensitivity of mass spectrometry

Direct injection of samples dissolved in organic solvents

Can withstand higher flow rates due to the lower viscosity of the mobile phase

Frequently Asked Questions about HILIC

Compared to reversed-phase chromatography, the most common problem in hydrophilic interaction chromatography is probably suboptimal peak shape (leading or tailing). This reflects the fact that HIC as a new separation method has not been widely used as RPC. HIC may still use type A silica as packing materials. We also need to continue to improve HILIC columns and find ways to improve peak shape.

When encountering peak shape problems, the first thing to try should be to increase the buffer concentration in the mobile phase. Some samples require buffer concentrations as high as 100 mmol/L to obtain good peak shape. In general, the sample should be dissolved in the mobile phase, but sometimes in order to improve the peak shape, the proportion of the organic solvent used to dissolve the sample can be increased. If peak shape still does not improve, then consider replacing the column or adjusting the pH of the mobile phase. The mobile phase should always contain a certain amount of water, and the proportion of water should be at least 3%-5%.

Some bonded-phase columns experience column bleed when running mass detection. The usual solution is to use a silica column or another bonded-phase column. It has been pointed out in the literature that when using hydrophilic interaction chromatography, some sample components will irreversibly bind to the silica column. In this case, a bonded-phase HILIC column can be considered.

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