Hydrophilic interaction chromatography (HILIC) is a variation of normal-phase LC. It has a similar elution profile to normal-phase chemistry in that analytes elute in the order of increasing polarity. However, the difference is that normal-phase methods use an entirely organic solvent mixture, while HILIC employs an aqueous-organic solvent blend.

HILIC HPLC columns are typically packed with polar material such as silica, cyano, amino and diol stationary phases. Analyte retention is governed by the underlying chemistry of the stationary phase, as well as by a liquid-liquid partitioning mechanism. The water from the aqueous-organic solvent mixture forms a thin layer on the surface of the stationary phase in which analytes are retained.

Commercially available HILIC columns have become rather plentiful and are available in different particle sizes and chemistries. They are quite useful for analyzing ionic and highly polar analytes, such as small polar molecules, glycans, simple sugars and peptides. They are also used for analyzing amino acids, nucleotides, oligonucleotides, alkaloids, phospholipids, carbohydrates, drug metabolites, and other small polar or ionizable compounds. HILIC is an orthogonal technique to reverse-phase chemistry and is often exploited in separating difficult samples. Chromatographers will generally switch to a HILIC column instead of drastically altering reverse-phase conditions or utilizing different reverse-phase columns to achieve the desired separation.

Merck KGaA, a leading vendor for HILIC columns, offers the SeQuant ZIC HPLC columns, which are based on a stationary phase with a covalently bonded, highly polar zwitterionic functional group. The company recently released its cHILIC HPLC column, which provides complementary selectivity to optimize separations of polar hydrophilic compounds. The columns carry a densely bonded phosphorylcholine functional group that features more accessible positive charges, enabling selectivity tuning by altering the interaction with anionic and cationic molecular moieties.

Nacalai Tesque recently introduced the COSMOSIL 2.5HILIC column that employs a 2.5 µm particle for UHPLC applications. The column features a triazole-bonded stationary phase and provides alternative selectivity for other HILIC columns. Thermo Fisher Scientific recently added the urea and 150-amide HILIC columns to its Accucore brand. The urea HILIC column is used for low ion exchange activity, while the 150-amide column is designed to separate hydrophilic biomolecules and glycans. Sigma-Aldrich Supelco recently released three HILIC phases for its Ascentis Express Fused-Core HPLC column line, consisting of the Express F5, Express ES-CN and Express OH5.

The market for HILIC HPLC columns was estimated at around $40 million in 2011 and is expected to expand rapidly, particularly with the growing number of manufacturers that are integrating HILIC chemistry.

HILIC Columns at a Glance:

Leading Suppliers

• Merck KGaA

• Waters

• Phenomenex

Largest Markets

• Academia

• Biotechnology

• Pharmaceuticals

Instrument Cost

• $500,000–$800