HILIC (Hydrophilic Interaction Chromatography) is a mode of HPLC that is complementary to reversed phase (RP). The technique is used to separate substances that are difficult to retain in RP, including carbohydrates, sugars, amino acids, organic acids, nucleotides, and other polar hydrophilic neutral and charged molecules. The use of HILIC has grown rapidly during the last two decades and is currently employed within research & development, and in quality control in several sectors, including pharmaceutical, food & beverage, and biotechnology.
The Diduco team has decades of experience with HILIC analysis using a wide variety of stationary phases and can assist with anything from method development to troubleshooting.
HILIC is a mixed mode separation process where the retention mechanism includes components of hydrophilic partitioning, hydrogen bonding, dipole interactions, and for charged molecules also ion exchange. Columns for HILIC are designed to be very hydrophilic and may simply contain bare silica porous particles. However, most often the silica carries a bonded stationary phase such as amide, diol or amine, or the popular zwitterionic structures originally invented by Prof. Knut Irgum at Umeå University and founder of Diduco.
Eluent compositions for HILIC contain high amounts of organic solvents (<50%), typically acetonitrile, mixed with aqueous buffers or modifiers to control the pH and ionic strength. These conditions will establish an enriched layer of water at the stationary phase surface and is thus normally critical to obtain sufficient retention and separation. HILIC eluent strength is mainly controlled by the ratio of water to acetonitrile and since retention typically decrease rather fast with small increases in water content, gradients tend to be shallow and hardly end above more than 50% water.
Separations in HILIC mode are straightforward to combine with most detectors used for RP, including UV light absorption. However, HILIC is ideal for interfacing with detection techniques relying on evaporation of the eluent, such as mass spectrometry (MS), evaporative light scattering (ELSD), and charged aerosol (CAD). HPLC instrument design requirements for HILIC are identical to RP and it is therefore rather easy to switch between the two techniques by only changing column and eluent composition. Note, however, that the requirements on sample solvents and wash solvents typically will differ between the two modes. Some of the major challenges when developing HILIC analysis methods are to recognise the drastic differences in selectivity with different column chemistries and to realize the effects of retained matrix components which otherwise may result in low reproducibility.
To learn more, browse the application examples below or contact Diduco to discuss your specific analysis.