Technique: HILIC

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.

Columns and mechanism

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.

Mobile phase conditions

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.

Instrumentation

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.

Learn more

To learn more, browse the application examples below or contact Diduco to discuss your specific analysis.

Applications

HILIC

Separation of selected amino acids

Amino acids are the fundamental building blocks of all proteins and therefore essential constituents of many nutritional supplements and can also be present in pharmaceutical formulations. Due to the highly hydrophilic nature and zwitterionic charge at neutral pH of many amino acids and their structurally related degradation products, hydrophilic interaction chromatography is an attractive mode of separation that require no derivatization, while evaporative light scattering enables rather sensitive detection of these poorly light-absorbing compounds.

HILIC

Separation of simple sugars and carbohydrates

Simple carbohydrates are frequent ingredients in drinks, food, dietary supplements and pharmaceutical formulations. One approach to their analysis is hydrophilic interaction chromatography combined with evaporative light scattering detection, although this might be challenging due to the possibility of reducing sugars to mutarotate and thereby change their conformation into other anomers with different retention.

HILIC

Separation of nucleosides and nucleobases

Being one of the basic components of all cells as building blocks of DNA and RNA, there is a high and growing need for bioanalytical methods to determine nucleosides, nucleobases and related compounds in a variety of samples. Hydrophilic interaction chromatography is one of the most suitable approaches for this, due to the hydrophilicity of these compounds as demonstrated by their negative logP and logD values.

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I’ll be happy to answer any questions you might have regarding our products and services.

Tobias Jonsson
CEO, PhD in chemistry
Mobile: +46 705783490
E-mail: tobias@diduco.com