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The Basics of HILIC

Hydrophilic interaction chromatography (HILIC) is applicable to the separation of very polar, highly water soluble analytes which are often poorly retained (if at all) under reversed phase HPLC conditions.  Although reversed phase HPLC stationary phases, such as those denoted ‘aqueous’, have been developed to work with highly aqueous mobile phases in order to promote retention of these very polar species, often they still do not work.

HILIC is based on a mixed-mode retention mechanism and can be considered as a type of normal phase chromatography, employing a polar stationary phase (for example, silica or a polar bonded phase) and an aqueous-organic mobile phase in which the aqueous content is the ‘strong’ solvent.  Typically initial eluent composition will be 98:2 organic:water.

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Figure 1: Hydrophilic interaction chromatography (HILIC) mechanism.

HILIC can be used in certain situations where reversed phase chromatography fails or is not efficient:

  • Samples with limited solubility in water or highly aqueous mobile phases
  • Samples that contain very polar analytes which are not retained adequately in revered phase
  • Hydrophilic water-soluble analytes, which are intractable to reversed phase and/or ion exchange chromatography

HILIC presents the added advantage of using acetonitrile, which has low UV absorbance (for better detection sensitivity) and low viscosity (for high chromatographic efficiency).  The highly organic mobile phase also makes HILIC amenable to hyphenation with mass spectrometric detection (MS).

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Figure 2: Hyphenation of HILIC with MS.

Retention Mechanism

Analyte retention in HILIC can be thought of as the opposite of that used in reversed phase HPLC - in fact HILIC is sometimes referred to as ‘reversed-reversed phase HPLC’.  A polar stationary phase and an aqueous-organic mobile phase are employed.  In contrast to reversed phase HPLC the aqueous component of the mobile phase (water or buffer) serves as the strong solvent, whereas, the organic component (often acetonitrile) is the weak solvent.  Analytes are eluted in order of increasing hydrophilicity.

Due to the polar nature of the stationary phase, water molecules concentrate at the surface.  It has been proposed that in HILIC mode, retention occurs as the analyte partitions between the bulk mobile phase and the water layer which hydrates the hydrophilic stationary phase (Figure 3, mechanism 1).  Some authors have suggested that the HILIC retention mechanism is similar to that in normal phase chromatography, although it is still not fully understood.  As well as partitioning, it is proposed that other molecular interactions such as hydrogen bonding and electrostatic interactions play a part in the retention of analytes in HILIC, with analytes which are able to undergo these types of interaction interacting directly with the stationary phase (Figure 3, mechanism 2). 

Electrostatic interactions can be attractive or repulsive, depending on the charge state of both the analyte and stationary phase.  Retention times of analytes which undergo attractive electrostatic interactions (positively charged compounds and negatively charged stationary phase and vice versa) will be increased, whereas, repulsive interactions will result in decreased retention times.

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Figure 3: HILIC separation mechanisms.

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You may also like the following CHROMacademy webcast

Hydrophilic Interaction Chromatography (HILIC)

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Dr. Dawn Watson
 

This article was written by Dr. Dawn Watson.

Dawn received her PhD in synthetic inorganic chemistry from the University of Strathclyde, Glasgow. The focus of her PhD thesis was the synthesis and application of soft scorpionate ligands. As well as synthetic skills, this work relied on the use of a wide variety of analytical techniques, such as, NMR, mass spectrometry (MS), Raman spectroscopy, infrared spectroscopy (IR), UV-visible spectroscopy, electrochemistry, and thermogravimetric analysis.

Following her PhD she spent two years as a postdoctoral research fellow at Princeton University studying the reaction kinetics of small molecule oxidation by catalysts based on Cytochrome P450. In order to monitor these reactions stopped-flow kinetics, NMR, HPLC, GC-MS, and LC-MS techniques were utilized.

Prior to joining the Crawford Scientific and CHROMacademy technical team she worked for Gilson providing sales and support for the entire product range including, HPLC (both analytical and preparative), solid phase extraction, automated liquid handling, mass spec, pipettes, and laboratory consumables.

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