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How to Choose a Stationary Phase for HILIC

HILIC requires a hydrophilic stationary phase to adsorb a water layer in order to allow the analyte partitioning process to take place (Figure 1). 

The hydrophilicity of the column packing material will influence the thickness to the water layer which will directly impact analyte retention. 

Other interactions between the analyte and stationary phase, such as, electrostatic and hydrogen bond interactions can also be manipulated by selecting stationary phase chemistries which contain polar or charged functional groups; these will also alter analyte retention and selectivity (Figure 2). 

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Figure 1: Hydrophilic interaction chromatography (HILIC) mechanism.

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Figure 2: Analyte interaction with HILIC stationary phase.

Modern HILIC stationary phases are either bare silica or polar chemically bonded phases which contain ionic (or ionizable) ligands bonded to the silica surface (Figure 3).  The use of ligands capable of undergoing electrostatic interactions can add an extra dimension to the separation when analyzing ionizable compounds.
Polar chemically bonded HILIC stationary phases can be divided into three main categories:

  1. Neutral - polar surface with no electrostatic interactions

  2. Charged - strong electrostatic interaction.  The stationary phase contains anionic or cationic functional groups

  3. Zwitterionic - weak electrostatic interaction.  The stationary phase contains both cationic and anionic functional groups
HILIC stationary phase classifications

Figure 3: HILIC stationary phase classifications.


Bare silica phases can be used for the separation of small polar compounds, such as, carbohydrates, peptides, and proteins.  Silica surfaces contain acidic surface silanol groups which have a pKa of 3.5, this means that above pH values equal to the pKa these groups will become ionized allowing the silica stationary phase to work as a cation exchanger which can interact with and retain positively charged basic analytes (Figure 4).

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Figure 4: The effect of mobile phase pH on the extent of ionization of surface silanol species.
 

Neutral HILIC phases predominantly comprise polar functional groups, for example, amides, aspartamide, diol, cross-linked diol, cyano and cyclodextrin.  These groups are in their neutral form in the pH ranges usually employed for HILIC mobile phases (pH 3-8).  The retention mechanism with these types of phases is via hydrophilic interactions.  Neutral HILIC phases have been applied to the separation of monosaccharides, peptides, amino acids, and small polar molecules (Figure 5).

The most common functional group used in charged phases is an aminopropyl.  The primary amino group is positively charged, therefore, it exhibits high affinity for anionic acid compounds, i.e. amino acids, peptides, antibiotics, carboxylic acids, and nucleosides.  The retention mechanism is based on anion-exchange.  These stationary phases are also highly hydrophilic; therefore, they are amenable to the retention and separation of neutral polar compounds (Figure 5).

Zwitterionic HILIC phases are possibly the most versatile, and are often the first choice of column during method development.  Zwitterionic phases contain functional groups which are permanently positively and negatively charged - for example, quaternary ammonium (positive) and sulfonic acid (negative) functional groups.  The hydrophilic nature and weak ion exchange properties allow these phases to be used to separate neutral, acidic and basic analytes, polar and hydrophilic compounds and inorganic ions (Figure 5).

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Figure 5: HILIC stationary phases.

When selecting an appropriate stationary phase the chemical properties of both the column and analytes should be considered.  HILIC is designed for use with analytes which have a log D < 0, (the more negative the log D value for an analyte, the greater the degree of stationary phase polarity required to retain it).  Log P, pKa, and solubility also need to be considered when designing methods (information on chemical properties of compounds can be found at www.chemicalize.org). 

Figure 6 shows a flow chart for selecting an appropriate HILIC stationary phase.  Phases which have little ion-exchange activity are suitable for the separation of neutral zwitterionic, and acid base mixtures.  Phases which have considerable ion exchange activity are suitable for analyzing acids (anion exchange phases) and bases (cation exchange phases).  A zwitterionic stationary phase is often a very good starting point.  If there is not enough information about the analytes screening several stationary phases may be required.

HILIC stationary phase selection

Figure 6: HILIC stationary phase selection.


Column Geometry

For standard HILIC applications use a 100 or 150 mm column with 3-5 µm particles and a 4.6 mm i.d. when using UV detection.  For applications which use MS or CAD detection smaller columns e.g. 100 x 2.1 mm or 50 x 2.1 mm, 3-5 µm are optimum (it should be noted that reducing column length will decrease the number of analytes which can be separated due to the decrease in efficiency).  Using sub-2 µm fully porous or superficially porous materials can increase efficiency and resolution.

See the following CHROMacademy pages for further information.

Hydrophilic Interaction Chromatography (HILIC) Webcast »

The Basics of HILIC »

HILIC - Hydrophilic Interaction Liquid Chromatography »

 
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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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