Hydrophilic interaction chromatography

See also: Chromatography

Hydrophilic interaction chromatography (or hydrophilic interaction liquid chromatography, HILIC)^[1] is a type of liquid chromatography that uses a hydrophilic stationary phase and a high-organic mobile phase for the separation of analytes by polarity.^[2] While it is not as popular as some other types of liquid chromatography, the number of scientific publications using HILIC have greatly increased since the early 2000s.^[3] HILIC is similar to reverse phase chromatography in its mobile phase composition, and also to normal phase chromatography, with its polar stationary phase. ^[4][5] It also has overlap with ion exchange chromatography. ^[4] Sometimes, HILIC is considered to be a hybrid of these techniques. ^[6]

HILIC was named in 1990 by Andrew Alpert, who described it as a type of liquid-liquid partition chromatography.^[7] He suggested that analytes elute in order of increasing polarity,^[7] a conclusion supported by review and re-evaluation of published data.^[8] The mechanism for HILIC is still not entirely understood, but it is thought to rely on analytes partitioning between the organic-rich mobile phase and a water-enriched layer that forms of the surface of the polar stationary phase, in a liquid-liquid extraction system.^[7][5] More polar analytes will have stronger interactions with the water-enriched layer and with the column itself, therefore being retained on the column for longer. ^[3]

HILIC Partition Technique Useful Range

Stationary Phase

One of the key factors influencing HILIC separations is the chemical nature of the stationary phase that is packed into the column.^[2][9] Stationary phases on HILIC columns not only provide physical support for the water layer which analytes separate into, but also interact with the analytes through hydrogen bonding and electrostatic interactions, affecting their retention and therefore the mechanism of separation. ^[3][5]

Typical HILIC stationary phases are polar, made of classical bare silica or silica gels modified with various polar groups.^[2][10] Some commonly used stationary phases include bare silica, or silica chemically bonded to amino-,^[4] amide-,^[11] cyano-, or diol- groups.^[9][10] Ion exchanger groups, both cationic ^{[citation needed]}and anionic ^{[citation needed]}, as well as zwitterionic^[3][12] groups are also commonly used. ^[10]

While most HILIC phases are polar, there have also been exceptions where non-polar bonded silicas are used with extremely high organic solvent composition. In this case, interactions are affected by exposed patches of silica in between the bonded ligands on the support. ^[13]

Mobile phase

The mobile phase, or the liquid phase that runs across the column during separation, for HILIC is typically composed of a high amount of water-miscible, polar organic solvent and a low amount of water.^[6] Typically, acetonitrile ("MeCN", also designated as "ACN") is used for the organic solvent, though other aprotic water-miscible solvents, such as alcohols at higher concentration, tetrahydrofuran, or dioxane, can also be used.^[3]

As with other methods of chromatography, the mobile phase can be delivered isocratically or with a gradient starting at high-organic progressing towards increasing aqueous content.^[3] If using a mobile phase gradient, the mobile phase will progressively increases in polar-aqueous content, causing increasingly polar analytes to be eluted. ^{[7] [14]}

All ions partition into the stationary phase to some degree, so an occasional "wash" with water is required to ensure a reproducible stationary phase.^{[citation needed]}

Additives

Mobile phase pH and electrostatic interactions, as well as analyte polarity, are regulated by the addition of ionic additives, commonly ammonium acetate and ammonium formate, to the mobile phase. ^[3] These additives improve separation efficiency, including more symmetric peaks, less peak tailing, and better recovery from the stationary phase. ^[3][15]

When considering additive addition, compatibility with detectors is important to consider. HILIC is often used with a mass spectrometry (MS), which cannot handle non-volatile salts like sodium perchlorate, which may suppress ion signal in the instrument, though it may increase mobile phase polarity and assist with elution in HILIC.^[3][16]

Applications

The HILIC mode of separation is used extensively for separation of some biomolecules, organic and some inorganic molecules^[17] by differences in polarity. Its utility has increased due to the simplified sample preparation for biological samples, when analyzing for metabolites, since the metabolic process generally results in the addition of polar groups to enhance elimination from the cellular tissue. This separation technique is also particularly suitable for glycosylation analysis^[18] and quality assurance of glycoproteins and glycoforms in biologic medical products.^[19] For the detection of polar compounds with the use of electrospray-ionization mass spectrometry as a chromatographic detector, HILIC can offer a ten fold increase in sensitivity over reversed-phase chromatography^[17] because the organic solvent is much more volatile.

Choice of pH

With surface chemistries that are weakly ionic, the choice of pH can affect the ionic nature of the column chemistry. Properly adjusted, the pH can be set to reduce the selectivity toward functional groups with the same charge as the column, or enhance it for oppositely charged functional groups. Similarly, the choice of pH affects the polarity of the solutes. However, for column surface chemistries that are strongly ionic, and thus resistant to pH values in the mid-range of the pH scale (pH 3.5–8.5), these separations will be reflective of the polarity of the analytes alone, and thus might be easier to understand when doing methods development.

ERLIC

In 2008, Alpert coined the term, ERLIC^[20] (electrostatic repulsion hydrophilic interaction chromatography), for HILIC separations where an ionic column surface chemistry is used to repel a common ionic polar group on an analyte or within a set of analytes, to facilitate separation by the remaining polar groups. Electrostatic effects have an order of magnitude stronger chemical potential than neutral polar effects. This allows one to minimize the influence of a common, ionic group within a set of analyte molecules; or to reduce the degree of retention from these more polar functional groups, even enabling isocratic separations in lieu of a gradient in some situations. His subsequent publication further described orientation effects^[21] which others have also called ion-pair normal phase^[22] or e-HILIC, reflecting retention mechanisms sensitive to a particular ionic portion of the analyte, either attractive or repulsive. ERLIC (eHILIC) separations need not be isocratic, but the net effect is the reduction of the attraction of a particularly strong polar group, which then requires less strong elution conditions, and the enhanced interaction of the remaining polar (opposite charged ionic, or non-ionic) functional groups of the analyte(s). Based on the ERLIC column invented by Andrew Alpert, a new peptide mapping methodology was developed with unique properties of separation of asparagine deamidation and isomerization. This unique properties would be very beneficial for future mass spectrometry based multi-attributes monitoring in biologics quality control.^[23]

Cationic eHILIC

For example, one could use a cation exchange (negatively charged) surface chemistry for ERLIC separations to reduce the influence on retention of anionic (negatively charged) groups (the phosphates of nucleotides or of phosphonyl antibiotic mixtures; or sialic acid groups of modified carbohydrates) to now allow separation based more on the basic and/or neutral functional groups of these molecules. Modifying the polarity of a weakly ionic group (e.g. carboxyl) on the surface is easily accomplished by adjusting the pH to be within two pH units of that group's pKa. For strongly ionic functional groups of the surface (i.e. sulfates or phosphates) one could instead use a lower amount of buffer so the residual charge is not completely ion paired. An example of this would be the use of a 12.5mM (rather than the recommended >20mM buffer), pH 9.2 mobile phase on a polymeric, zwitterionic, betaine-sulfonate surface to separate phosphonyl antibiotic mixtures (each containing a phosphate group). This enhances the influence of the column's sulfonic acid functional groups of its surface chemistry over its, slightly diminished (by pH), quaternary amine. Commensurate with this, these analytes will show a reduced retention on the column eluting earlier, and in higher amounts of organic solvent, than if a neutral polar HILIC surface were used. This also increases their detection sensitivity by negative ion mass spectrometry.

Anionic eHILIC

By analogy to the above, one can use an anion exchange (positively charged) column surface chemistry to reduce the influence on retention of cationic (positively charged) functional groups for a set of analytes, such as when selectively isolating phosphorylated peptides or sulfated polysaccharide molecules. Use of a pH between 1 and 2 pH units will reduce the polarity of two of the three ionizable oxygens of the phosphate group, and thus will allow easy desorption from the (oppositely charged) surface chemistry. It will also reduce the influence of negatively charged carboxyls in the analytes, since they will be protonated at this low a pH value, and thus contribute less overall polarity to the molecule. Any common, positively charged amino groups will be repelled from the column surface chemistry and thus these conditions enhance the role of the phosphate's polarity (as well as other neutral polar groups) in the separation.