Hydrophilic interaction liquid chromatography (HILIC)—a powerful separation technique
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Hydrophilic interaction liquid chromatography (HILIC) provides an alternative approach to effectively separate small polar compounds on polar stationary phases. The purpose of this work was to review the options for the characterization of HILIC stationary phases and their applications for separations of polar compounds in complex matrices. The characteristics of the hydrophilic stationary phase may affect and in some cases limit the choices of mobile phase composition, ion strength or buffer pH value available, since mechanisms other than hydrophilic partitioning could potentially occur. Enhancing our understanding of retention behavior in HILIC increases the scope of possible applications of liquid chromatography. One interesting option may also be to use HILIC in orthogonal and/or two-dimensional separations. Bioapplications of HILIC systems are also presented.
KeywordsHydrophilic interaction liquid chromatography Stationary phase Separation mechanism Bioapplication
A theoretical description of analyte retention in high-performance liquid chromatography (HPLC) has been the subject of various publications. There are several ways to model the separation mechanism: partition, adsorption, ion exchange, and size exclusion. This mechanism is based on specific and nonspecific interactions [1, 2, 3, 4]. Nevertheless, there is currently no detailed quantitative retention model that would allow the chromatographic parameters for individual analytes separated under given conditions to be accurately predicted.
Normal or reversed-phase liquid chromatography can be used for analysis. In normal phase liquid chromatography (NP-LC), the stationary phase is more polar than the mobile phase. The retention increases as the polarity of the mobile phase decreases, and thus polar analytes are more strongly retained than nonpolar ones. The opposite situation occurs in reversed-phase liquid chromatography (RP-LC) . NP-LC has been widely used to separate various compounds, from nonpolar to highly polar compounds (note that chromatography was first introduced as a method used in separation science). Although RP-LC systems were previously commonly used by scientists, NP-LC methods are currently undergoing a renaissance.
HILIC has many specific advantages over conventional NP-LC and RP-LC. For example, it is suitable for analyzing compounds in complex systems that always elute near the void in reserved-phase chromatography. Polar samples always show good solubility in the aqueous mobile phase used in HILIC, which overcomes the drawbacks of the poor solubility often encountered in NP-LC. Expensive ion pair reagents are not required in HILIC, and it can be conveniently coupled to mass spectrometry (MS), especially in the electrospray ionization (ESI) mode. In contrast to RP-LC, gradient elution HILIC begins with a low-polarity organic solvent and elutes polar analytes by increasing the polar aqueous content . A desirable mobile phase would contain high organic content for better sensitivity and also show good on-column retention for polar ionic compounds. Hydrophilic interaction liquid chromatography has established itself as the separation mode of choice for uncharged highly hydrophilic and amphiphilic compounds that are too polar to be well retained in RP-LC but have insufficient charge to allow effective electrostatic retention in ion-exchange chromatography. HILIC separation is currently attracting a lot of interest since it solves many previously difficult separation problems, such as the separation of small organic acids, basic drugs, and many other neutral and charged substances. It has been successfully applied to the analysis of carbohydrates [14, 15], peptides [8, 16, 17] and polar pharmaceuticals [11, 18], etc.
This paper covers fundamental developments in hydrophilic interaction liquid chromatography. The objective of the present work is to review options for the characterization of HILIC stationary phases and their applications to separations of polar compounds in complex matrices. Gaining a thorough understanding of retention behavior in HILIC enhances the scope of applications of liquid chromatography. The separation mechanism can depend on many factors, such as the physicochemical properties of the stationary phase and hydroorganic mobile phase, and the structures of the samples investigated. Precisely defining which mechanism prevails is currently a difficult and complicated task. This phenomenon is still waiting for theoretical elucidation.
Stationary phases for the HILIC mode
Any polar chromatographic surface can be used for HILIC separations. Typical HILIC stationary phases consist of classical bare silica or silica gels modified with many polar functional groups. Polymer-based stationary phases can also be used.
The first generation of HILIC mode separations started in 1975. Linden et al.  separated carbohydrates by an amino-silica phase, Bondapak (Waters, Milford, MA, USA) in a mixture of acetonotrile and water (75:25 v/v). The next generation of stationary phases for HILIC used DIOL- and amide-silica. The DIOL-silica column has mainly been used for the separation of proteins [20, 21]. According to Tosoh, producer of TSKgel Amide-80, amide-silica columns have been available since at least 1985. This particular phase is described as consisting of nonionic carbamoyl groups that are chemically bonded to the silica gel, but it is commonly known as an amide-bonded silica. After Yoshida  applied these phases to the separation of peptides, the amide-silica phase soon found common usage in HILIC. Chemically bonded stationary phases with specific structural properties have been prepared by Buszewski et al. [23, 24, 25]. One of them contains aminopropyl ligands bonded to silica (SG–NH2); others are an alkylamide packing phase (SG-AP) and a mixed phase (SG-MIX) containing different types of ligands (–NH2, –CN, –Ph, –C8, –C18) bonded to the support.
Unmodified bare silica gel has some advantages for HILIC, in contrast to chemically bonded stationary phases. Type A silica gels, prepared by precipitation from the solutions of silicates, are acidic because they are polluted with certain metals that activate surface silanol groups and form complexes with some chelating solutes, causing strong retention or asymmetric peaks. Type B silica gels are formed by the aggregation of silica sols in air, contain very low amounts of metals, and are more stable at intermediate and higher pH values (up to at least pH 9) than xerogel-type materials. They generally provide better separations, especially for basic samples, because they are highly purified, less acidic “sol-gel” spherical silica particles . At higher pH values, silanol groups are ionized and cation exchange plays a important role in retention, especially for positively charged basic compounds. Suppressing silanol ionization through the addition of TFA may promote the ion-pairing mechanism. Similar effects have also been observed in HILIC on monolithic silica gel columns, which offer higher permeability than the particle-packed HILIC columns . Silica gel type C with a hydrosilated surface populated with nonpolar silicon hydride Si–H groups instead of silanol groups may have up to 95% of its original silanols removed, making it less polar than silica gels with higher populations of silanol groups . It can be used to separate acids or bases in the HILIC mode in buffered mobile phases containing more than 50–70% organic solvent (acetonitrile).
DIOL, amino, amide and other bonded phases used in HILIC are usually prepared by chemically modifying the silica gel surface, like the C18 phases used for RP-LC [8, 60]. Chemically bonded DIOL phases demonstrate high polarity and hydrogen bonding properties, and do not contain ionizable groups other than unreacted residual silanols, meaning that they are appropriate for the HILIC mode . Bonded amino-silica columns are relatively often used in the HILIC mode. While basic analytes are in general strongly retained on silica gel by hydrogen bonding and ion-exchange interactions with silanol groups, acidic compounds show increased affinities to amino-silica columns, which can sometimes even lead to irreversible adsorption . Chemically bonded phases with other functionalities, such as polyethylene glycol or alkyls with embedded amide or carbamate groups, are generally proposed for RP applications in water-rich mobile phases. On the other hand, when the percentage of organic solvent is high, the retention of many compounds increases with increasing concentration of acetonitrile, showing typical NP behavior [6, 47, 62]. Cyclodextrin-silica stationary phases that possess several linked glucopyranoside units and have chiral recognition properties are useful for HILIC chiral separations .
Zwitterionic sulfoalkylbetaine stationary phases have also been introduced for HILIC separations. The active layer, which is grafted onto wide-pore silica gel or a polymer support, contains both strongly acidic sulfonic acid groups and strongly basic quaternary ammonium groups separated by a short alkyl spacer. Ion-exchange interactions of the zwitterionic stationary phase are assumed. The sulfoalkylbetaine bonded phases strongly adsorb water by hydrogen bonding, and the bulk layer of water, which forms part of the stationary phase, then largely controls the retention mechanism. Zwitterionic columns are commercially available under the tradenames ZIC-HILIC (on a silica gel support) and ZIC-pHILIC (on a polymer support) .
The separation of neutral compounds on ion exchangers under typical HILIC conditions has been known about for a very long time. On both cation-exchange and anion-exchange styrene-divinylbenzene resins, only the retentions of some polar compounds (e.g., carbohydrates and related substances) increase with increasing ethanol concentration in the mobile phase. For other compounds, the opposite effects have been observed [62, 63, 64]. Due to the presence of ion-exchange groups, a mixed-mode HILIC/ion-exchange mechanism controls the retention, which may cause specific selectivity effects. The mixed anion-exchange/cation-exchange/HILIC mechanism that occurs on silica-based, small-pore, weak ion-exchange resins was found to be useful for the analysis and purification of compounds from natural products .
Mobile phase selection
A typical mobile phase for HILIC chromatography includes water-miscible polar organic solvents such as acetonitrile with a small amount of water . However, any aprotic solvent that is miscible with water (e.g., tetrahydrofuran, THF, and/or dioxane) can be used. Alcohols can also be adopted, although a higher concentration is needed to achieve the same degree of retention of the analyte relative to an aprotic solvent–water combination .
It is commonly believed that in HILIC, the mobile phase forms a water-rich layer on the surface of the polar stationary phase vs. the water-deficient mobile phase, creating a liquid/liquid extraction system. The analyte is distributed between these two layers [6, 33].
Mobile phase additives
Ionic additives, such as ammonium acetate and ammonium formate, are typically used to control the mobile phase pH and ion strength. In HILIC, they can also contribute to the polarity of the analyte, resulting in differential changes in retention. For ionizable analytes, such as aminoglycoside antibiotics, the pH must be adjusted to ensure that the analyte will be in a single ionic form. Increasing the buffer concentration decreases the retention if ion exchange controls the retention, while the opposite effect may occur, affecting the solvation, in the absence of ion exchange under HILIC conditions. If this is not done, an asymmetric peak shape, chromatographic peak “tailing,” and/or poor recovery from the stationary phase will be observed. No buffer is needed to separate neutral polar analytes (e.g., carbohydrates). The use of other salts (such as 100–300 mM sodium perchlorate) that are soluble in high organic solvent mixtures (ca. 70% acetonitrile) can be used to increase the polarity of the mobile phase in order to achieve elution. These salts are not volatile, so this technique is less useful with a mass spectrometer as a detector [6, 65].
Separation mechanism in HILIC mode
The mechanism and theoretical description of analyte retention in HPLC has been the subject of many articles. Different research groups and scientific schools still disagree about the most realistic retention mechanism and the best theory to describe and predict it [66, 67].
When the acetonitrile volume fraction is between 30 and 70%, the adsorbed phase consists of at least three adsorbed layers of acetonitrile and water mixture. This agrees with previous studies that demonstrated the formation of an adsorbed multilayer of water on bare silica. The number of adsorbed monolayers of pure acetonitrile is around 2, while that of pure water is close to 3 .
To sum up, when the concentration of water in the mobile phase is lower than 20%, water adsorption can be multilayer in nature, and it can create an excess of adsorbed water in comparison with the concentration of water in the eluent. McCalley and Neue  concluded that about 4–13% of the pore volume of the silica phase is occupied by a water-enriched layer when there is 75–90% acetonitrile in the eluent. The solvent adsorption governed by these specific interactions may have a large influence on the selectivity of the separation in the discussed HILIC systems .
Although it is well established that a hydrophilic surface holds water when exposed to mixtures of organic solvent and water, the HILIC partitioning theory is based on only circumstantial evidence. HILIC is more than just simple partitioning, and includes hydrogen donor interactions between neutral polar species, as well as weak electrostatic mechanisms under the high organic solvent conditions used for retention. This indicates that the mechanism of HILIC is distinct from that of ion-exchange chromatography. There are studies that point towards a more multimodal separation mechanism [6, 7]. Alpert  considered dipole–dipole interactions, and hydrogen bonds may also contribute to partitioning into the stationary phase layer. He noted charged that basic groups in a solute lead to pronounced hydrophilicity and retention, so these interactions make important contributions to the mechanism of separation. Yoshida  similarly considered that HILIC retention encompassed both hydrogen bonding (which depends on Lewis acidity/basicity) and dipole–dipole interactions (dependent on dipole moments and the polarizability of molecules).
It was shown the elution pattern was similar to that in (nonaqueous) normal phase liquid chromatography, so it was proposed that the mechanism must be similar too. Hydrogen bonding, especially when using low-water mobile phases, probably also contributes to the retention mechanism in HILIC .
The interactions of basic and acidic analytes with the stationary phase are expected to be based on both hydrophilic interactions and electrostatic forces. However, the final separation mechanism of the elution process is most probably a superposition of partitioning and electrostatic interactions or hydrogen bonding to the stationary phase [6, 7].
The extent to which each mechanism dominates is dependent on the actual type of stationary phase used and the buffer conditions, including the level and type of organic solvent, the type and concentration of salt, and the pH .
The presence of buffering salts in the mobile phase can decrease electrostatic interactions through disruption [42, 65]. Additionally, the same salt-based disruption can decrease the retentions of analytes, and can be useful during elution . However, in some instances, a higher salt concentration might drive the more solvated salt ions into the water-enriched layer formed on the particles, yielding an increase in the volume of the water layer and therefore an increase in retention [42, 65, 84]. A thickening of the water layer on the stationary phase through hydration takes place at the same time . Another possible cause has been hypothesized: electrostatic repulsion between the stationary phase and the analytes is weakened by the higher salt concentration .
Another factor that influences the retention characteristics in HILIC is the pH of the buffer. Whether the buffer pH is above or below the pK a of the analyte determines its charge state, which in turn affects the hydrophilicity of the analyte and likewise the interaction with the stationary phase [7, 33]. For example, acidic solutes had low retention or showed exclusion in ammonium formate buffers, but were strongly retained when trifluoroacetic acid (TFA) buffers were used. This is possibly due to suppression of the repulsion of the solute anions from ionized silanol groups at the low pH values of TFA solutions of aqueous acetonitrile. At high buffer pH, the ionization of weak bases was suppressed, reducing ionic (and possibly hydrophilic) retention, leading to further opportunities to manipulate selectivity .
Charged stationary phases, such as the above mentioned anion or cation exchangers and deprotonated silanol groups, are most likely to display some degree of electrostatic interaction .
The effect of column temperature on HILIC separations is often rather small (considerably less than in RP-LC), but ultimately this depends on the nature of the retained molecule . The retention in the RP mode decreases at increasing temperature and is principally controlled by the enthalpic contribution. However, the retention factors in the HILIC range of the mobile phase are almost independent of the temperature. In HILIC mode, the retention is probably controlled by entropic contributions, possibly originating in different levels of sample solvation in the stationary and bulk mobile phases .
It was demonstrated recently that the mechanism of HILIC separation involves various combinations of hydrophilic interactions, ion exchange, and reversed-phase retention by the siloxane on the silica surface, which contribute to various degrees depending on the particular conditions employed [33, 34].
Characterization and selection of HILIC separation systems
The selectivity depends not only on the stationary phase but also strongly on the mobile phase .
The relative polarity of the mobile phase with respect to the stationary phase is a distinguishing feature that can be used to classify NP and RP systems. Hence, a dual retention mechanism, where the NP and RP effects contribute simultaneously to the retention, is a rather common phenomenon in hydrophilic interaction liquid chromatography .
The main problem with QSRR prediction models is that the results are affected by the composition of the mobile phase, which may become more or less adsorbed in the stationary phase and thus change its true nature. Therefore, comparing the properties of HILIC stationary phases in order to predict the retention of polar compounds would be probably more complicated than comparing RP columns . Additional study in this direction is necessary to show the real merits of the QSRR approach in HILIC. Possibly, comparing the retention data, either isocratic or obtained in gradient experiments with increasing salt and/or water concentration(s), may prove useful for the selection of appropriate stationary–mobile phase combinations in HILIC of different sample types .
Orthogonal and two-dimensional separations of HILIC
True orthogonality is technically difficult to achieve, as orthogonality depends not only on the separation mechanisms but also on the properties of the solutes and the separation conditions. Successful orthogonal combinations can be achieved when the appropriate stationary and mobile phases are carefully chosen with respect to the physicochemical properties of the sample constituents, including size, charge, polarity, hydrophobicity, etc. A variety of stationary phases are presently available, with differences in surface chemistries, support material, carbon load, pore size, etc., whereas the characteristics of the mobile phase can be altered by changing the modifier, pH, temperature, or by adding ion pair agents. These parameters play important roles in the mixed-mode HILIC retention mechanism and can be flexibly tuned to suit specific separation problems.
Because of its selectivity is highly complementary to RP-LC, HILIC is ideally suited to 2D LC separations. Therefore, an attractive feature of HILIC is its 2D separation capabilities when used together with RP-LC [94, 99]. However, the high-organic mobile phases used in HILIC systems have very high elution strengths in RP systems, and vice versa: mobile phases with high concentrations of water (common in RP systems) are very strong eluents in HILIC systems. This causes serious problems when attempting to use on-line 2D HILIC × RP-LC setups with direct fraction transfer via a switching valve interface. The first-dimension mobile phase in which the fractions are transferred to the second dimension is an excessively strong eluent in the second dimension, which often causes low resolution, peak asymmetry, and even split peaks in the second dimension .
Other examples are the separation of peptides in 2005 by Gilar et al. , and of flavonoid glycosides in both RP-LC and HILIC modes on a β-cyclodextrin column by Feng et al. in 2010 . 2D techniques combining RP and HILIC chromatography represent powerful tools in the analysis of pharmaceuticals and their degradation products, and in the metabolic profiling of physiological fluids . Two-dimensional liquid chromatography is often used to reduce the complexity of a proteomic sample prior to tandem mass spectrometry analysis.
An elegant way to utilize the advantageous orthogonality of HILIC and RP-LC is to use solid-phase extraction (SPE) to fractionate the sample before separation. The two fractions are then separated in either the HILIC or the RP-LC mode, depending on the polarity . Other techniques, such as IC and size exclusion chromatography (SEC), result in more peak grouping or poorer resolution in comparison to HILIC .
Applications of HILIC
Several examples of applications of HILIC systems
Type of packing
Groups of detected compounds
Salt gradient in TEAP buffer with ACN
Peptides, amino acids
Isocratic elution, (A) ACN and (B) 6.5 mM ammonium acetate (pH 5.5)
Electrospray ion trap mass spectrometry (ESI-MS)
Metabolites occurring in different plant species
Isocratic elution, (A) ACN and (B) 13 mM ammonium acetate buffer, pH 9.1
Ion trap mass spectrometry (MS)
Cancer biomarker in urine (proteomics approach)
Spherisorb silica gel
ACN/methanol/phosphoric acid (99/3/1 v/v)
Phospholipids from pulmonary surfactant
Cyclodextrin stationary phases
Isocratic elution, (A) ACN and (B) 6.5 mM ammonium acetate, pH 5.5 buffer
Chiral separations of drug substances and underivatized amino acids
Cyanopropyl stationary phases
Methanol/ACN (60:40) with or without 50–200 mM ammonium acetate or ammonium formate; or 100% toluene
Electrospray ionization tandem mass spectra with collision-induced dissociation (CID)
Free folic acid in human plasma
Gradient elution: 13–27% water in 45 min and 27–40% water in 5 min; (A) 90% ACN and ammonium acetate buffer (pH 7.0) and (B) 60% ACN and ammonium acetate buffer, pH 7.0
Electrospray ionization mass spectrometry (ESI-MS)
Small polar compounds in food analysis
Diazolidinyl urea, urea, and allantoin in cosmetic samples
Gradient elution, 5% mobile phase A increasing linearly to 95% over a period of 15 min; (A) ammonium acetate modified with 0.1% (v/v) formic acid (pH 4), (B) ACN with 0.1% (v/v) formic acid
Mass spectrometry (MS)
Metabolomic fingerprint in human urine
Isocratic elution, ammonium formate
Adrenoreceptor agonists and antagonists—used as therapeutic agents in the treatment of hypertension, cardiac arrest, and other medical conditions
(A) ACN with 0.05% formic acid and (B) water
Mass spectrometry (MS)
Sulfonamide antibacterial residues in milk and egg
Gradient elution, (A) ACN with aqueous ammonium acetate (pH 6.80) (95/5, v/v) and (B) ACN with aqueous ammonium acetate (pH 6.80) (75/25, v/v)
Tandem mass spectrometry (MS/MS)
Free estrogens and their conjugates in river water
(A) ACN and (B) potassium phosphate, pH 6.5 acetonitrile (20:80, v/v)
Polar pharmaceuticals and impurities
Gradient elution, 0 min: 1.1% B; 1 min: 2% B; 30 min: 4% B; 45 min: 5% B; 75–160 min: 9.9% B; (A) n-hexane and (B) ethyl alcohol
Orange essential oil and juice carotenoids
(A) ACN with 0.1% v/v formic acid; (B) 5 mM ammonium acetate with 0.1% v/v formic acid (pH 4.0)
Mass spectrometry (MS)
Neutral sugars, sugar phosphates, sugar alcohols
(A) ACN; (B) 10 mM ammonium acetate (pH 5.5)
Ion trap mass spectrometry (MS)
Comprehensive analysis of the microbial metabolome
HILIC separations are very easy to combine with several detection techniques, such as ultraviolet light absorbance (UV), fluorescence (FL), refractive index (RI), evaporative light scattering (ELSD), charged aerosol (CAD), and mass spectrometry (MS) [127, 128, 129]. In addition it is ideally suited to the sensitive LC-MS analysis of water-soluble polar compounds, because the high organic content in the mobile phase leads to rapid evaporation of the solvent during electrospray ionization. HILIC can offer a tenfold increase in sensitivity over reversed-phase chromatography because the organic solvent is much more volatile.
Recent years have witnessed an increased interest in HILIC. More versatile and diverse stationary phases have become available, leading to reports of an exciting and broad range of applications. The unique separation ability of HILIC and its orthogonality towards RP make it an ideal method for multidimensional chromatography, which can extend separation power. As far as selectivity is concerned, HILIC can complete well with RP, which is one of the main chromatographic techniques applied today.
Its applications now encompass most categories of polar compounds, charged as well as uncharged, although HILIC is particularly well suited to solutes that lack charge (meaning that Coulombic interactions cannot be used to mediate retention). This approach has recently gained a great deal of attention because of the increased need to analyze polar compounds in complicated mixtures. Another reason for its popularity is the widespread use of MS coupled to HPLC, as HILIC mobile phases are very compatible and give high sensitivity. Recently, HILIC has found great popularity in bioanalytical applications because drugs and their metabolites are often polar structures.
The work was supported by European Social Fund, the Polish National Budget, Kujawsko-pomorskie Vovidship Budget—“Stypendia dla doktorantów 2008/2009—ZPORR.” Financial support from the Foundation for Polish Science (Professor’s Subsidy “Mistrz”) is gratefully acknowledged.
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
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