Anal Bioanal Chem (2012) 402:231–247

DOI 10.1007/s00216-011-5308-5

REVIEW

Hydrophilic interaction liquid chromatography (HILIC)—a

powerful separation technique
Bogusław Buszewski & Sylwia Noga

Received: 24 June 2011 / Revised: 29 July 2011 / Accepted: 1 August 2011 / Published online: 31 August 2011
# The Author(s) 2011. This article is published with open access at Springerlink.com

Abstract Hydrophilic interaction liquid chromatography specific and nonspecific interactions [1–4]. Nevertheless,
(HILIC) provides an alternative approach to effectively there is currently no detailed quantitative retention model
separate small polar compounds on polar stationary phases. that would allow the chromatographic parameters for
The purpose of this work was to review the options for the individual analytes separated under given conditions to be
characterization of HILIC stationary phases and their accurately predicted.
applications for separations of polar compounds in complex Normal or reversed-phase liquid chromatography can be
matrices. The characteristics of the hydrophilic stationary used for analysis. In normal phase liquid chromatography
phase may affect and in some cases limit the choices of mobile (NP-LC), the stationary phase is more polar than the mobile
phase composition, ion strength or buffer pH value available, phase. The retention increases as the polarity of the mobile
since mechanisms other than hydrophilic partitioning could phase decreases, and thus polar analytes are more strongly
potentially occur. Enhancing our understanding of retention retained than nonpolar ones. The opposite situation occurs in
behavior in HILIC increases the scope of possible applications reversed-phase liquid chromatography (RP-LC) [5]. NP-LC
of liquid chromatography. One interesting option may also be has been widely used to separate various compounds, from
to use HILIC in orthogonal and/or two-dimensional separa- nonpolar to highly polar compounds (note that chromatogra-
tions. Bioapplications of HILIC systems are also presented. phy was first introduced as a method used in separation
science). Although RP-LC systems were previously commonly
Keywords Hydrophilic interaction liquid chromatography . used by scientists, NP-LC methods are currently undergoing a
Stationary phase . Separation mechanism . Bioapplication renaissance.
Hydrophilic interaction liquid chromatography (HILIC) is
an alternative high-performance liquid chromatography
Introduction (HPLC) mode for separating polar compounds. For historical
reasons, it has been reported that HILIC is a variant of normal
A theoretical description of analyte retention in high- phase liquid chromatography, but the separation mechanism
performance liquid chromatography (HPLC) has been the used in HILIC is more complicated than that in NP-LC. While
subject of various publications. There are several ways to the acronym HILIC was first suggested by Alpert in 1990 [6],
model the separation mechanism: partition, adsorption, ion the number of publications on HILIC has increased
exchange, and size exclusion. This mechanism is based on substantially since 2003 (Fig. 1), as outlined in the well-
constructed review by Hemström and Irgum [7].
Like NP-LC, HILIC employs traditional polar stationary
Published in the 10th Anniversary Issue. phases such as silica, amino or cyano [8–12], but the mobile
B. Buszewski (*) : S. Noga phase used is similar to those employed in the RP-LC mode
Chair of Environmental Chemistry and Bioanalytics, [7, 11, 12]. HILIC also allows the analysis of charged
Faculty of Chemistry, Nicolaus Copernicus University,
substances, as in ion chromatography (IC). Figure 2 shows
7 Gagarin St.,
87-100 Toruń, Poland how HILIC complements other areas of chromatography and
e-mail: bbusz@chem.uni.torun.pl extends the range of separation options.
232 B. Buszewski, S. Noga

Fig. 1 SciFinder Scholar search results documenting the continuously growing research area of HILIC, based on the numbers of publications per year

HILIC has many specific advantages over conventional compounds that are too polar to be well retained in RP-LC
NP-LC and RP-LC. For example, it is suitable for analyzing but have insufficient charge to allow effective electrostatic
compounds in complex systems that always elute near the retention in ion-exchange chromatography. HILIC separa-
void in reserved-phase chromatography. Polar samples always tion is currently attracting a lot of interest since it solves
show good solubility in the aqueous mobile phase used in many previously difficult separation problems, such as the
HILIC, which overcomes the drawbacks of the poor solubility separation of small organic acids, basic drugs, and many other
often encountered in NP-LC. Expensive ion pair reagents are neutral and charged substances. It has been successfully
not required in HILIC, and it can be conveniently coupled to applied to the analysis of carbohydrates [14, 15], peptides [8,
mass spectrometry (MS), especially in the electrospray 16, 17] and polar pharmaceuticals [11, 18], etc.
ionization (ESI) mode. In contrast to RP-LC, gradient elution This paper covers fundamental developments in hydro-
HILIC begins with a low-polarity organic solvent and elutes philic interaction liquid chromatography. The objective of the
polar analytes by increasing the polar aqueous content [13]. A present work is to review options for the characterization of
desirable mobile phase would contain high organic content HILIC stationary phases and their applications to separations
for better sensitivity and also show good on-column retention of polar compounds in complex matrices. Gaining a thorough
for polar ionic compounds. Hydrophilic interaction liquid understanding of retention behavior in HILIC enhances the
chromatography has established itself as the separation mode scope of applications of liquid chromatography. The separa-
of choice for uncharged highly hydrophilic and amphiphilic tion 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.
Fig. 2 HILIC combines the characteristics of the three major methods The first generation of HILIC mode separations started
in liquid chromatography in 1975. Linden et al. [19] separated carbohydrates by an
HILIC—a powerful separation technique 233

amino-silica phase, Bondapak (Waters, Milford, MA, USA) pounds. Suppressing silanol ionization through the addition
in a mixture of acetonotrile and water (75:25 v/v). The next of TFA may promote the ion-pairing mechanism. Similar
generation of stationary phases for HILIC used DIOL- and effects have also been observed in HILIC on monolithic
amide-silica. The DIOL-silica column has mainly been used silica gel columns, which offer higher permeability than the
for the separation of proteins [20, 21]. According to Tosoh, particle-packed HILIC columns [58]. Silica gel type C with
producer of TSKgel Amide-80, amide-silica columns have a hydrosilated surface populated with nonpolar silicon
been available since at least 1985. This particular phase is hydride Si–H groups instead of silanol groups may have
described as consisting of nonionic carbamoyl groups that up to 95% of its original silanols removed, making it less
are chemically bonded to the silica gel, but it is commonly polar than silica gels with higher populations of silanol
known as an amide-bonded silica. After Yoshida [22] applied groups [59]. It can be used to separate acids or bases in the
these phases to the separation of peptides, the amide-silica HILIC mode in buffered mobile phases containing more
phase soon found common usage in HILIC. Chemically than 50–70% organic solvent (acetonitrile).
bonded stationary phases with specific structural properties DIOL, amino, amide and other bonded phases used in
have been prepared by Buszewski et al. [23–25]. One of HILIC are usually prepared by chemically modifying the
them contains aminopropyl ligands bonded to silica (SG– silica gel surface, like the C18 phases used for RP-LC [8, 60].
NH2); others are an alkylamide packing phase (SG-AP) and Chemically bonded DIOL phases demonstrate high polarity
a mixed phase (SG-MIX) containing different types of and hydrogen bonding properties, and do not contain
ligands (–NH2, –CN, –Ph, –C8, –C18) bonded to the support. ionizable groups other than unreacted residual silanols,
Over the last 15 years, HILIC has progressed into second meaning that they are appropriate for the HILIC mode
and third generation implementations, most of which involve [61]. Bonded amino-silica columns are relatively often used
mixed or multiple-interaction solid phases. Many column in the HILIC mode. While basic analytes are in general
vendors sell both traditional HILIC and its more sophisticated strongly retained on silica gel by hydrogen bonding and ion-
relatives. Some novel separation materials for HILIC have exchange interactions with silanol groups, acidic compounds
attracted increasing attention in recent years [7, 18, 26–31]. show increased affinities to amino-silica columns, which
Hence, the structural variations of HILIC-type stationary can sometimes even lead to irreversible adsorption [8].
phases are wider than those found in reversed-phase systems. Chemically bonded phases with other functionalities, such as
HILIC phases can be grouped into neutral polar or ionic polyethylene glycol or alkyls with embedded amide or
surfaces. Table 1 shows the different structures of stationary carbamate groups, are generally proposed for RP applica-
phases that are applicable to HILIC-mode separation. These tions in water-rich mobile phases. On the other hand, when
special separation materials for HILIC show good selectiv- the percentage of organic solvent is high, the retention of
ity and reproducibility for the separation of polar com- many compounds increases with increasing concentration of
pounds. Although the number of commercially available acetonitrile, showing typical NP behavior [6, 47, 62].
columns designed especially for HILIC is growing, there is Cyclodextrin-silica stationary phases that possess several
still no versatile stationary phase like C18 in RP-LC. linked glucopyranoside units and have chiral recognition
However, different types of separation materials for HILIC properties are useful for HILIC chiral separations [40].
have different retention characteristics and separation Zwitterionic sulfoalkylbetaine stationary phases have also
selectivities. been introduced for HILIC separations. The active layer, which
Unmodified bare silica gel has some advantages for is grafted onto wide-pore silica gel or a polymer support,
HILIC, in contrast to chemically bonded stationary phases. contains both strongly acidic sulfonic acid groups and strongly
Type A silica gels, prepared by precipitation from the basic quaternary ammonium groups separated by a short
solutions of silicates, are acidic because they are polluted alkyl spacer. Ion-exchange interactions of the zwitterionic
with certain metals that activate surface silanol groups and stationary phase are assumed. The sulfoalkylbetaine bonded
form complexes with some chelating solutes, causing phases strongly adsorb water by hydrogen bonding, and
strong retention or asymmetric peaks. Type B silica gels the bulk layer of water, which forms part of the stationary
are formed by the aggregation of silica sols in air, contain phase, then largely controls the retention mechanism.
very low amounts of metals, and are more stable at Zwitterionic columns are commercially available under the
intermediate and higher pH values (up to at least pH 9) tradenames ZIC-HILIC (on a silica gel support) and ZIC-
than xerogel-type materials. They generally provide better pHILIC (on a polymer support) [12].
separations, especially for basic samples, because they are The separation of neutral compounds on ion exchangers
highly purified, less acidic “sol-gel” spherical silica under typical HILIC conditions has been known about for a
particles [57]. At higher pH values, silanol groups are very long time. On both cation-exchange and anion-exchange
ionized and cation exchange plays a important role in styrene-divinylbenzene resins, only the retentions of some
retention, especially for positively charged basic com- polar compounds (e.g., carbohydrates and related substances)
234 B. Buszewski, S. Noga

Table 1 Selected stationary phases used in HILIC separations

Packing materials Structure of stationary phase Application

underivatized silica
stationary phases that
contain functional groups
such as siloxanes, silanols
[7, 26, 32-34]
with (or without) a small
quantity of metals

DIOL bonded phases [8, 20, 21, 35]

cyano bonded phases [36]

amino bonded phases [19, 26, 37-39]

alkylamide [23-24]

amide bonded phases [22, 26, 40-42]

mix-mode [23, 25]

polymeric structures of
poly(succinimide) [14, 26, 35, 43]
derivatives

increase with increasing ethanol concentration in the mobile groups, a mixed-mode HILIC/ion-exchange mechanism con-
phase. For other compounds, the opposite effects have been trols the retention, which may cause specific selectivity
observed [62–64]. Due to the presence of ion-exchange effects. The mixed anion-exchange/cation-exchange/HILIC
HILIC—a powerful separation technique 235

Table 1 (continued)

Packing materials Structure of stationary phase Application

polyethylene
[44]
glycol/silica (HS PEG)

“click” ß-cyclodextrin [40, 45, 46]

“click” saccharides
[47]
(“click” maltose)

“click” dipeptide [48]

zwitterionic sulfobetaine
bonded phases [11, 18, 49-53]
(ZIC-HILIC)

cationic exchangers
[54, 55]
bonded phases

mix-mode RP/anionic
exchangers bonded [50, 56]
phases

mechanism that occurs on silica-based, small-pore, weak ion- achieve the same degree of retention of the analyte relative
exchange resins was found to be useful for the analysis and to an aprotic solvent–water combination [7].
purification of compounds from natural products [18]. An eluotropic row is useful for selecting a suitable
organic modifier for the mobile phase. This lists solvents
according to increasing elution strength. Relative solvent
strengths in HILIC can be approximately summarized as
Mobile phase selection
follows:
A typical mobile phase for HILIC chromatography includes
acetone < isopropanol
propanol < acetonitrile
water-miscible polar organic solvents such as acetonitrile
with a small amount of water [6]. However, any aprotic < ethanol < dioxane < DMF
methanol < water
solvent that is miscible with water (e.g., tetrahydrofuran,
THF, and/or dioxane) can be used. Alcohols can also be HILIC separations are performed either in isocratic mode
adopted, although a higher concentration is needed to with a high percentage of organic solvent or with gradients
236 B. Buszewski, S. Noga

starting with a high percentage of organic solvent and Present theory proposes that HILIC retention is caused
ending with a high proportion of aqueous solvent [6]. by partitioning. This phenomenon still lacks a thorough
It is commonly believed that in HILIC, the mobile phase theoretical explanation. In this mode, the separation
forms a water-rich layer on the surface of the polar stationary mechanism is based on the differential distribution of the
phase vs. the water-deficient mobile phase, creating a liquid/ injected analyte solute molecules between the acetonitrile-rich
liquid extraction system. The analyte is distributed between mobile phase and a water-enriched layer adsorbed onto the
these two layers [6, 33]. hydrophilic stationary phase [6, 7] (see Fig. 3). The more
hydrophilic the analyte, the more the partitioning equilibrium
is shifted towards the immobilized water layer on the
Mobile phase additives stationary phase, and thus, the more the analyte is retained.
In other words, a separation based on the polarities of the
Ionic additives, such as ammonium acetate and ammonium compounds and the degree of solvation takes place. As a result
formate, are typically used to control the mobile phase pH of the preferential solvation of the stationary phase surface, it
and ion strength. In HILIC, they can also contribute to the is very likely that the gradient of a given solvent concentration
polarity of the analyte, resulting in differential changes in from the adsorbent surface into a bulk mobile phase is formed.
retention. For ionizable analytes, such as aminoglycoside When the concentration of acetonitrile (or some other
antibiotics, the pH must be adjusted to ensure that the organic solvent) increases, water interacts more strongly with
analyte will be in a single ionic form. Increasing the buffer the surface of the polar stationary phase (e.g., bare silica). In
concentration decreases the retention if ion exchange this case, acetonitrile cannot interact with residual silanols on
controls the retention, while the opposite effect may occur, the stationary phases, so they are uncovered and water
affecting the solvation, in the absence of ion exchange molecules can be adsorbed onto them [74–78]. Figure 4a
under HILIC conditions. If this is not done, an asymmetric shows the largest excess adsorption of acetonitrile on the
peak shape, chromatographic peak “tailing,” and/or poor surface area of an HILIC adsorbent. It was found for water-
recovery from the stationary phase will be observed. No buffer rich mobile phases containing about 80% water by volume.
is needed to separate neutral polar analytes (e.g., carbohy- This mirrors the preferential adsorption of acetonitrile onto
drates). The use of other salts (such as 100–300 mM sodium siloxane bridges. The largest deficit of acetonitrile is found
perchlorate) that are soluble in high organic solvent mixtures for a water concentration of close to 20% (v/v). This
(ca. 70% acetonitrile) can be used to increase the polarity of corresponds to the maximum excess adsorption of water onto
the mobile phase in order to achieve elution. These salts are the silica, and illustrates the preferential adsorption of water
not volatile, so this technique is less useful with a mass molecules onto free, accessible silanol groups (≡Si–OH) [79].
spectrometer as a detector [6, 65]. The water adsorption is higher when the proportion of water
molecules in the mobile phase decreases [78]. Figure 4b
shows the plots obtained when following the convention that
Separation mechanism in HILIC mode total adsorption isotherms have a horizontal plateau or rather
inflection point. This convention represents the minimum
The mechanism and theoretical description of analyte reten- adsorbed amount of eluent, because the total amount
tion in HPLC has been the subject of many articles. Different adsorbed is a strictly monotonously increasing function of
research groups and scientific schools still disagree about the its bulk concentration [79].
most realistic retention mechanism and the best theory to When the acetonitrile volume fraction is between 30 and
describe and predict it [66, 67]. 70%, the adsorbed phase consists of at least three adsorbed
There are essentially three possible ways to model the layers of acetonitrile and water mixture. This agrees with
separation mechanism. The first is analyte partitioning previous studies that demonstrated the formation of an
between the mobile and stationary phases [68, 69]; the adsorbed multilayer of water on bare silica. The number of
second is the adsorption of the analyte onto the surface of adsorbed monolayers of pure acetonitrile is around 2, while
the adsorbent [70, 71]; the third assumes the preferential that of pure water is close to 3 [79].
adsorption of the organic mobile phase modifier onto the To sum up, when the concentration of water in the mobile
adsorbent surface, followed by the partitioning of this phase is lower than 20%, water adsorption can be multilayer in
analyte into the adsorbed layer [72]. The retention nature, and it can create an excess of adsorbed water in
phenomenon in HPLC simultaneously depends on various comparison with the concentration of water in the eluent.
types of intermolecular interactions between the solute and McCalley and Neue [78] concluded that about 4–13% of the
the stationary phase, the solute and the mobile phase, and pore volume of the silica phase is occupied by a water-
the stationary and mobile phases. Known intermolecular enriched layer when there is 75–90% acetonitrile in the
interaction types are given in Table 2. eluent. The solvent adsorption governed by these specific
HILIC—a powerful separation technique 237

Table 2 Types of interactions between the analyte, stationary phase, and mobile phase [73]

Energy Example
Type of interaction Characterization
[kJ·mol-1] system
formed between hydrogen of the protodonor
Hydrogen group and atom being hydrogen acceptor, can be
- (4 - 17)
bonding formed within the same molecule, as well as
Chemical between two different
interactions
Donor – acceptor occurs between pairs of electron donor (Lewis
- (4 - 17)
interactions base) and acceptor (Lewis acid)

ion acts on electrically neutral molecule; leads to

Ion – dipole - (4 - 17)
a multicomponents

force between two electrically neutral particles,

Dipole – dipole - (4 - 17)
but having determined dipole moments

Physical
interactions
Dipole-induced forces between the molecule possesses clear
- (4 - 17)
dipole dipole moment and non-polar molecule

Temporary two electrically neutral particles, if they come

dipole-induced close together, will attract on the electrostatic - (4 - 17)
dipole forces way

weak forces of attraction between the fragments

Intermolecular interactions of individual molecules, declining rapidly with
- (2 - 4)
(Van der Waals forces) increasing distance between the interacting
particles, do not lead to permanent connections

forces operating in the aquatic environment

Hydrophobic interactions -4
between molecules with low affinity for water

interactions may have a large influence on the selectivity of for retention. This indicates that the mechanism of HILIC is
the separation in the discussed HILIC systems [76]. distinct from that of ion-exchange chromatography. There are
Although it is well established that a hydrophilic surface studies that point towards a more multimodal separation
holds water when exposed to mixtures of organic solvent and mechanism [6, 7]. Alpert [6] considered dipole–dipole
water, the HILIC partitioning theory is based on only interactions, and hydrogen bonds may also contribute to
circumstantial evidence. HILIC is more than just simple partitioning into the stationary phase layer. He noted charged
partitioning, and includes hydrogen donor interactions that basic groups in a solute lead to pronounced hydro-
between neutral polar species, as well as weak electrostatic philicity and retention, so these interactions make important
mechanisms under the high organic solvent conditions used contributions to the mechanism of separation. Yoshida [16]
238 B. Buszewski, S. Noga

silica surface, which cannot be removed or blocked due to

steric effects of bonded ligands, are partially ionized.
Residual silanols may influence the separation of polar
analytes, especially basic compounds and biopolymers,
because of the strong polar (hydrogen-bonding and dipole–
dipole) interactions between their basic groups and the
ionized residual silanols of the support. Adsorbed water
molecules suppress the ionization of surface silanols, which
leads to negative charging of the silica gel surface. This layer
of negative charges on the silica gel surface creates a negative
electric field in the contacting mobile phase. Positive charges
attracted by the field create the second layer of the electric
double layer (Fig. 5) [81].
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
Fig. 3 Schema of the separation mechanism in a HILIC system solvent, the type and concentration of salt, and the pH [47].
Increasing the concentration of water in the eluent decreases
similarly considered that HILIC retention encompassed both retention when—as is typical in HILIC—high acetonitrile
hydrogen bonding (which depends on Lewis acidity/ concentrations are used (Fig. 6) [10, 57]. Increasing the
basicity) and dipole–dipole interactions (dependent on admixture of an organic component in a partially aqueous
dipole moments and the polarizability of molecules). mobile phase generally leads to a decrease in the relative
It was shown the elution pattern was similar to that static permittivity of the mixed mobile phase. This is indeed
in (nonaqueous) normal phase liquid chromatography, so the case with acetonitrile, the solvent typically used in HILIC.
it was proposed that the mechanism must be similar too. Lower relative static permittivity is accompanied by decreased
Hydrogen bonding, especially when using low-water ion stabilization, and this is manifested in increased pKa
mobile phases, probably also contributes to the retention values for solutes [65, 82]. Above 60% v/v acetonitrile,
mechanism in HILIC [80]. strong deviations start to take place due to homo- and
On the other hand, electrostatic interactions can play an heteroconjugation when the amount of water available to form
important role in HILIC, due to either ionic groups that have hydrogen bonds with the buffer compounds and solutes
been intentionally incorporated onto the stationary phase or decreases [65].
less intentional residual charges, such as those of dissociated The presence of buffering salts in the mobile phase can
silanol groups [65]. These residual silanol groups on the decrease electrostatic interactions through disruption [42, 65].

Fig. 4 Surface excesses of ace-

tonitrile and water on the HILIC
adsorbent (a). Total adsorbed
amounts of acetonitrile and
water per unit surface area of
HILIC adsorbent as a function
of the volume fraction of aceto-
nitrile in the binary eluent (b).
Based on data from [79]
HILIC—a powerful separation technique 239

Fig. 5 Model of the electric

double layer on the stationary
bonded-phase surface. Based on
data from [81]

Additionally, the same salt-based disruption can decrease the retained when trifluoroacetic acid (TFA) buffers were used.
retentions of analytes, and can be useful during elution [6]. This is possibly due to suppression of the repulsion of the
However, in some instances, a higher salt concentration solute anions from ionized silanol groups at the low pH
might drive the more solvated salt ions into the water-enriched values of TFA solutions of aqueous acetonitrile. At high
layer formed on the particles, yielding an increase in the buffer pH, the ionization of weak bases was suppressed,
volume of the water layer and therefore an increase in reducing ionic (and possibly hydrophilic) retention, leading
retention [42, 65, 84]. A thickening of the water layer on to further opportunities to manipulate selectivity [57].
the stationary phase through hydration takes place at the same Charged stationary phases, such as the above mentioned
time [57]. Another possible cause has been hypothesized: anion or cation exchangers and deprotonated silanol groups,
electrostatic repulsion between the stationary phase and the are most likely to display some degree of electrostatic
analytes is weakened by the higher salt concentration [85]. interaction [7].
Another factor that influences the retention characteristics The effect of column temperature on HILIC separations
in HILIC is the pH of the buffer. Whether the buffer pH is is often rather small (considerably less than in RP-LC), but
above or below the pKa of the analyte determines its charge ultimately this depends on the nature of the retained
state, which in turn affects the hydrophilicity of the analyte molecule [86]. The retention in the RP mode decreases at
and likewise the interaction with the stationary phase [7, 33]. increasing temperature and is principally controlled by the
For example, acidic solutes had low retention or showed enthalpic contribution. However, the retention factors in the
exclusion in ammonium formate buffers, but were strongly HILIC range of the mobile phase are almost independent of

Fig. 6 Effect of the mobile-phase aqueous buffer concentration on the HILIC retentions of the test compounds obtained using HILIC monolithic
columns. Adapted from the data in [83]
240 B. Buszewski, S. Noga

the temperature. In HILIC mode, the retention is probably the constant m increases with decreasing polarity of the
controlled by entropic contributions, possibly originating in organic solvent and with increasing size of the sample
different levels of sample solvation in the stationary and molecule. Equation 1 was assumed to apply generally to the
bulk mobile phases [87]. systems where partition or nonlocalized adsorption controls
Polar analytes show very different selectivities under HILIC the retention, except for mobile phases with low concen-
and RP-LC conditions [88]. Sometimes polar compounds are trations of organic solvents. Because the retention in HILIC
well retained, and the elution order in HILIC is one of is proportional to the polarity of the solute and inversely
increasing hydrophilicity, which is roughly the reverse of that proportional to the polarity of the mobile phase (opposite to
seen in RP-LC. For example, Olsen [10] has compared the the retention in RP systems), Eq. 1 with φ taken to mean
properties of various stationary phases used for the separation the volume fraction of water rather than that of the organic
of pyrimidines and purines (polar compounds of significant solvent can be used to describe the effects of the mobile
biological and pharmaceutical interest). Figure 7 shows phase on the retention in HILIC systems, where the
changes in the selectivity of the separation of these types of partition between the adsorbed water and the bulk mobile
polar compound on the HILIC and RP-LC columns. phase is assumed to control retention [94].
It was demonstrated recently that the mechanism of For conventional nonaqueous normal phase systems,
HILIC separation involves various combinations of hydro- where retention is based on surface adsorption, the
philic interactions, ion exchange, and reversed-phase retention following equation was found to adequately describe the
by the siloxane on the silica surface, which contribute to retention model [95, 96]:
various degrees depending on the particular conditions 0 0
log k ¼ log k0  m  log 8 ; ð2Þ
employed [33, 34].
where φ is the volume fraction of the polar solvent in the
binary organic mobile phase, k′0 is the retention factor in pure
Characterization and selection of HILIC separation polar solvent, and m is the stoichiometric coefficient charac-
systems terizing the number of molecules of the strong solvent that are
needed to displace one adsorbed molecule of the analyte.
The selectivity depends not only on the stationary phase but The relative polarity of the mobile phase with respect to the
also strongly on the mobile phase [89]. stationary phase is a distinguishing feature that can be used to
In reversed-phase systems, the effects of the volume classify NP and RP systems. Hence, a dual retention
fraction of a strong organic solvent (φ) in a binary mobile mechanism, where the NP and RP effects contribute simulta-
phase on the retention factor k over a limited concentration neously to the retention, is a rather common phenomenon in
range of acetonitrile–water can be described by a widely hydrophilic interaction liquid chromatography [89].
used semi-empirical equation [90–93]: The retention factor (k) includes the solvophobic and the
polar contributions, which depend on the polarity of the
log k ¼ log k0  m1  8 ¼ a  m  8 : ð1Þ
solute, the chemistry of the stationary phase, and the
The constant a in Eq. 1 should theoretically be equal to the composition of the mobile phase [94]. Plots of retention
logarithm of the retention factor of the solute in pure water; factor versus binary aqueous–organic mobile phase com-

Fig. 7 Comparison of the selectivities obtained for polar pharmaceuticals using a reversed-phase ion pairing (LiChrospher RP-Select B column)
and b HILIC (Zorbax NH2) separation modes. Compound numbering is the same for both chromatograms. Adapted from the data in [10]
HILIC—a powerful separation technique 241

position show a “U shape” [97, 98]. Minimum retention at

the “U turn” composition of the mobile phase corresponds
to the transition from the RP to the NP (HILIC in an
aqueous–organic mobile phase) mechanism, and the reten-
tion at this point is usually very low [94, 99]. The U shape
of k versus the volume fraction of an aqueous buffer φ
(BUF) can be described by the following equation:
log k ¼ a1 þ m1  8 ðBUFÞ  m2  log 8 ðBUFÞ: ð3Þ
The parameters a1, m1 and m2 of Eq. 3 can be determined
by nonlinear regression of the experimental retention
factors measured for varying volume fractions of water in
the mobile phase. The parameter m1 characterizes the effect of
increasing the water concentration in the mobile phase on the
contribution of the RP mechanism to the retention, whereas
the parameter m2 is a measure of the opposite effect, Fig. 8 Comparison of columns through the cluster analysis of retention
data for peptide separation. Adapted from data in [106]
characteristic of the HILIC contribution to the retention in
highly organic mobile phases. a1 is an empirical constant [100].
To characterize the retention in liquid chromatography, the real merits of the QSRR approach in HILIC. Possibly,
quantitative structure–retention relationships (QSRRs) are comparing the retention data, either isocratic or obtained in
widely used. QSRRs are the most extensively studied gradient experiments with increasing salt and/or water con-
manifestations of linear free-energy relationships (LFERs). centration(s), may prove useful for the selection of appropriate
These are the statistically derived relationships that model the stationary–mobile phase combinations in HILIC of different
analyte retention as a function of properties (descriptors) sample types [94].
related to the molecular structure of the analyte and the
physicochemical properties of both the stationary and mobile
phase [73]. Using the QSRR approach, the chromatographic Orthogonal and two-dimensional separations of HILIC
column can be regarded as a “free-energy transducer,”
translating differences in the chemical potentials of solutes In 2D (LC×LC) (Fig. 9), the orthogonality between the two
resulting from differences in their structures into the dimensions is of paramount importance. It is generally
chromatographic retention parameter [101]. The QSRR understood that a 2D separation is orthogonal if the two
method can assist in elucidating the molecular mechanism separation mechanisms used are independent of each other
for separation that occurs in a given chromatographic system, and show distinct retention profiles; or, in other words, they
and can aid in the optimization of this process [102]. QSRR provide different selectivities [107–110].
models can be used to characterize and compare the The concept and meaning of orthogonality is presented
suitability of columns for both RP [103] and HILIC schematically in Fig. 10 using practical examples of NP-LC×
separations [104, 105]. Buszewski et al. [106] grouped RP-LC and RP-LC×RP-LC separations of lemon oil and
analytical columns based on the retention data for peptides. steroids, respectively. Low correlation (high orthogonality) is
They showed that different columns for RP-LC and HILIC observed in Fig. 10a, in which the compounds are randomly
can be clearly distinguished using the QSRR method (Fig. 8). distributed across the entire separation space, whereas there
Jandera et al. [99] investigated the retention of the antiox- is a high linear correlation for the separation shown in Fig. 10b.
idants phenolic acid and flavone under HILIC and RP In this case, the addition of an extra separation dimension
conditions, and characterized the selectivities of various HILIC leads to an unnecessary increase in the complexity of the
systems using a linear solvation energy relationship model system [112].
with molecular structure descriptors. True orthogonality is technically difficult to achieve, as
The main problem with QSRR prediction models is that orthogonality depends not only on the separation mechanisms
the results are affected by the composition of the mobile but also on the properties of the solutes and the separation
phase, which may become more or less adsorbed in the conditions. Successful orthogonal combinations can be
stationary phase and thus change its true nature. Therefore, achieved when the appropriate stationary and mobile phases
comparing the properties of HILIC stationary phases in are carefully chosen with respect to the physicochemical
order to predict the retention of polar compounds would be properties of the sample constituents, including size, charge,
probably more complicated than comparing RP columns polarity, hydrophobicity, etc. A variety of stationary phases are
[73]. Additional study in this direction is necessary to show presently available, with differences in surface chemistries,
242 B. Buszewski, S. Noga

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 asym-
metry, and even split peaks in the second dimension [99].
For this reason, off-line setups are generally used in
HILIC×RP-LC systems, with trapping columns and/or
make-up flow post-column adjustment of the mobile-phase
elution strength employed [114], which is often time-
consuming and not very convenient. Another possibility for
orthogonal HILIC×RP separations on a single polar column
is to use repeated sample injection on a single column with
alternating gradients of increasing (RP) and decreasing
Fig. 9 Scheme of the LC×LC sample transfer interface with two (HILIC) organic solvent concentration in the mobile phase
sampling loops or two X-Terra trapping columns. Adapted from the
[99]. Figure 11 illustrates the concentration profile of ACN
data in [111]
in a sequence of 10 min alternating gradient runs over the full
range of mobile phase composition (HILIC with 100–0%
support material, carbon load, pore size, etc., whereas the ACN and RP with 0–100% ACN), with 10 min equilibration
characteristics of the mobile phase can be altered by changing periods inserted between the alternating gradient runs.
the modifier, pH, temperature, or by adding ion pair agents. It has been concluded on several occasions that compounds
These parameters play important roles in the mixed-mode which display large k values on HILIC are not well retained
HILIC retention mechanism and can be flexibly tuned to suit by RP-LC and vice versa [112]. HILIC×RP-LC is the most
specific separation problems. orthogonal combination of liquid separation techniques when

Fig. 10 Theoretical representation of orthogonality in comprehensive LC: a NP-LC×RP-LC separation of lemon oil, b RP-LC×RP-LC
separation of steroids. Adapted from the data in [113]
HILIC—a powerful separation technique 243

Fig. 11 Schematic diagram of the ACN concentration profile in a sequence of alternating HILIC and RP gradients on a single column. Adapted
from the data in [99]

orthogonality is defined as the fraction of coverage of a 2D Two-dimensional liquid chromatography is often used to
separation space [115]. The benefits of the orthogonality of reduce the complexity of a proteomic sample prior to tandem
HILIC×RP-LC can also be utilized by running the same mass spectrometry analysis.
sample in both HILIC and RP-LC modes and combining the An elegant way to utilize the advantageous orthogonality
data. This provides more information on the total sample and of HILIC and RP-LC is to use solid-phase extraction (SPE)
can, for example, be used for enhanced protein identification to fractionate the sample before separation. The two
via database searches or in approaches with sample fingerprint fractions are then separated in either the HILIC or the RP-
analysis. For example, selected LC modes including RP-LC LC mode, depending on the polarity [34]. Other techniques,
and HILIC were combined in different 2D LC setups for the such as IC and size exclusion chromatography (SEC), result
separation of the natural antioxidants phenolic acid and in more peak grouping or poorer resolution in comparison
flavone (Fig. 12) by Jandera et al. [116]. to HILIC [115].
Other examples are the separation of peptides in 2005
by Gilar et al. [115], and of flavonoid glycosides in both
RP-LC and HILIC modes on a β-cyclodextrin column by Applications of HILIC
Feng et al. in 2010 [46]. 2D techniques combining RP and
HILIC chromatography represent powerful tools in the There are many examples of the application of HILIC to the
analysis of pharmaceuticals and their degradation products, analysis of small polar molecules, including biomarkers,
and in the metabolic profiling of physiological fluids [61]. nucleosides, nucleotides/oligonucleotides, amino acids,

Fig. 12 Three-dimensional chromatogram for the comprehensive LC×LC separation of phenolic acid and flavone test standards with an
optimized two-dimensional parallel gradient. Adapted from the data in [116]
244 B. Buszewski, S. Noga

peptides and proteins, saccharides, glycosides, oligosac- metabolomics, glycomics, medical science, agricultural
charides, hydrophilic drugs, alkaloids, carbohydrates, and and food chemistry. The HILIC mode is used intensively
other small polar or ionizable compounds, which contribute to separate some biomolecules through differences in
to the fields of pharmaceutical chemistry, proteomics, polarity, as well as organic and some inorganic molecules.

Table 3 Several examples of applications of HILIC systems

Type of packing Mobile phase Detection Groups of detected Reference

compounds

Polyhydroxyethyl A Salt gradient in TEAP UV-Vis Peptides, [6]

buffer with ACN amino acids
Polyhydroxyethyl A Isocratic elution, (A) ACN Electrospray ion trap Metabolites occurring [46]
and (B) 6.5 mM ammonium mass spectrometry in different plant species
acetate (pH 5.5) (ESI-MS)
Luna NH2 Isocratic elution, (A) ACN Ion trap mass Cancer biomarker in urine [37]
and (B) 13 mM ammonium spectrometry (proteomics approach)
acetate buffer, pH 9.1 (MS)
Spherisorb ACN/methanol/phosphoric UV/DAD Phospholipids from [117]
silica gel acid (99/3/1 v/v) pulmonary surfactant
Cyclodextrin Isocratic elution, (A) ACN UV/DAD Chiral separations of [118]
stationary and (B) 6.5 mM ammonium drug substances and
phases acetate, pH 5.5 buffer underivatized amino acids
Cyanopropyl Methanol/ACN (60:40) with or Electrospray ionization Free folic acid in human plasma [9]
stationary without 50–200 mM ammonium tandem mass spectra
phases acetate or ammonium formate; with collision-induced
or 100% toluene dissociation (CID)
TSKgel Gradient elution: 13–27% water Electrospray ionization Small polar compounds [119]
Amide-80 in 45 min and 27–40% water mass spectrometry in food analysis
in 5 min; (A) 90% ACN and (ESI-MS)
ammonium acetate buffer (pH 7.0)
and (B) 60% ACN and ammonium
acetate buffer, pH 7.0
Triazole-bonded ACN–water (90:10) UV-Vis Diazolidinyl urea, urea, and [120]
silica allantoin in cosmetic samples
ZIC-HILIC Gradient elution, 5% mobile phase A Mass spectrometry Metabolomic fingerprint [13]
increasing linearly to 95% over a (MS) in human urine
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
DIOL Isocratic elution, ammonium formate UV-Vis Adrenoreceptor agonists and [121]
antagonists—used as therapeutic
agents in the treatment of
hypertension, cardiac arrest,
and other medical conditions
Luna NH2 (A) ACN with 0.05% formic Mass spectrometry Sulfonamide antibacterial [122]
acid and (B) water (MS) residues in milk and egg
ZIC-pHILIC Gradient elution, (A) ACN with Tandem mass Free estrogens and their [123]
aqueous ammonium acetate spectrometry conjugates in river water
(pH 6.80) (95/5, v/v) and (B) (MS/MS)
ACN with aqueous ammonium
acetate (pH 6.80) (75/25, v/v)
Polyhydroxyethyl A (A) ACN and (B) potassium phosphate, UV-Vis Polar pharmaceuticals and impurities [10]
pH 6.5 acetonitrile (20:80, v/v)
Supelcosil LC-SI Gradient elution, 0 min: 1.1% B; UV-Vis Orange essential oil and juice [124]
1 min: 2% B; 30 min: 4% B; 45 min: carotenoids
5% B; 75–160 min: 9.9% B; (A)
n-hexane and (B) ethyl alcohol
ZIC-HILIC (A) ACN with 0.1% v/v formic acid; Mass spectrometry Neutral sugars, sugar phosphates, [125]
(B) 5 mM ammonium acetate with (MS) sugar alcohols
0.1% v/v formic acid (pH 4.0)
TSKgel Amide-80 (A) ACN; (B) 10 mM ammonium Ion trap mass Comprehensive analysis of the [126]
acetate (pH 5.5) spectrometry (MS) microbial metabolome
HILIC—a powerful separation technique 245

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