Introduction to HILIC chromatography

Hydrophilic Interaction Chromatography (HILIC), is a chromatographic mode used to

exploit differences in analyte polarity. It can especially be used when reversed phase
separations do not give satisfactory retention or separation. It is not aqueous normal
phase, but something uniquely suited to the analysis of polar molecules in an aqueous,
aqueous - miscible organic mobile phase.

The HILIC mode of chromatography can be advantageous for the following reasons:

• Improved retention of polar and ionisable analytes which show poor retention
under reversed phase conditions
• A useful alternative (orthogonal) selectivity where separation under reversed
phase conditions requires verification or enhancement
• Enhanced detector sensitivity when using electrospray mass spectrometry due
to the improvement in spraying and droplet desolvation with highly organic
eluent systems and lowering of ion suppression via lower buffer ion
concentrations (see Figure 1)
• Possibility of direct injection of solid phase extraction eluates which are
typically high in organic content without suffering peak shape effects (or
elimination of SPE of biological
samples by a precipitation with 80% acetonitrile, centrifuge and inject
protocol)
• Increased eluent flow rates due to lower eluent viscosity (90% acetonitrile
solutions are typically half of the viscosity of 30% solutions)
• Faster mass transfer at lower eluent viscosity giving, better efficiency at higher
eluent flow rates
• Improved peak shape for compounds that show tailing in reversed phase
separations
• Analysis of polar organic analytes and their (typically) inorganic counter ions
in a single separation

The term HILIC was first coined by Alpert1 in the early 1990’s to differentiate this
chromatographic mode against reversed and normal phase separations. Typically, in
simple HILIC mode, the elution pattern resembles the inverse of a reversed phase
separation. As a first approximation of what you expect to see, flip over your reversed
phase chromatogram! But, HILIC will additionally show separation of neutral polar
analytes which might co-elute in reversed phase mode.

HILIC mode eluents will typically contain organic (e.g. acetonitrile) and aqueous
components. This helps overcome some of the challenges associated with normal
phase eluent systems, including limited polar (and polar biological) analyte solubility,
difficulty in swapping HPLC equipment from reversed phase to normal phase modes
and the cost and environmental impact associated with disposal of large amounts of
organic solvent.

Figure 1 shows the relative sensitivity of ESI-MS detection of various HPLC modes
against analyte polarity. For example, if one uses methanol as the aqueous miscible
organic modifier, one might typically achieve 100x the sensitivity of reversed phase
chromatography.
Figure 1: HILIC mode relative to reversed and normal phase chromatography in terms
of analyte polarity and ESI-MS response (Reproduced with permission of Waters Inc.,
Milford Massachusetts, USA)

Retention mechanism in HILIC

The HILIC retention mechanism is a complex combination of liquid-liquid

partitioning, dipole-dipole and hydrogen bonding interactions as well as Coulombic
(electrostatic) interactions. As such, the HILIC mode provides a useful alternative
selectivity to that obtained with reversed phase separations.

Understanding the possible interactions between the analyte and stationary phase
surface and the effects of eluent pH and ionic strength on these interactions is
important in developing and optimizing robust and effective HILIC separations.

Figure 2 (below): Under reversed phase conditions the retention and response for the
polar ranitidine molecule is poor whilst both retention and response are good under
typical HILIC conditions. (Reproduced with permission of Agilent Technologies,
Santa Clara, California, USA)
The fundamental HILIC separation characteristics can be outlined as follows:

• Any polar stationary phase can be used in a HILIC mode. Polarity can come
from surface silanol moieties or from a bonded phase's polar groups.
Functionalised silicas are extensively used due to their durability and
reproducibility and will be outlined later in this Resolver edition.
• The polar stationary phases can adsorb a ‘water enriched’ layer at the silica
surface which is more highly aqueous (hydrophilic) than the general mobile
phase composition flowing through the column
• Retention is proportional to the polarity of the analyte and inversely
proportional to the polarity of the mobile phase
• As the stationary phase is polar, mobile phases generally compose a high
degree of water miscible organic solvent with a smaller amount of water (the
opposite to reversed phase HPLC)
• The difference in polarity between the mobile and stationary phases dictates
retention of the generally polar analytes
 • At low mobile phase water concentrations, polar analytes favour the polar
stationary phase environment and retention is increased
• As the amount of water in the mobile phase is increased, the mobile phase
polarity increases and this facilitates elution of the analyte molecules
• Both coulombic (electrostatic) and Partitioning mechanisms are important in
HILIC
• Any electrostatic interaction in HILIC is analogous to aqueous, ion exchange
chromatography where the analyte and ‘strong solvent’ mobile phase
molecules (water in this case), compete for the polar (charged) stationary
phase sites (silanol species in Animations 1 & 2). Where buffer ions are
included in the eluent, these will also compete for any ionised stationary phase
sites.
• The partitioning mechanism is a unique and interesting feature of HILIC
chromatography which is driven by the relative solubility of the analyte in the
mobile phase and the hydrophilic layer of bound water at the stationary phase
surface. Partitioning processes analogous to liquid-liquid extraction occur at
the interface of the eluent and bound water layer and analytes partitioning
strongly into the bound layer exhibit increased retention 2 (See Animation 1)
• It is generally accepted that a minimum of 3% water is required in order to
obtain a significant and usable hydration layer at the stationary phase surface 3
• Secondary retention mechanisms such as dipole-dipole and hydrogen bonding
interactions undoubtedly play a part in the fundamental HILIC separation
mechanism. However the most important secondary mechanism is
electrostatic interaction between any column surface charged species and
charged analyte moieties – this will be studied in greater depth subsequently
and will be dependent upon the mobile phase pH, the buffer strength, the pKa
of the column surface and that of the analyte. Manipulation of these
parameters allows the orientation of an analyte vis a vis a stationary phase
selection, allowing enhanced selectivity options, and/or elution with low
buffer strength of ionic analytes (i.e., eHILIC, ERLIC).

Figure 3 highlights some interesting aspects of the HILIC separation mode and the
various mechanisms of interaction for various analytes.

For retention in the HILIC mode – it is generally accepted that the mobile phase
should contain greater than 70% acetonitrile. The example above indicates a rapid
increase in retention as the organic increases from 70 to 95%. 4

However, at low % acetonitrile a different effect is seen. Now the more hydrophobic
nortriptyline molecule is less soluble in the mobile phase and is more likely to interact
with the stationary phase surface. We will see later that it is possible for the cationic
nortriptyine molecule to interact via electrostatic interactions with the anionic surface
(silanol) species. The nicotinic acid is more polar and as such is more soluble at high
water content and therefore is less likely to interact with the stationary phase surface
than the nortriptyline. Further, the analyte is anionic, and therefore may suffer
repulsion effects between the anionic carboxylate function and any surface anionic
sites.
 Figure 3: Relationship between retention and % acetonitrile in the mobile phase
when using pH 5.5 and <20mM buffer HILIC separation conditions.
(Reproduced with permission of Waters Inc., Milford Massachusetts, USA)

The interplay between adsorption, partitioning and hydrophobic retention mechanisms

comes to the fore when using bonded phase ligands for HILIC separations and these
will be studied in more detail subsequently.

Figure 4 shows a general schema for the relationship between analyte LogP and the
corresponding separation mode of choice. It should be noted that this is a general
guideline and the user should not be dissuaded from attempting HILIC separations
with analytes whose Log P > 0 where reversed phase retention / separation is poor.

Figure 4: General schema for analyte Log P value versus separation mode of choice.
(Adapted with permission from SIELC Technologies, Prospect Heights, Illinois, USA)
HILIC Mobile phase solvent choice

As has been stated, HILIC mobile phases typically contain between 3 and 70% of the
strong solvent (water), with retention tending to increase exponentially at <10% of the
strong solvent.

Figure 5: Effect of increasing acetonitrile eluent composition on the retention of

cyanuric acid using a silica stationary phase (Reproduced with permission of Agilent

Technologies, Santa Clara, California, USA)

The most popular weak solvent is acetonitrile, due to its aprotic (intermediate polarity,
lacking an acidic proton) characteristic which encourages retention of polar analytes.
Figure 5 illustrates the increase in retention of the polar analyte cyanuric acid as
acetonitrile concentration increases using a silica stationary phase.

Other ‘weak’ solvents may be used in HILIC mode and the general solvent strength
series for HILIC separations is:

Acetone can be used in place of acetonitrile where extra retention is required or there
is a need to reduce the amount of acetonitrile. However, it should be noted that the
selectivity of the separation will be different and as acetone has a UV cut-off of
330nm it is generally unsuitable for UV detection. Most MS and evaporative light
scattering detectors can be successfully used with acetone as the organic solvent if the
detector operating parameters are optimized.

The protic solvents (methanol, ethanol, isopropanol etc.) tend to result in much
shorter retention times (Figure 6), as the polar nature of the solvent results in
increased competition for polar stationary phase sites and a disruption of the adsorbed
water layer. 5
 Figure 6: Retention factor k as a function of the concentration of acetonitrile (closed
symbols) or methanol (open symbols) as organic modifier in the eluent. Legend:
(triangles) cytosine (squares) adenine.

If it is necessary to use an alternative solvent (to intentionally increase retention

times or alter the separation selectivity), it is more usual to replace a portion of the
water content with one of the alternative solvents, rather than substituting the
acetonitrile – this concept is illustrated in Figure 7. Retention times increase due to
the substitution of water with the less polar alcohol, however retention will be shorter
than substitution with acetonitrile alone. This approach can be helpful when fine
control of retention times is required, when a change in selectivity is necessary or
when analyte solubility is an issue.

Figure 7: Changes in selectivity and retention through the use of mixed ‘strong
solvents’ in HILIC mode separations. (All phases contain - 10mM ammonium acetate
with 0.02% acetic acid)
1 – Methacrylic acid
2 – Cytosine
3 – Nortryptyline
4 – Nicotinic acid

(Reproduced with permission of Waters Inc., Milford, Massachusetts, USA)

Effect of pH in HILIC separations

There are several aspects that we need to consider regarding the analyte, stationary
phase chemistry and local solvent environment at the silica surface as mobile phase
pH is altered and these considerations are outlined here:

Acidic analytes – as the mobile phase pH is lowered (relative to the pka value) the
analyte becomes increasingly non-ionic:

Nicotinic acid - acidic functional group pKa – 4.8

Eluent pH of 2.8 the analyte will be totally non-ionised
Eluent pH of 6.8 the analyte will be totally ionised (de-protonated)

Basic analytes – as the mobile phase pH is raised (relative to the pKa value) the
analyte becomes increasingly non-ionic:

Nortriptyline - Basic functional group pKa – 9.7

Eluent pH of 12.0 the analyte will be totally non-ionised
Eluent pH of 8.0 the analyte will be totally ionised (protonated)

• As the degree of analyte ionization decreases, analyte polarity decreases,

analytes will be more soluble in the mobile phase relative to the adsorbed
surface water-enriched layer, retention will tend to decrease
• As the degree of analyte ionisation decreases, they are less able to
electrostatically interact with cationic sites (for acids) or anionic sites (for
bases) on the stationary phase surface and retention will tend to decrease
 • As the degree of analyte ionization decreases, it will be less repelled by any
anionic (for acids) or cationic (for bases) stationary phase surface sites and
retention will tend to increase

Stationary phase surface – we will begin by considering silica as the stationary phase
and explore the other bonded HILIC and Mixed Mode chromatography phases
subsequently:

Figure 8: Surface silanol equilibrium on a bare silica HILIC stationary phase

There are several forms of surface silanol group on silica stationary phases, however
the pKa of most forms lies between 4 and 5. Therefore, as the eluent pH is raised
above a value of 5, the acidic silica surface becomes increasingly charged (anionic).
This has three important effects in HILIC mode:

1. The surface becomes increasingly polar

2. The increasingly anionic surface will lead to increased electrostatic
interactions with charged basic analytes, and hence tend to increase retention
3. The anionic surface will tend to decrease retention of acidic species through
electrostatic repulsion

These three parameters will have a complex interplay of effects for a particular
analyte (or separation) and it cannot be said, for example, that acidic analytes have
poor retention on bare silica at high pH in HILIC mode, as the partitioning into the
adsorbed polar hydrophilic layer, may counterbalance any electrostatic repulsion
effects. Any predictions of analyte behavior should be empirically tested.

Of course the opposite is also true, and as the eluent pH is lowered below pH 3 or so,
the silica surface becomes increasingly neutral and:

1. The stationary phase surface will become increasingly less polar

2. The surface will become increasingly neutral and therefore less likely to retain
cationic analytes through electrostatic attraction
3. Anionic analyte retention will tend to increase due to a lowering of the
electrostatic repulsion from the silica surface.

Figure 9 shows a simple example which can be used to investigate the behavior of the
analyte and stationary phase surface under different pH conditions
 Figure 9: Influence of eluent pH on selectivity and retention in HILIC using a silica
stationary phase
(Reproduced with permission of Waters Inc., Milford, Massachusetts, USA)

Figure 10: Interplay between analytes and stationary phase surface at pH3
pH-3 Commentary

The silica surface is extensively non-ionised and therefore has a very limited ability to
undertake electrostatic interactions. It is thus a neutral HILIC surface at this pH.

Methacrylic acid (1), whilst being moderately polar (Log P 1.81) at neutral pH, at pH
3, the carboxyl will be protonated . Compared to amine containing analytes it will be
uncharged, less polar and therefore show shorter retention time compared to basic
analytes in the mixture.

Nortriptyline (2), is more hydrophobic (Log P 4.74) and therefore less attracted to the
hydrophilic adsorbed water layer and will be limited to very few opportunities for
electrostatic retention due to the ion-suppressed silica surface, giving rise to poor
retention

Nicotinic acid (3) will be partially charged from the pyridinium nitrogen and also has
the neutral protonated carboxyl (and therefore is moderately more polar than either
the single polar group of nortriptyline and the methacrylic acid) thus it shows
reasonable retention.

Cytosine (4) is highly polar (doubly charged at this pH) and shows good retention via
partitioning into the adsorbed hydrophilic layer and hydrogen bonding to the
protonated silanols

pH9 Commentary

The silica surface is completely ionised, readily undergoes electrostatic interactions

with cationic analytes and shows a degree of repulsion towards anions.

Methacrylic acid (1), shows increased retention due to complete ionization of the
carboxyl and a more polar surface chemistry. Note: A change in the amount of
organic in the mobile phase may be used to counterbalance repulsion between the
anionic surface and a charged analyte.

Nortriptyline (2), shows slightly improved retention from the increased polarity of the
now anionic surface. The pH is close to its pKa so there will be a diminished
electrostatic attraction of the opposite charges.

Nicotinic acid (3) the two polar groups are completely ionized at this pH, and shows
much improved retention through partitioning into the adsorbed hydrophilic water
layer. It should be noted that this is not always the case and there will be cases where
retention of acidic analytes at high pH will be poor due to electrostatic repulsive
effects from an anionic surface.

Cytosine (4) shows improved retention due to the more polar surface and possible
electrostatic interaction. But due to the reduction in degree of ionization (polarity) as
the pH is now higher than the pKa of the ring nitrogen, it is less retained than
nicotinic acid.
Figure 11: Interplay between analytes and stationary phase surface at pH9

It should be obvious from the commentary above that the relationships between the
analytes and stationary phase surface are complex at varying pH. To illustrate this
further, Figure 12, shows an example in which strong acids (7,8) are completely
unretained at higher pH on a silica stationary phase. 3 This is assumed to be due to
extensive repulsion of the strong acids by the extensively anionic silica surface.

In general, retention of stronger basic species should increase with increasing pH due
to the increasingly charged silanol surface and this is seen with the nortryptyline (pKa
9.7) retention in Figure 12. However, it is interesting to note that retention of the
quaternary diphenhydramine (pKa = 9.0), the weakest base in the test mixture, does
not increase in retention as the degree of analyte ionisation is significantly reduced
between pH 7.5 and pH 8.2. This illustrates the need to consider changes in the degree
of ionization of both the stationary phase surface as well as the analyte with eluent pH
and the profound effect these ionisation (polarity) changes can have on the selectivity
of HILIC mode separations.

It is recommended that whilst an understanding of pH effects is useful to help explain

empirical results, the first approach should always be experimentation based on an
understanding of the HILIC retention mechanism.
Figure 12: Analysis of test solutes on Atlantis silica.
Mobile phase ACN–0.1 M, HCOONH4, ww pH 8.2–10.2 (85:15, v/v).

1 = phenol
2 = caffeine
3 = nortriptyline
4 = diphenhydramine
5 = benzylamine
6 = procainamide
7 = 2-naphthalenesulfonic acid (2-NSA)
8 = p-xylenesulfonic acid (p-XSA)
(Reproduced with permission from reference 3)
 Effect of Buffers and additives

Changes in eluent buffer ion concentration have a profound effect on retention in

HILIC chromatography due to their influence on the degree of analyte and stationary
phase ionization and the polarity of the eluent. 7,8

Having a sufficiently high ionic strength counter ion is often essential to achieving
good peak shape and satisfactory / reproducible retention in the HILIC mode. The
type and concentration of additive or buffer chosen can figure as a primary method
development parameter for altering the retention and selectivity characteristics of
HILIC methods. The importance of having a counter ion is likely to be related to
changes in the molarity (and hence the polarity) of the adsorbed water layer and
increased competition for ion exchange sites at the silica surface, as well as regulation
of the degree of ionization of analyte species under various pH conditions. These
effects are further explained below.

Ammonium formate and acetate are popular buffers for use in HILIC separations as
they are readily soluble (20mM is typical for HILIC separations), in highly organic
solvent systems and are volatile enough to permit their use with MS detection.
Crucially, they also provide important counter ions necessary for good peak shape in
many HILIC separations, through improved kinetics of diffusion and surface
interactions. 7

There are many effects that need to be considered regarding the effects of buffer
addition and we might briefly consider these using an ammonium acetate buffer in the
separation from Figure 9.

Ammonium Acetate at pH9 - Commentary

The addition of the ammonium acetate buffer serves to increase the molarity of the
eluent system, especially the adsorbed water layer. The increase in buffer strength
results in a decrease in retention. It does however result in orientation or pairing
effects as illustrated which can shift the retention order. 12 Points A, B and C in the
figure are worthy of special note.

A – the ammonium ion can act to neutralize the charge on the silica surface which
potentially leads to two phenomena
1. reduction in retention of the basic (cationic) species which may otherwise undergo
electrostatic interactions with the silica surface
2. increase in retention of acidic (anionic) species due to ‘shielding’ of the charged
surface which may otherwise act to electrostatically repel the analyte. Of course other
bonded phase ligands may act in different ways and we will study these in a
subsequent section

B – the formate counter ion may ‘ion-pair’ with the basic analyte species, acting to
reduce retention through reduced electrostatic interactions with the silica surface
combined with a general lowering of the analyte polarity
C – the ammonium ion may ion-pair with acidic analytes, resulting in a reduction in
analyte polarity (tending to reduce analyte retention) whilst the reduction in silica
surface polarity will tend to lead to increased acidic analyte retention.

Figure 13: Effects of ammonium acetate buffer addition to a HILIC separation using
a silica column at pH 9

As can be seen from the commentary regarding the effects of buffer addition to this
separation, these effects can be highly interactive and one must, as always, use
‘predictions’ of the effects on a separation alongside empirical results in order to
optimize our separations.

One recent study using a silica column in a HILIC mode, shows the results of varying
buffer concentration on the retention and separation of several test compounds (Figure
14). 8
Figure 14: Plots of retention factor vs. 1/[counter-ion concentration] for silica HILIC
columns.
Mobile phase: ACN–water (90:10, v/v) containing ammonium formate (concentration
varied from 2 to 10 mM) pH 3.0.
(Reproduced with permission from reference 8)

One should note from Figure 14 that under the experimental conditions, the strong
acid (p-xylenesulfonic acid) shows poor retention as does the neutral species
(caffeine). The acidic species may be subject to repulsion (actual solution pH ~ 5.1)
from a partially charged silica surface. However the behaviour of the basic species
does follow the predictions above, and as buffer strength is increased (note: from right
to left on the figure), the retention of the basic species decreases, primarily due to a
reduction in analyte and surface charge (from ion pairing with the buffer ions) as the
concentration of counter ions increases, leading to an overall reduction in analyte
polarity and lowering of the electrostatic interaction between the analytes and the
silica surface. In this example, the selectivity of the separation (relative spacing of the
various lines within the plot), does not change appreciably, however, this is not
always the case, especially with some of the cationic bonded phases used for HILIC
separations or when acidic and basic species are present in the sample. Use of a
"neutral" polar bonded phase for HILIC, with sufficient buffer, overcomes the
variable surface conundrum. A minimum of 20mM achieves this for most molecules.

Table 1 shows data which correlates the degree to which the overall retention of the
test compounds is electrostatic as opposed to partitioning or other mechanisms. 8

Procainamide Benzylamine Diphenhydramine Nortriptyline

2 2
10 mM 10 mM 2 mM 10 mM 2 mM 10 mM
mM mM
Silica 55 28 50 25 70 43 70 42

The table clearly shows that the electrostatic retention mechanism is very important
when using bare silica stationary phases in HILIC to separate basic solutes - 50-70%
of the total interaction is electrostatic at 2mM ammonium formate. Further, and as
predicted, as buffer concentration increases, the contribution of electrostatic retention
mechanisms to overall basic analyte retention is lowered considerably, reflecting the
overall reduction in surface and analyte charge as described above.

Some column manufacturers recommend the use of formic and trifluoroacetic acids as
additives for HILIC – which for certain examples may be useful, and the use of a
counter-ion is recommended for reproducibility. Figure 15 demonstrates the use of
0.1% TFA to improve the retention of acidic analytes and alter separation selectivity
using a silica stationary phase. It should be noted that the eluent solution pH values
are 1.35 for 0.1% TFA and around 5.2 for the ammonium formate buffer solution.
Clearly the TFA is acting to ion-suppress the analyte and silica surface charge,
reducing electrostatic repulsion and improving retention. It should be noted that TFA
is not a buffer of choice for MS detection due to ion suppression effects and that
retention of bases may reduce through the formation of less polar ion pairs with the
acid counter ion.

TOP:
Analysis of test compounds on Atlantis silica.
Mobile phase ACN–water (95 /10 v/v) containing overall 0.1% TFA

BOTTOM:
Analysis of test compounds using Atlantis silica.
Mobile phase 10% ACN in 1.5 mM HCOONH4, pH 3.0.
Peak identities:
1 = phenol
2 = caffeine
3 = nortriptyline
4 = diphenhydramine
5 = benzylamine
6 = procainamide
7 = 2-naphthalenesulfonic acid (2-NSA)
8 = p-xylenesulfonic acid (p-XSA)
Flow 1 mL min−1; injection volume 5μL,
all solutes 50 mg L−1 except 2- NSA 12.5 mg L−1;
Detection UV 215 nm.
(Reproduced with permission from reference 3)

One should be aware that when operating gradients in HILIC mode, if the aqueous
component alone is buffered, the overall buffer concentration may change as the %
water changes with the gradient. It should be borne in mind that if the ionic strength
affects selectivity, then selectivity changes may occur when developing gradient
HILIC methods. In general, the higher the aqueous buffer concentration, the lower the
impact on selectivity. Of course, once a suitable method has been developed, provided
the mobile phase components are prepared in the same fashion, the separation should
be absolutely reproducible.

Stationary phases

So far we have concentrated on the use of high organic content mobile phases and
bare silica stationary phases for HILIC mode separations. The information gleaned
will stand us in good stead to understand HILIC separations using a variety of
commercially available bonded phases, however we need to look at some of these
phases in more detail to understand their advantages, disadvantages and potential
applications.

Shown below are just some of the bonded phases available which can operate in the
HILIC separation mode. 8

Figure 16: Representations of some typical bonded phases available for HILIC and
‘mixed mode’ chromatography

1 &2 – Generic structures for Amide and Diol phases (or analogues) offered by a
variety of manufacturers
3, 4 & 5 – Mixed-mode column structures with terminal functional chemistry based
on the Acclaim® column series from Dionex (Sunnyvale, California UA)

6 ,7 – HILIC Mixed-mode columns with embedded functional chemistry based on the

Primesep® series from SILEC Technologies (Prospect Heights, Illinois, USA)

8 – Zwitterionic phase based on ZIC HILIC® (MerckKGaA, Darmstadt, Germany)

9 & 10 – Mixed-mode zwitterionic columns - zwitterionic phases based on the

Obelisk® series from SILEC Technologies (Prospect Heights, Illinois, USA),
typically used with more conventional reversed phase solvent systems

9. Zwitterionic – Long Hydrophobic 10. Zwitterionic – Long Hydrophilic

Chain Chain
Amide phases – are designed for the extended retention of acidic analytes in HILIC
via electrostatic interaction with the cationic surface at mid pH where the stationary
phase and acidic analytes will both be ionised. Basic species may be less well
retained, relative to retention on bare silica for example, due to moderate electrostatic
repulsion especially at low buffer concentrations.

Diol Phases – are designed to maximize the partition and adsorption interactions of
the HILIC mode and to reduce the degree of electrostatic interaction between the
analyte and stationary phase surface. It should be noted however that most bonded
phases will have some degree of acidic surface silanol species that will be ionised at
mid pH. A characteristic feature of the diol bonded phases is their ability to orient in
order to shield the surface charge, and therefore show good retention for acidic
species.

Mixed mode phases – contain anionic or cationic groups embedded into (or bonded
onto the stationary phase surface alongside) a hydrophobic chain. This allows
electrostatic interaction with the ionised group at the correct eluent pH as well as the
classic adsorption of a water layer at the stationary phase surface (which will also
occur to the polar silanol groups which will be present on the underlying silica
surface). The main difference with these phases is the hydrophobic chain which is
capable of undergoing hydrophobic type retention with analytes of higher LogP at
increased eluent water content. Whilst some hydrophobic retention occurs with silica
phases, this only occurs at very high water content (>70% aq.). With mixed mode
phases, some degree of hydrophobic retention is possible with as little as 10 - 15%
water. Manufacturers embed the different charged functional groups at different
positions within the alkyl chains in order to alter the retention characteristics and
degree of stationary phase ionisation at various pH values and eluent water content.

Zwitterionic phases – are classified into two groups. Those with shorter alky chains
are designed such that the synthetic placement of ligands results in a net charge of
zero or near zero on the silica surface. This effectively reduces the degree to which
the analyte will undergo ion-exchange interactions, however the polar surface will
attract a significant adsorbed water layer for partitioning and the pH of the eluent
solution may be adjusted to alter the degree of ionization of the analyte molecule to
explore the retention and selectivity differences that this creates. Further, a net surface
charge of zero results in a reduction of electrostatic repulsion as seen with acids on a
silica stationary phase or bases on an amide phase.

Phases with longer alkyl chain spacers are typically used with much higher water
content than HILIC where the alkyl moities are able undertake hydrophobic
interaction with analyte molecules. Here, crucially, the spacing between the charges
on the ligands are designed so that both charged groups can function as ion
exchangers and the net surface charge is not zero – hence both anionic and cationic
groups may be retained via ion-exchange and the hydrophobic portion of the analyte
molecule is retained via the non-specific hydrophobic retention mechanism (see
Figures 18 and 19 for more details). These ‘mixed-mode’ columns will feature more
heavily in a future Resolver newsletter.
Figure 17 shows these effects on various HILIC columns. 8 Note that the exact surface
chemistry of the columns used are not necessarily represented by the structures in
Figure 16 above.

Figure 17: Chromatograms of probe compounds on five different HILIC phases.

Detection: UV at 215 nm
Column temperature: 30 ◦C.
Peak identities:
1 phenol
2 2-naphthalenesulfonic acid
3 p-xylenesulfonic acid;
4 caffeine;
5 nortriptyline
6 diphenhydramine
7 benzylamine
8 procainamide.

Flow rate: 1mL/min.

Mobile phase: (a) ACN–water (85:15 ,v/v) containing 5 mM ammonium formate pH
3.0. (b) ACN–water (95:5, v/v) containing 5 mM ammonium formate pH 3.0.

(Reproduced with permission from reference 8

It should be noted firstly that the actual eluent pH at 95% organic was 6.1 and at 85%
was 5.2.
 The differences in selectivity of the phases for the various test probes is obvious and
in general the results are in good agreement with the behaviors described above for
each of the phase types, however some specific points are worthy of note.

The diol phase shows particularly good retention of acidic compounds (2 -

naphthalenesulfonic acid, p-xylenesulfonic acid) with reasonable retention also shown
on the amide and zwitterionic phases. In particular the selectivity of the separations
using the diol and amide phases changes markedly at different eluent compositions (%
water) indicating their usefulness for method development, especially when analyzing
mixtures of acidic and basic species.

The selectivity of the basic species changes markedly using the zwitterionic column
as the eluotropic strength is altered, again perhaps indicating the usefulness of these
phases for the analysis of bases.

In most cases the hydrophilic bases (benzylamine, procainamide) are eluted after the
more hydrophobic bases (nortriptyline, diphenhydramine) with the exception of the
mixed mode phase, where all bases elute over a narrow retention window and
nortriptyline is retained longest at higher eluent water content. Presumably this is due
to the increased contribution of hydrophobic retention at higher water content and
highlights the multi-modal potential of these columns.

Once again, it should be clear that the retention mechanism in HILIC mode is
complex and does not rely solely on the partitioning of analytes into and out of an
adsorbed water layer at the silica surface. 9 Further information regarding the degree
of electrostatic interaction involved in the retention of the basic analytes on the
various phases shown above is contained in Table 2.

Procainamide Benzylamine Diphenhydramine Nortriptyline

2 mM 10 mM 2 mM 10 mM 2 mM 10 mM 2 mM 10 mM
Diol 18 7 7 3 39 17 29 11
Zwitterion 58 32 58 32 78 55 78 55
Mixed-Mode 54 27 56 27 55 27 49 22
Amide 49 24 41 19 73 46 72 47
Table 2: % contribution of ion exchange to k at two different levels of counter-ion
concentration for 4 basic solutes.

From Table 2, the diol phase shows a very low contribution to retention via ion-
exchange, which is explained by the non-ionisable nature of the bonded phase ligand.
Presumably most of the electrostatic interactions are with acidic surface silanol
species.

The zwitterionic phase shows very similar results for each of the hydrophilic and
hydrophobic bases, with the hydrophobic bases being retained via electrostatic
interaction rather than partitioning or adsorption due to their lower polarity. This may
give an indication of the ability to differentiate between more or less hydrophobic
species based on both Log D and pKa differences.

Mixed mode phase (note – of type 6 and 7 shown in Figure 16), show very similar
results for all of the bases – again perhaps indicating that the relative retention
(selectivity) for these species may be better optimized by exploiting differences in
hydrophobicity, although it is fair to say that only one pH and buffer concentration
was used to obtain the results in Table 2 and these conditions would require further
investigation.

The amide phase shows differences in the degree of ion-exchange interaction between
the more hydrophilic and more hydrophobic bases within the test mix – which is
perhaps surprising given that the analyte and surface will both be cationic. One
possible explanation is retention of the bases on the acidic silanols which remain after
bonded phase application.

As a brief introduction to Mixed Mode chromatography under Non-HILIC conditions,

Figures 18 and 19 demonstrate the usefulness of mixed-mode zwitterionic columns
with longer alkyl chains and their ability to retain polar and ionisable compounds
whilst working in the hydrophobic (reversed phase) rather than HILIC mode

Figure 18: Separation of amino acids, bases, acids, and neutrals on Obelisc R (see
structure 9 in Figure 16)

Column: Obelisc R, 150 x 4.6 mm

Mobile phase: MeCN 35%, Ammonium Acetate10 mM pH 4.0,
Flow: 1.0 ml/min,
Detection: UV 250 nm

1. Phenylalanine
2. Trypthophan
3. Phenol
4. Benzonitrile
5. Pyridine
6. Toluene
7. 2,6-Lutidine
8. Benzylamine
9. Benzoic acid

Figure 19: Separation of hydrophilic basic drugs at various eluotropic strengths to

illustrate mixed mode behaviour

Column: Obelisc R (see structure 9 in Figure 16), 150 x 4.6 mm

Mobile phase: As indicated,
Flow: 1.0 mL/min,
Detection: UV 250 nm

1.Epinephrine
2.Phenylephrine
3. Norepinephrine

More information on the usefulness of mixed mode and zwitterionic phases will be
presented in the Part 2 of this publication.

Practical Aspects
Diluent Solvent Choice

One of the primary reasons for failure to develop suitable methods or to properly
adopt HILIC as a technique is the inappropriate choice of sample diluent, which, as in
reversed phase HPLC, can have serious effects on peak shape, peak efficiency, signal
response and retention reproducibility. 10

To match the eluotropic environment in HILIC separations, the sample should ideally
contain no less than 90% of the acetonitrile necessary to retain the first peak of
interest, and be identical in buffer ions to the mobile phase. Otherwise one can get
ion-pairs of two different counter ions of the analyte if it is ionizable.

One difficulty with this approach lies with the potential for limited sample solubility.
To aid with sample dissolution, methanol may be used to replace the aqueous portion
of the diluent and in some cases 0.2% formic acid or ammonium hydroxide have been
added to further assist sample solubility. 11

Figure 20 shows various peak shape effects obtained using a range of sample diluents
and 75% acetonitrile with 25% methanol is recommended as good general sample
diluent which balances improved sample solubility with good chromatographic
performance in HILIC mode.

Figure 20: Influence of sample diluent on solubility and peak shape.

Column: ACQUITY UPLC BEH HILIC 2.1 x 100, 1.7μm

Conditions: 10 mM ammonium acetate with 0.02% acetic acid in 90% acetonitrile

(Reproduced with permission of Waters Inc., Milford, Massachusetts, USA)

It is also worthy of note here that any auto-sampler needle wash solvents should also
be regarded in the same way, and depending upon instrument design a wash solvent
containing too much aqueous may lead to broad or split peaks. A wash solvent of 50%
acetonitrile with 50% water tends to give a good balance between cleaning and
chromatographic properties.

Column Equilibration

HILIC columns generally take longer to equilibrate than reversed phase HPLC
columns, primarily due to the need to establish ionic strength / ion exchange equilibria
on the stationary phase surface as well as the time required to re-equilibrate the
adsorbed aqueous layer.

Most manufacturers will have their own equilibration guidelines, however as a

general recommendation, a new column should be flushed with at least 50 column
volumes of the mobile phase being used and 20 column volumes daily in routine use.

Gradient Elution in HILIC Mode

Unlike reversed phase HPLC, HILIC columns tend to be much less suitable for fast
(ballistic) gradients and shallow gradients are much preferred and will give more
reproducible results, again primarily due to the complex nature of the separation and
the many equilibria that are operating to affect the separation.

A re-equilibration of 10 EMPTY column volumes (π x radius2 x length, in mm) is

recommended between each injection for gradients and an occasional water wash is
recommended to remove retained ions when operating in an isocratic mode.

When running gradient separations, which is usually achieved by gradually increasing

the aqueous portion of the eluent over a limited range (say 5-25%), it is recommended
that a consistent approach is taken. If the method has been developed using a buffered
aqueous system (only) then this should be maintained during routine operation as
changes in ionic strength during the gradient may affect the selectivity of the
separation. If both solvents are buffered (to maintain constant buffer strength), again
this approach should be maintained.

Summary

The HILIC mode of chromatography is very useful for the retention and separation of
polar and ionisable compounds. The mechanism of retention is orthogonal to that of
reversed phase HPLC and the strong solvent is water, usually with acetonitrile as the
weak solvent, although alternatives to both have been given.

The mechanisms of retention include partitioning into an adsorbed water layer at the
stationary phase surface, adsorption and ion-exchange in a complex interplay which
depends, in some instances, upon the eluent pH and buffer ion concentration. We
strongly advise on experimentation backed with a more thorough understanding of the
retention mechanisms to explore the various affects of altering mobile phase
composition.
Various stationary phases are available and can be readily selected depending upon
the analyte type and the effects of changing eluent composition with the various
phases using a variety of acidic, basic and neutral analytes have been briefly
discussed. Practically, the technique is slightly more complex than reversed phase
HPLC, however the benefits of the technique far outweigh the extra effort required in
both understanding and implementing the technique.

References

1. Alpert, A.J., J. Chromatogr. 1990, 499, 177-197

2. Alpert, A.J., Anal. Chem. 2008, 80, 62-76
3. McCalley, D.V., J. Chromatogr. A 2007, 1171, 46-56
4. McCalley, D.V., Neue, U.D., J. Chromatogr. A 2008, 1192, 225-229
5. Hao, Z., Xiao, B., Weng, N., J. Sep. Sci. 2008, 31, 1449-1464
6. Fountain, K.J., Neue, U.D., Diehl, D.M., Morrison, D., J. Sep. Sci., 2010, 33,
740-751
7. Gou, Y., Gaiki, S., J. Chromatogr. A 2005, 1074, 71-80
8. McCalley, D.V., J. Chromatogr. A 2010, 1217, 3408–3417
9. Bicker, W., Wu J., Lämmerhofer, M., Lindner, W., J. Sep. Sci. 2008, 31, 2971
10. Chauve, B., Guillarme, D., Cleon, P., Veuthey, J., J. Sep. Sci. 2010, 33, 752-
764
11. Grumbach, E.S., Wagrowski-Diehl, D.M., Mazzeo, J.R., Alden, B., Iraneta, P.,
LCGC N Am. 2004, 22, 1010-1023
12. Alpert, A.J. et. al., Anal. Chem., 2010, 82, 5253 - 5259

Essential CHROMacademy Reading on HILIC

Hydrophilic Interaction Chromatography *** CHROMacademy Registered

users only ***
David V. McCalley (2008)

A Comparison of HILIC and Aqueous NP-Chromatography ***

CHROMacademy Registered users only ***
Joseph Pesek and Maria T. Matyska (2007)

Is HILIC Permanently Changing the Scene for HPLC Separations? ***

CHROMacademy Registered users only ***
Finar Pontein, Patrik Appleblad, Tobia Jonsson (2010)

Introduction to HILIC - CHROMacademy Learning Module ***

CHROMacademy Registered users only ***

Other HILIC Resources

Comprehensive Guide to HILIC – Waters Corporation
A Practical Guide to HILIC – Merck Sequant
An Introduction to HILIC – SIELC Technologies