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The CHROMacademy Essential Guide to Hydrophilic Interaction Chromatography (HILIC) and Related Techniques - WEBCAST

In this session industry expert Amos Heckendorf of the Nest Group and Crawford Scientific Technical Director Tony Taylor explore the key aspects of HILIC separations

Tony Taylor Tony Taylor
Technical Director
Crawford Scientific
Dr Amos Heckendorf Dr Amos Heckendorf
The Nest Group
Peter Houston Peter Houston
Editorial Director
LCGC Europe

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The CHROMacademy Essential Guide to Understanding Hydrophilic Interaction Chromatography (HILIC) – Part 1

Industry experts Dr Amos Heckendorf (The Nest Group) and Tony Taylor (Crawford Scientific & CHROMacademy), present a comprehensive review and tutorial on Hydrophilic Interaction Chromatography (HILIC).
This technique is very useful for retention and separation of polar and ionisable compounds and provides a very useful orthogonal separation mechanism for chromatographers.
The tutorial includes a comprehensive review of the mechanisms of HILIC separations as well as considering the major parameters which affect the separation selectivity including eluent solvent composition, pH as well as buffer type and strength.

There are a host of practical tips and tricks to ensuring a successful separation as well as a guide to stationary phases for HILIC separations. A must for everyone using, or considering the use of HILIC to improve the separation of polar analytes.

group  subs
Tony Taylor Tony Taylor
Technical Director
Crawford Scientific
Dr Amos Heckendorf Dr Amos Heckendorf
The Nest Group


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.

Fig 1Figure 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)

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)

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Fig 2.  
Fig 2.

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).

Animations 1 & 2 show the fundamental HILIC retention mechanisms in operation.

Animation 1: HILIC retention mechanisms. More highly hydrophilic species (purple) partition into the water rich layer at the stationary phase surface Interaction at the surface occurs through dipole-dipole and hydrogen bonding between the surface's functional groups and polar functional groups of the analyte   Animation 2: HILIC retention mechanism with electrostatic interactions. With an unbonded silica surface, and the eluent at pH ~ 5-9 and <~20mM buffer, the surface silanol species will be ionized and therefore able to undergo coulombic interaction with cations (bases) in solution. Note that the surface will now be less attractive to anions (acids) in solution through electrostatic repulsion. Unless there is also an analyte cation moiety, retention can decrease relative to a separation carried out at an eluent of pH<<5 .

Fig 3

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)


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.

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.

Fig. 4Figure 4: General schema for analyte Log P value versus separation mode of choice. (Adapted with permission from SIELC Technologies, Prospect Heights, Illinois, USA)
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.
Fig. 5  

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)Fig. 5

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:


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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

Fig. 6   Fig. 6
  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. (Reproduced with permission from Merck Sequant, Umeå, Sweden)
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.
Fig. 6    

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)

As with reversed phase separations, the eluent pH can affect retention and selectivity by influencing the degree of ionization of both the analyte and stationary phase surface. The electrostatic aspect of the separation may, in some instances, be secondary to the partitioning mechanism in HILIC separations, however these secondary effects can be profound and effective in optimizing (or inadvertently confounding) separations. 3, 12

There are some important points to note ahead of any study of the effects of mobile phase pH on HILIC separation

  1. Due to the highly organic nature of the mobile phases in question, the pH to which the aqueous portion of the mobile phase is adjusted will not be representative of the pH of the actual eluent solution. Whilst the direct measurement of pH in aqueous organic mixtures using a traditional pH meter is not particularly accurate or useful, it is worthy of note that the eluent solution pH will be around 1 to 1.5 units closer to NEUTRAL than the value obtained for the aqueous component alone.6 These values are often written as wwpH 4.5 (for the aqueous pH) which would give an approximate corresponding mobile phase pH of swpH 5.8 when the organic component is added. The amount of organic solvent within the mixture will affect the degree to which the pH changes from the original aqueous solution.
  2. Analyte pKa values will differ from literature or calculated values when in highly organic solution.
  3. The relationships between mobile phase pH, the degree of ionization of the analyte and stationary phase surface and the adsorbed water layer are highly complex and empirical investigation / optimization of the separation is highly recommended.
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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:

mol 3   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:
mol 4   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:

Fig. 8 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

Fig. 9

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)


Fig. 10pH3 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 layerand 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


Figure 10: Interplay between analytes and stationary phase surface at pH3


Fig. 11pH9 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.

Fig. 12  

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)

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.

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.

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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.


Fig. 13Ammonium 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

Fig. 14Figure 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

2 mM
10 mM
2 mM
10 mM
2 mM
10 mM
2 mM
10 mM

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.

Fig. 15TOP:
Analysis of test compounds on Atlantis silica.
Mobile phase ACN–water (95 /10 v/v) containing overall 0.1% TFA

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.

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


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bonded phases
Fig. 16 Fig. 16

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 Chain
10. Zwitterionic – Long Hydrophilic 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.

Fig. 17

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.

2 mM
10 mM
2 mM
10 mM
2 mM
10 mM
2 mM
10 mM









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)

Fig. 18Column: 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



Fig. 19Figure 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

3. Norepinephrine

Fig. 19

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


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.

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Fig. 20

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.


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.

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  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


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