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10 Tips for HILIC Mobile Phase Optimization

1. Organic Solvent

After stationary phase choice, organic solvent content has the greatest impact on analyte retention and separation in HILIC

Organic solvent content > salt content ≈ pH > column temperature

In contrast to reversed phase HPLC, water is the strongest eluting solvent (Table 1).  HILIC mobile phases typically contain between 3 and 70% of the strong solvent (water or aqueous buffer), with retention tending to increase exponentially at < 10% of the strong solvent.  The ideal organic content is between 60-97% with a minimum of 3% water to hydrate the stationary phase.  The most popular weak solvent is acetonitrile, due to its aprotic (intermediate polarity, lacking an acidic proton) characteristic which encourages retention of polar analytes.

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 330 nm 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, as the polar nature of the solvent results in increased competition for polar stationary phase sites and a disruption of the adsorbed water layer.

Solvent Chemical Formula UV cut off
Aprotic Solvents
Tetrahydrofuran Tetrahydrofuran 220 nm
Acetone Acetone 330 nm
Acetonitrile Acetonitrile 190 nm
Protic Solvents
Isopropanol (IPA) Isopropanol (IPA) 210 nm
Ethanol Ethanol 210 nm
Methanol Methanol 210 nm
Water Water 191 nm

Table 1: HILIC mobile phase organic solvents.


 
2. Buffer Choice

HILIC uses buffers and additives to achieve high column efficiency and reproducibility.  In general terms, the buffer concentration should be in the range 10-20 mM and additives should be used in concentrations usually not exceeding 1.0% (Table 2).  As a general rule, negatively or positively charged stationary phases require higher concentrations of buffers compared to neutral or zwitterionic phases.

Ammonium salts of formic and acetic acid are used for buffers due to their good solubility in organic solvents.  These buffers have the added advantage of being volatile making them compatible with mass spectrometric (MS) and charged aerosol (CAD) detectors.  As a result of their poor solubility in highly organic mobile phases, phosphate buffers are not recommended for HILIC applications.  Phosphate buffers are also incompatible with MS and CAD detection due to being non-volatile.

Buffer/Additive pKa Used for HILIC Further Information
Trifluoroacetic acid (TFA) 0.52 Yes Ion pair additive, can suppress mass spectrometer (MS) signal. 
Used in the range 0.01-0.1%
Formic acid 3.75 Yes Used in the range 0.1-1.0%
Acetic acid 4.756 Yes Used in the range 0.1-1.0%
Ammonium formate 3.74, 9.24 Yes Used in the range 10-20 mM
Ammonium acetate 4.76, 9.24 Yes Used in the range 10-20 mM. 
Sodium or potassium salts are not volatile
Phosphate 2.15, 7.20, 12.15 No Will reduce column lifetime. 
Not volatile so not MS compatible

Table 2: Buffers and additives for HILIC.

Electrostatic interactions can play an important role in the retention of analytes under HILIC conditions.  If analytes and the stationary phase are charged at the mobile phase pH attractive or repulsive secondary interactions can occur, the presence of a buffer/salt can reduce these interactions improving peak shapes (tailing) and altering retention.
Remember to always select the correct buffer for your pH range (buffers are only effective within 1 pH unit of their pKa).


 
3. pKa

A knowledge of analyte pKa can be used to manipulate ionization state and in turn retention.  HILIC favors retention of hydrophilic species, ionized species are more hydrophilic than their neutral counterparts, hence, altering the mobile phase pH to render analytes in their ionized form will increase retention.  For acidic analytes, raise the pH 2 units above the pKa to give the negatively charged species, conversely, when working with basic species lower the pH 2 units below the pKa to give the positively charged analyte (Figure 1).

Effect of pH on ionization state of nortriptyline and methacrylic acid

Figure 1:  Effect of pH on ionization state of nortriptyline (left) and methacrylic acid (right).


4. Log D

Alternatively mobile phase pH can be selected by assessing the log D value of analytes.  Compounds which have ionizable groups exist in solution as a mixture of different ionic forms.  The ionization state of those groups, and hence, the ratio of ionic forms depends on the pH.  Since log P describes the hydrophobicity of one form only, the apparent log P value can be different. 

The octanol-water distribution coefficient, log D represents the compounds at any pH value.  Log D gives a good measure of analyte hydrophilicity, with the lower the value of log D the more hydrophilic the molecule (Equation 1, Figure 2).  Selecting a mobile phase pH where the log D value is lower will increase analyte retention.  This approach may be particularly useful when working with analytes which have several ionizable groups and associated pKa’s. 

Log D acetylsalicylic acid

Figure 2:  Log D acetylsalicylic acid.

Information on log D, log P, and pKa values can be found for free at www.chemicalize.org and www.chemspider.com


 
5. pH

The charge state of the stationary phase can affect retention of ionizable analytes; electrostatic interactions between a charged stationary phase and a charged analyte can be critical, leading to drastic variations in retention.  This may be due to attractive interactions between oppositely charged species (increased retention) or repulsive interactions between species with the same charge (decreased retention).

When working with silica based stationary phases surface silanols will be ionized as the mobile phase pH increase above their pKa (~3.5).  At elevated pH values negatively charged silanol species can interact with positively charged analytes which can result in secondary interactions causing peak tailing, changes in selectivity, or increased retention. 

The pH stability of the column support should also be considered in relation to mobile phase pH.  Silica based columns have a working pH range of 2-8, with silica dissolution occurring at high pH and hydrolysis of silyl ether linkage between the bonded phase and silica surface at low pH (resulting in column bleed, poor peak shape, and loss of efficiency).  Modern hybrid and polymeric phases allow the use of extremes of pH.  The column user guide should always be consulted for the working pH range.


 
6. 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.

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.

Figure 3 shows various peak shape effects obtained using a range of sample diluents.  75% acetonitrile with 25% methanol is recommended as a good general sample diluent which balances improved sample solubility with good chromatographic performance in HILIC mode.

Influence of sample diluent on solubility and peak shape

Figure 3:  Influence of sample diluent on solubility and peak shape.

Column: ACQUITY UPLC BEH HILIC 2.1 x 100 mm, 1.7 μm 
Conditions: 10 mM ammonium acetate with 0.02% acetic acid in 90% acetonitrile.

It is also worthy of note here that any autosampler needle wash solvents should 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.


 
7. Gradient Elution

Unlike reversed phase HPLC, HILIC columns tend to be much less suitable for fast gradients; therefore, shallow gradients are preferred and will result in more reproducible results.  This is 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 (VM, Equation 2) is recommended between each injection for gradients, and an occasional water wash is suggested to remove retained ions when operating in an isocratic mode.

Gradient Elution

Where:
VM = column volume (μL)
r = radius (mm)
l = column length (mm)

When running a gradient method buffer both mobile phases, do not run buffer gradients.  Do not run gradients from 100% organic to 100% aqueous, a good working range is 97-60% organic.


 
8. 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.


 
9. Eluent Preparation

To maintain maximum HILIC separation performance, always use high quality eluent components, buffers, and additives. 

Good eluent preparation practice should also be adhered to:

  1. Filter all aqueous buffers prior to use
  2. Particulate solvents will generally clog the column
  3. Bacterial growth can be prevented by adding small amounts of organic modifier to the buffer system
  4. Degas all solvents before use
  5. Use freshly prepared eluent systems wherever possible

 
10. Premix Eluent

HILIC retention varies significantly for small variations of water content. The use of premixed solutions of acetonitrile/water helps to increase the reproducibility of the analyses.


 

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Dr. Dawn Watson
 

This article was written by Dr. Dawn Watson.

Dawn received her PhD in synthetic inorganic chemistry from the University of Strathclyde, Glasgow. The focus of her PhD thesis was the synthesis and application of soft scorpionate ligands. As well as synthetic skills, this work relied on the use of a wide variety of analytical techniques, such as, NMR, mass spectrometry (MS), Raman spectroscopy, infrared spectroscopy (IR), UV-visible spectroscopy, electrochemistry, and thermogravimetric analysis.

Following her PhD she spent two years as a postdoctoral research fellow at Princeton University studying the reaction kinetics of small molecule oxidation by catalysts based on Cytochrome P450. In order to monitor these reactions stopped-flow kinetics, NMR, HPLC, GC-MS, and LC-MS techniques were utilized.

Prior to joining the Crawford Scientific and CHROMacademy technical team she worked for Gilson providing sales and support for the entire product range including, HPLC (both analytical and preparative), solid phase extraction, automated liquid handling, mass spec, pipettes, and laboratory consumables.

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