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Troubleshooting HILIC

Insufficient Retention

Insufficient Retention

Incorrect stationary phase

The analyte log P or log D values should be matched to the stationary phase polarity.  For acidic analytes, a stationary phase with anion exchange properties will provide increased retention; conversely, cation exchange phases will exhibit greater retention for basic analytes.

Incorrect mobile phase pH

Analyte retention increases as the polarity of the analyte increases.  Ionized analytes are more polar; therefore, adjust the pH to give the ionized analyte - two pH units above analyte pKa for acids and 2 pH units below the analyte pKa for bases (Figure 1).

2 pH rule

Figure 1: 2 pH rule.

Too much water in the mobile phase

Increase organic content, although ensure that there is a minimum of 3% water to maintain analyte partitioning and stationary phase hydration.

Retention Time Drift
Retention Time Drift

Column insufficiently equilibrated

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

A re-equilibration of 10 EMPTY column volumes (VM, Equation 1) is recommended between each injection for gradients, and an occasional water wash is suggested to remove retained ions when operating in an isocratic mode.

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

Calculation for a 4.6 x 150 mm column

Re-equilibration time at a flow rate of 1.5 mL/min = 50/1.5 = 33.3 minutes.


Column contamination

Shifting retention times or resolution in conjunction with an increase in system pressure indicates column contamination or a blocked inlet frit.  The column can be reversed (unconnected from the detector to avoid washing particulates into the flow cell) and flushed with the mobile phase to remove particulates from the inlet frit.
To remove strongly retained components from the column, flush the column in the reverse direction with strong solvents such as 50:50 methanol:water.  Always consult the column user guide for any specific column washing instructions.

Incorrect mobile phase pH

If the mobile phase pH is close to the analyte pKa this can cause retention time drift.  Adjust the pH of the buffer to two pH units above or below the analyte pKa (depending on whether it is an acid or base, Figure 1).  Make sure the correct buffer has been selected for the pH range - buffers have optimum buffering capacity ±1 pH unit from their pKa (Table 1).

Buffer pKa pH Range UV Cut Off (nm)
Phosphate 2.1
Acetate* 4.8 3.8-5.8 210 (10 mM)
Citrate 3.1
Carbonate 6.1
Formate* 3.8 2.4-4.8 210 (10 mM)
Ammonium bicarbonate 7.6 6.6-8.6 230
Borate 9.3 8.3-10.3 N/A

Table 1: Common HPLC buffers. * denotes volatile buffer suitable for use with mass spectrometry.

Poor Peak Shapes
Peak fronting   Peak broadening   Peak tailing

Peak fronting


Peak broadening


Peak tailing


Injection solvent/mobile phase mismatch

It is recommended that the injection (sample) solvent composition is as close to the initial mobile phase composition as possible, or at least has an organic content greater than 50%.  Using highly aqueous injection solvents with high elution strength leads to peak broadening as it impairs analyte partitioning into the stationary phase.  This can also result in column overload, reduced retention, and loss of resolution.  For polar analytes which exhibit low solubility in organic solvents, using methanol instead of water is recommended.  When there are extreme solubility issues even the aqueous portion of the mobile phase can be replaced by polar non-aqueous solvents; this technique is referred to as non-aqueous HILIC.

Injection volume too high

If the injection volume is too large fronting, broad, tailing, or flattened peaks will be seen. 
The recommended injection volumes are 0.5-5 μL for a 2.1 mm i.d. column and 5-50 μL for a 4.6 mm i.d. column.

Needle wash/mobile phase mismatch

Needle wash solutions should match the mobile phase composition, but with the buffer portion replaced by water.  If too much water is present in the wash solution broadened peaks will be evident.  Pure organic solvents should also be avoided as they will not be polar enough to remove the analytes which will result in contamination, carryover, ghost peaks etc.

Insufficient mobile phase buffering

Peak tailing can be caused by insufficient buffer capacity as analytes will undergo secondary interactions with the stationary phase.  Increased buffer concentration promotes hydrogen bonding between the analyte and stationary phase, overcoming other secondary interaction, and improving peak shapes.  When increasing buffer concentration, be aware of the potential to suppress ion signal with mass spectrometry.  Furthermore, always use volatile buffers for HILIC-MS applications. 


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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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