HILIC Resource Library
See this collection of resources to help you learn more about HILIC columns, their use and application.
This section is not an exhaustive guide to HPLC column and instrument troubleshooting but an aid to help you understand and correct some of the more common chromatographic problems that could arise during HILIC separations.
Common problems, possible causes, and possible solutions
| Possible Cause | Corrective Action |
|---|---|
| Incorrect choice of stationary phase | In order to get the optimum retention of charged analytes, it is important to match the analyte log P or log D values to the degree of polarity of the stationary phase. For acidic analytes, a stationary phase with anion exchange properties will give increased retention. For basic analytes, a stationary phase with cation exchange properties will give increased retention. |
| Mobile phase buffer pH | To increase analyte retention, select a mobile phase buffer pH at which the analyte is charged. Mobile phase buffer pH can also affect the charge on the stationary phase. |
| Mobile phase water content is too great | Increase the organic percentage in the mobile phase. A minimum water content of 3% is recommended in order to maintain the partitioning effect in HILIC. |
| Possible Cause | Corrective Action |
|---|---|
| Column not fully equilibrated | HILIC columns are less tolerant of short equilibration times than RPLC columns, mainly because the water in the aqueous layer within the stationary phase derives from the mobile phase and takes time to establish itself. This is particularly true for gradient conditions; post-gradient re-equilibrations of approximately 20 column volumes are recommended. Fast HILIC gradients and gradients that run from 100% organic to 100% aqueous are to be discouraged. New columns need conditioning; running approximately 20 column volumes of mobile phase is recommended. |
| Column contamination | A shift in peak retention or resolution, associated with a sudden increase in operating pressure beyond expected levels indicate column contamination. Reversing the flow direction of the column may be attempted to remove debris on the inlet frit (column outlet should go to waste, to avoid HPLC system contamination). To remove strongly retained materials from the column, flush the column in the reverse direction with very strong solvents such as 50:50 methanol:water. |
| Mobile phase | A mobile phase buffer pH that is close to the pKa of an analyte can cause retention time drift. Adjust the pH of the buffer or choose an alternative buffer. |
| Possible Cause | Corrective Action |
|---|---|
| Injection solvent (sample solvent) too strong for the mobile phase | To ensure good chromatographic performance, it is recommended that sample solvent conditions be as close as possible to the initial mobile phase conditions, or at least to have an organic content greater than 50%. Using aqueous sample solvent with high elution strength—which impairs the partitioning of the analytes into the stationary phase—is detrimental and results in peak broadening. This in turn could lead to column overload, reduced retention, and loss in resolution. Unfortunately, polar analytes often have low solubility in organic solvents; in this case it is recommended to replace water with methanol. In extreme cases of solubility issues, even the aqueous portion of the mobile phase can be replaced by polar non-aqueous solvents, in which case the technique is referred to as ‘non-aqueous HILIC chromatography.’ |
| Syringe/needle wash does not match mobile phase | The solution used for washing the syringe and the injection needle should match the mobile phase composition but without the buffers. Undesired band broadening will result if too much water is used in the wash. Pure organic solvents should be avoided as well, as these not polar enough to remove the analytes. |
| Injection volume too large | Injection of excessive sample volumes will cause column overloading, resulting in broad/tailing peaks or in extreme cases flattened peaks. The recommend injection volumes are 0.5-5µL, for a 2.1mm id column and 5-50µL, for a 4.6mm id column. |
| Insufficient buffering of mobile phase | Insufficient buffer concentration can result in increased secondary interactions that cause peak tailing. Increasing the buffer concentration can enhance hydrogen bonding between analyte and stationary phase, masking other secondary interactions and improving peak shape. When increasing buffer concentration, be aware of the potential to suppress ion signal with mass spectrometry. |
| Possible Cause | Corrective Action |
|---|---|
| Incorrect choice of stationary phase | In order to get the optimum retention of charged analytes, it is important to match the analyte log P or log D values to the degree of polarity of the stationary phase. For acidic analytes, a stationary phase with anion exchange properties will give increased retention. For basic analytes, a stationary phase with cation exchange properties will give increased retention. |
| Mobile phase buffer pH | To increase analyte retention, select a mobile phase buffer pH at which the analyte is charged. Mobile phase buffer pH can also affect the charge on the stationary phase. |
| Mobile phase water content is too great | Increase the organic percentage in the mobile phase. A minimum water content of 3% is recommended in order to maintain the partitioning effect in HILIC. |
| Possible Cause | Corrective Action |
|---|---|
| Column not fully equilibrated | HILIC columns are less tolerant of short equilibration times than RPLC columns, mainly because the water in the aqueous layer within the stationary phase derives from the mobile phase and takes time to establish itself. This is particularly true for gradient conditions; post-gradient re-equilibrations of approximately 20 column volumes are recommended. Fast HILIC gradients and gradients that run from 100% organic to 100% aqueous are to be discouraged. New columns need conditioning; running approximately 20 column volumes of mobile phase is recommended. |
| Column contamination | A shift in peak retention or resolution, associated with a sudden increase in operating pressure beyond expected levels indicate column contamination. Reversing the flow direction of the column may be attempted to remove debris on the inlet frit (column outlet should go to waste, to avoid HPLC system contamination). To remove strongly retained materials from the column, flush the column in the reverse direction with very strong solvents such as 50:50 methanol:water. |
| Mobile phase | A mobile phase buffer pH that is close to the pKa of an analyte can cause retention time drift. Adjust the pH of the buffer or choose an alternative buffer. |
| Possible Cause | Corrective Action |
|---|---|
| Injection solvent (sample solvent) too strong for the mobile phase | To ensure good chromatographic performance, it is recommended that sample solvent conditions be as close as possible to the initial mobile phase conditions, or at least to have an organic content greater than 50%. Using aqueous sample solvent with high elution strength—which impairs the partitioning of the analytes into the stationary phase—is detrimental and results in peak broadening. This in turn could lead to column overload, reduced retention, and loss in resolution. Unfortunately, polar analytes often have low solubility in organic solvents; in this case it is recommended to replace water with methanol. In extreme cases of solubility issues, even the aqueous portion of the mobile phase can be replaced by polar non-aqueous solvents, in which case the technique is referred to as ‘non-aqueous HILIC chromatography.’ |
| Syringe/needle wash does not match mobile phase | The solution used for washing the syringe and the injection needle should match the mobile phase composition but without the buffers. Undesired band broadening will result if too much water is used in the wash. Pure organic solvents should be avoided as well, as these not polar enough to remove the analytes. |
| Injection volume too large | Injection of excessive sample volumes will cause column overloading, resulting in broad/tailing peaks or in extreme cases flattened peaks. The recommend injection volumes are 0.5-5µL, for a 2.1mm id column and 5-50µL, for a 4.6mm id column. |
| Insufficient buffering of mobile phase | Insufficient buffer concentration can result in increased secondary interactions that cause peak tailing. Increasing the buffer concentration can enhance hydrogen bonding between analyte and stationary phase, masking other secondary interactions and improving peak shape. When increasing buffer concentration, be aware of the potential to suppress ion signal with mass spectrometry. |
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