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Troubleshooting
This section is intended as an aid in troubleshooting specific chromatographic problems that could
occur during the running of HILIC separations. It is not meant to be an exhaustive guide to HPLC
column and instrument troubleshooting. Advice specific to your column will be found in the Product
Manual.
The following topics will be covered:
• Solubility
• Allowable buffers
• Retention reproducibility (peak retention drift)
• pH extremes affecting column stability
• Injection solvent
• Injection volume
• Syringe wash
Broad Peaks
Possible Cause
Corrective Action
Injection solvent (sample solvent) too
strong for the mobile phase
To ensure good chromatographic performance, it is recommended for the
sample solvent to 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
for the chromatography, resulting 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 substitute 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
be matched to 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 they are not
polar enough to remove the analytes.
Injection volume too large
Injection of excessive sample volumes may cause column overloading,
resulting in broad/tailing peaks or, in extreme cases flattened peaks. The
recommend injection volumes are 0.5−5 µL, for a 2.1 mm ID column
and 5−50 µL, for a 4.6 mm ID column.
