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Peak Retention Drift
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 too and we recommend to run
approximately 20 column volumes of mobile phase.
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 effluent 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 (HILIC) solvents such as 50:50
methanol:water. Strong buffers may be needed to remove ionic
contaminants.
Allowable Buffers
Possible Issues
Solubility/MS compatibility
We recommend the use of buffered eluents in HILIC, in order to minimize
electrostatic interactions between charged analytes and deprotonated
silanol groups of the stationary phase. A concentration range 5−20 mM
is generally sufficient for most analytes.
Ammonium acetate and ammonium formate are mostly recommended
due to their high solubility in mobile phases with high organic content;
their buffering range – 3.8−5.8 for ammonium acetate and 2.7−4.7 for
ammonium formate – are suitable for most HILIC applications and
additionally they are compatible with MS detection due to their volatility.
If higher pH is required, ammonium hydroxide and ammonium carbonate
can be considered.
Formic acid and acetic acid are valuable alternatives, due to their
solubility even in very high concentrations of organic solvents.
Phosphate buffers should not be used, as they are liable to precipitate,
and tend to foul MS sources.
