Diagnostic:
Diluent solvent strength
A good rule for
HPLC is "to do the best chromatography, disturb the equilibrium of the
system as little as possible". If the sample is dissolved in a solvent
that is (chromatographically) stronger than the mobile phase, the
analyte peaks can be driven past the head of the column before they
equilibrate with the stationary phase. The result can be a variety of
distorted peak shapes, including tailing, fronting, flat-top, shoulders
or split peaks. The symptoms can be mitigated to some extent by
extra-column volume between the injector and the column inlet which
allows the diluent to mix with the mobile phase before it encounters
the stationary phase. In some cases, subtle differences in plumbing
will cause the problem to show up on some instruments but not others.
If your sample is
dissolved in a solvent stronger than the mobile phase, try preparing a
five-fold concentrated sample in the usual solvent, then diluting by a
factor of 5 with the mobile phase. If the peak shape improves, consider
either dissolving the sample in the mobile phase or limiting the volume
of sample injected.
Note that in
gradient separations, what matters is the diluent strength relative to
the initial composition of the gradient.
Ref: LC-GC, 15(7) 612 (1997)
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