3.
Abnormally wide peaks (abnormally low plate count)
Peak broadening
in a chromatographic separation can be characterized by one of a number
of peak width measurements or by the plate number, with wider peaks (for a
given retention) having a lower plate number.
If all the peaks in
the chromatogram are abnormally broad, then the following possibilities
should be checked:
1. For isocratic
separations only, especially if retention times are too long:
A. Is the pump flow
rate is correctly set?
B. Check the system
carefully for evidence of leaks.
C. Check the system
for evidence of bubble problems or check valve failure; these often
will be accompanied by excessive fluctuations in the baseline or the
system pressure.
2. For both
isocratic and gradient separations:
A.
Is the temperature of the column compartment correctly set?
B. If the column
compartment temperature is correctly set, use a thermometer to confirm
the actual temperature.
C. Check whether the
dilution solvent is stronger than the mobile phase.
3. For gradient
separations only: check for excessive extra-column volume
4. For all
separations, chemical contamination or
attack on the stationary phase is always a possibility.
Replacing the column should fix the problem; in most cases, this is the
most cost-effective solution.
If only some of the
peaks in the chromatogram are abnormally broad, while other peaks meet
the peak width specifications, you should check the following
possibilities:
1. Sample
overload
(especially if the broad peaks have a significantly larger area than
the narrow peaks).
2. Incorrectly
prepared mobile phase (pay special attention to buffer concentration
and pH).
3. A
partially-resolved interference.
4. Sample
degradation.
Ref: LC-GC
17(9)
824 (1999)
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LC Resources, Inc. all rights reserved