11.
Repeatability problems
Peak size
repeatability problems can be caused by a number of chemical, physical,
or computational issues.
1. Excessive
baseline noise.
An excessively noisy baseline is probably the most
common cause of integration problems. Before addressing repeatability
problems, you should check to confirm that your baseline noise level is
within specification.
2. Poor
resolution from an interference. If resolution between the analyte and
an interfering peak is marginal, then even small amounts of noise can
affect the allocation of area between the peaks.
3.
Variation in injection volume. This will usually affect all of the
peaks in the chromatogram by the same proportion. The "pseudo internal standard" test can be a useful
diagnostic.
4. Variation in
sample concentration. This will usually manifest as a systematic
change in response (i.e., the peak size will steadily increase or
decrease as a function of time). Steady increases in the size of all
peaks in a chromatogram suggests evaporation of solvent from the sample
vial. Steady decrease in the size of some peaks (and possibly increase
in the size of others) suggest sample degradation.
4. Variation in
detector response. Variations in lamp output or photodetector
response usually manifest themselves as excessive baseline noise. The problem can be
exacerbated if your detection wavelength is located on a steeply
sloping portion of the absorbance spectrum, because small variations in
either detector wavelength or sample spectrum will result in large
changes in absorbance.
A certain amount of
fluctuation in lamp output is normal; the magnitude increases with the
age of the lamp. In most cases, the fluctuations are short-term, and
result in excessive baseline noise. Most UV detectors are effectively
"double beam", so that variations in lamp output should cancel.
5. Variation in
peak area integration. The final possibility is that the peaks are
actually the correct size, but that the data system is integrating
incorrectly. This is usually due to one of three things:
A. Changes in peak
shape. These are usually systematic (i.e., steady increase or
decrease in peak area) and can show up as excessive tailing, abnormally
wide peaks,
shoulders, flat top peaks or, in extreme cases, split
peaks.
They should be diagnosed in the same fashion as any other peak shape
problems
B. Excessive
baseline noise.
C. Incorrect
integrator settings. These usually result in larger errors for smaller
peaks or for noisier baselines.
Ref: LC-GC, 15(1) 24 (1997)
Ref: LC-GC, 15(6) 516 (1997)
Ref: LC-GC, 15(12) 1118 (1997)
Ref: LC-GC, 16(10) 910 (1998)
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