Column Contaminants - PerkinElmer 200 UV/VIS Series User Manual

Troubleshooting Detector Problems Maintenance

Column Contaminants

Due to the detector's high sensitivity, the HPLC column must be optimally maintained. In
some instances, contaminants may wash off the column and affect the flowcell. This condi-
tion often appears as a slow but steady upward or downward shift in the detector baseline
over the course of a run (especially during gradient elution). The HPLC column must be
clean at all times. Columns which work well with other detectors may fail with the detector
due to its high sensitivity. If this occurs, consult the column manufacturer for instructions
on regeneration or decontamination. If flowcell contamination is suspected, refer to the Rou-
tine Maintenance section for guidance on cleaning the flowcell.
Solvents
Mobile phase solvents (including water) and buffers should also be suspect in cases of con-
tamination. Organic contaminants in water, for example, may concentrate themselves on
reverse phase columns during the early part of a gradient run, then slowly wash off as the
concentration of the organic modifiers increases with time.
All solvents used for mobile phases and at all stages of sample preparation should be HPLC
grade. This indicates that they have been specially purified and filtered to meet the demands
of today's sophisticated chromatographic instruments. Special attention must be paid to UV
cutoff values and gas saturation, since both of these factors affect instrument operation. Cer-
tain strategies can be used to minimize their negative effects.
UV Cutoff
The UV cutoff of an HPLC grade solvent is usually defined as the wavelength at which the
solvent exhibits 1.0 AU absorbance, as determined in a 10 mm path flowcell. The absorbance
at wavelengths below the cutoff value is even higher. Essentially, the solvent should not be
used in mobile phases when the detector wavelength is near or below the cutoff value since
the background will be too high. Even though the detector's auto zero circuit can remove up
to 2 AU of background absorbance, this will inevitably cause a decrease in signal-to-noise
ratios. The difficulty of working near the UV cutoff of the solvents used in the mobile phase
is compounded by the fact that cutoffs usually occur in the low UV where lamp energy, grat-
ings and optical components are working at the limits of their design specifications.
In the unusual situation where there is no choice as to mobile phase composition, wave-
length, or sensitivity setting, then a certain amount of noise and drift are to be expected. If
possible, one or more of these parameters should be modified (i.e., run the detector at higher
wavelength or on a less sensitive setting). Another alternative would be to substitute sol-
vents of similar chromatographic characteristics but with lower UV cutoffs; an example of
this would be acetonitrile (190 nm cutoff) instead of methanol (205 nm cutoff). Additionally,
the solvent supplier may be contacted to supply a different lot of solvent with a lower cutoff
value. Most good HPLC grade solvents are individually lot tested, with specific UV absorp-
tion values appearing on the label.
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