Minimizing Baseline Effects; Drift - Thermo Scientific UltiMate 3000 Standard Applications Manual

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3 FAQs
3.5

Minimizing Baseline Effects

3.5.1

Drift

Page 90
The 3 nL flow cell (P/N 6074.0270) and 45 nL flow cell (P/N 6074.0280)
are designed to function in the same way as transfer tubing normally
used to connect a column outlet to a mass spectrometer. This allows UV
detection in nano and capillary LC without introducing detrimental post
column band broadening.
Typically, peptide UV detection is performed at a wavelength of 214 nm,
at which most organic compounds absorb quite strongly. A number of
actions can be taken to minimize baseline drift and noise for optimal use
of the UV detection.
Ensure that the UV lamp has been switched on for sufficient time in
order that the lamp temperature can stabilize. Chromeleon can detect
this and will give a warning during the 'Ready Check' whenever the UV
lamp temperature is not stable. In such cases, the UV detector can be
used but may not perform optimally.
Gradient RP nano LC typically involves a significant change in solvent
composition. The higher absorption from the organic modifier in the
B solvent will result in a rise of the baseline. Varying the ion-pairing
agent concentration (typically FA or TFA) in the A and B solvent can be
used to compensate the baseline rise. As a rule of thumb, the ion-pairing
agent concentrations indicated in
straight baseline.
Solvent A
0.1%
0.05%
Table 24: Ion pairing agent addition
The age of both lamp and flow cell can have a significant influence on
baseline drift. New lamps and flow cells may show some drift during the
so-called 'burn in' period. Allowing sufficient equilibration time for the
lamp is necessary for obtaining a stable baseline.
Lamps should be replaced after approximately 2000 hours. Older flow
cells can be cleaned by flushing overnight with organic solvent or for a
UltiMate 3000 RSLCnano Standard Applications Guide
Table 24 can be used to obtain a
Solvent B
0.08%
0.04%