UltiMate 3000 RSLCnano – Standard Applications
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3.3 The use of TFA and FA
The separation of peptides by reversed phase is typically done in the presence of an ion
pairing agent. The typical ion pairing agents serve a double function. First, these weak acids
bring the pH of the solvents down to pH 2-3, causing almost all peptides to have an overall
positive charge. Secondly, the negative counterion of the acid will serve as the ion pairing
agent with the peptides to create an overall neutral analyte that is separates on the RP column.
The double function of the ion pairing agent allows having an efficient separation with
minimal additives added to the solvents. Nonetheless, there is a choice of ion pairing agents,
where the most common choice is between Trifluoro Acetic Acid (TFA) and Formic Acid
(FA). In this manual, TFA is used in the application as this is the stronger ion pairing agent
and results in better chromatography. However, in LC-MS applications, often FA is preferred
to minimize the effects of ion suppression. When performing the applications mentioned in
this manual with FA, replace the volume (%) of TFA by double the volume (%) of FA; e.g.
0.05% TFA becomes 0.1% FA.
3.4 Minimizing Baseline Noise
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
post column band broadening.
Typically peptide UV detection is performed at a wavelength of 214 nm, in which most
organic compounds absorb quite strongly. There are some actions that can be taken to
minimize baseline drift and noise for optimal use of the UV detection.
3.4.1 Drift
Ensure that the UV lamp has been switched on for sufficient time in order to have it running
at a stable temperature. Chromeleon can detect this and will give a warning during the ‘Ready
Check‘ if the UV lamp temperature is not stable yet. The UV detector can be used, but it is
not at its optimal performance.
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. The ion pairing agent (typically FA or TFA) in the A and B solvent can be used to
compensate the baseline rise. As a rule of thumb, the compositions as indicated in Table 11
can be used to obtain a straight baseline.
Table 11: Ion pairing agent addition
A B
FA 0.1% 0.08%
TFA 0.05% 0.04%
Lamp and flow cell age can have a significant influence on baseline drift. New lamps and
flow cells may show some drift during the so-called ‘burn in’ period.
Lamps should be replaced after approximately 2000 hours and older flow cells can be cleaned
by flushing overnight with organic solvent or for a shorter period with a strong acidic
solution; see operating instructions for details.
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