GEN-MKT-18-7897-A
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Using the dual gradient system, the nanoLC 425 system can be configured such that the trap can be washed to waste rather than through the column and into the mass spectrometer. This workflow can be performed with either nanoflow or microflow chromatography.
Figure 1. Example of washing the late-eluting compounds to waste.
In a typical trap elute workflow, the isocratic load pump is used to load the sample onto the trap in the Load position and any salts are washed through to waste with aqueous. The trap valve is then switched to Inject position and the analytical gradient is used to elute the analytes off the trap, through the analytical column and then into the MS for analysis.
In this workflow, the second gradient channel (e.g Gradient 2) will be used instead of the isocratic Load pump to load the samples onto the trap with high aqueous. After loading, the trap valve switches to inject and the analytes are eluted using the analytical gradient. What is novel to this workflow is that after the bulk of the analytes have been eluted off the trap (say 35% B) then the trap valve is switched back to the Load position for washing. Because it is a gradient channel, this can now be used to run an organic wash step through the trap column in the Load position to waste at the end of the peptide elution gradient, such that hydrophobic analytes will be washed to waste. An example is shown below for running the PepCalMix sample with the normal microflow set-up (pink and blue traces) and the offline trap wash strategy (orange trace).
Figure 2. Trap elute configuration for off-line trap washing.
The only change in configuration that is required is that the second gradient channel (not being used for the analytical column) is converted to a microflow channel using the Low Micro Flow Module and the line to the AS valve is moved from the Load pump to this Gradient channel (e.g. Gradient 2 in this example).
See Appendix for a table of parts required for this configuration.
In this example, Gradient 2 will be used for trap loading and washing and Gradient 1 will be used as the analytical gradient. The Trap elute configuration will be basically the same as the normal trap elute configuration however the 50 µm ID PeekSIL line from the pump to the Autosampler valve will be connect to Gradient 2 rather than the Loading pump.
Autosampler method
Create a µL pick-up autosampler method according to the method shown below.
Gradient 2 pump method – trap washing
Gradient 1 method – peptide elution
Parts list.
Below is a table of all the parts needed to perform the nanoflow test. All part numbers for SCIEX unless otherwise indicated.
for 1-10 µL/min
RUO-MKT-18-8218-A
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