UltiMate 3000 RSLCnano – Standard Applications
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2.7.3 Installation Tips
• Follow the General Recommendations for Applications (→ page 6).
• Using a stainless steel loop (on a standard system) instead of a PEEK loop is
recommended in case of injecting highly diluted samples.
• With column oven temperatures below 45°C, TFA can be used instead of HFBA in the
loading solvent to load the sample onto the trap column.
• Due to the speed of the separation, the value for the detector's time constant (response)
must be reduced to 0.1s.
• Concentration of HFBA in the loading solvent can be increased to 0.1% in case trapping
problems are observed.
• The loss of hydrophilic peptides (for example, peaks 1 and 2 are lower than expected)
may (partially) be decreased by lowering the oven temperature.
2.7.4 Testing the Application
The pre-concentration setup can be tested using the following conditions:
Property Setting
Mobile phase A 100% water + 0.05% TFA
Mobile phase B 50%/50% water/ACN + 0.04% TFA
Loading solvent 100% water + 0.05% HFBA
Sample Cytochrome C digest 1 pmol/μL, prepared according to the instruction sheet
Note: The sample must be diluted in the loading solvent.
Injection volume 0.5 μL (partial loop)
UV detection 214 nm
Loading time 3 min (may vary with different injection volume/routine)
Gradient 1% to 70% B in 8 min
90% B for 2 min
8.5 min equilibration
Oven temperature
60°C
WPS temperature
5°C (WPS-3000(B)T only)
Loading flow 10 μL/min
Flow rate 3.0 μL/min (capillary flow selector)
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