UltiMate 3000 RSLCnano – Standard Applications
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2.6.3 Installation Tips
• Follow the General Recommendations for Applications (→ page 6).
• The design of the nano trap column provides the easiest connections, but must only be
used in forward flush operation. Please check the indicated flow direction when
installing a nano trap column.
• If the loss of hydrophilic peptides is observed, the concentration of acetonitrile in the
loading solvent can be decreased down to 99/1 Water/ACN + 0.05% TFA or the use of
a stronger ion-pairing agent such as heptafluorobutyric acid (HFBA) can be considered.
2.6.4 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 20%/80% (v/v) water/ACN + 0.04% TFA
Loading solvent 98%/2% (v/v) water/ACN + 0.05% TFA
Sample Cytochrome C digest 1 pmol/μL, prepared according to the instruction sheet
Injection volume 1 μL (partial loop fill of a 20µL loop)
UV detection 214 nm
Loading time 3 min (may vary with different injection volume/routine)
Gradient 4% to 55% B in 30 min
90% B for 5 min
25min equilibration
WPS temperature
5°C (WPS-3000(B)T only)
Loading flow 5 μL/min
Flow rate 300 nL/min (nano flow selector)
Figure 17: Typical chromatogram for Pre-concentration of 1 pmol Cyt C onto a nano column
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