UltiMate 3000 RSLCnano – Standard Applications
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2.4.4 Testing the Application
The direct injection 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
Sample Cytochrome C digest 1 pmol/μL, prepared according to the instruction sheet
Injection volume 1 μL
UV detection 214 nm
Gradient 4% to 55% B in 30 min
90% B for 5 min
25 min equilibration
WPS temperature
5°C (WPS-3000(B)T only)
Flow rate 300 nL/min (nano flow selector)
Figure 11: Typical chromatogram for a Direct Injection of 1 pmol Cyt C onto a nano column
For details on interpretation and troubleshooting of the Cytochrome C nano LC separation,
see the FAQ section on page 50.
2.4.5 Large Volume Injections
Typically direct injections in nano LC are performed with 1 µL loop sizes to minimize the
gradient delay. Larger volume injections are performed with a pre-concentration setup. The
WPS-3000PL autosampler series allows for a custom injection program (UDP) to switch back
the injection valve after sample loading to bypass the loop. This way a larger sample volume
can be injected and pre-concentrated directly onto the nano column, without using a pre-
concentration setup.
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