UltiMate 3000 RSLCnano – Standard Applications
41
2.10.4 Testing the Application
The Mic Nan SCX – RP application 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 pump A 5 mM HxPO4 pH 3 + 5% ACN
Loading pump B Loading pump A with 1 M NaCl
Loading pump C 98%/2% (v/v) water/ACN + 0.05% TFA
Sample Protein mix digest, prepared according to the included instruction sheet
Injection volume Sample: 10 μL
Fractions: 20 μL
Gradient SCX 0-50% B in 20 min
90%B for 5 min
10 min equilibration
SCX flow rate 50 µL/min
UV detection 214 nm
Fractions Every minute for 20 minutes
Loading time 7 min (may vary with different injection volume/routine)
Desalting time 5 min (counted after loading time has passed)
Gradient RP Isocratic 4% for 10min
4% to 55% B in 30 min
90% B for 5 min
15 min equilibration
WPS temperature
5°C
Loading flow 5 μL/min
Flow rate 300 nL/min (nano flow selector)
The automated off-line application allows, as any off-line application, optimization of the
individual separation dimensions. Thermo Fisher Scientific recommends testing and
optimizing the performance of each separation dimension (SCX and RP) individually in a
one-dimensional application.
This means that, for a nano RP second dimension, the test criteria are identical as described in
the part of Pre-concentration onto a Nano Column on page 21.
To Next Page
