Dionex UltiMate 3000 - Testing the Application

UltiMate 3000 RSLCnano – Standard Applications
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2.9.4 Testing the Application
The 2D salt plug 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 95%/5% (v/v) water/ACN + 0.025% TFA
Sample Protein mix digest, prepared according to the included instruction sheet
Salt plugs
concentration (mol/L)
1 mmol NaCl
2 mmol NaCl
5 mmol NaCl
10 mmol NaCl
20 mmol NaCl
50 mmol NaCl
100 mmol NaCl
200 mmol NaCl
500 mmol NaCl
1000 mmol NaCl
2000 mmol NaCl
Injection volume Sample: 10 μL
Salt plugs: 20 μL
UV detection 214 nm
Loading time 5 min (may vary with different injection volume/routine)
Desalting time 7 min (started after loading time has passed)
Gradient Isocratic 4% for 10min
4% to 55% B in 30 min
90% B for 5 min
18 min equilibration
WPS temperature
5°C
Loading flow 10 μL/min
Flow rate 300 nL/min (nano flow selector)
To evaluate the result of the experiment, focus on the following points:
• Injection profile should be reproducible.
• The peptides should be well distributed over the different fractions and among the
fractions (orthogonal separation).