Care and use manual – Waters Enzymate BEH Pepsin Columns User Manual
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[ CARE AND USE MANUAL ]
Enzymate BEH Pepsin Column
b. Column Installation and Equilibration
Install the Enzymate BEH Pepsin Column in the temperature controlled compartment located on the left side of the HDX Manager
(maintained between 10 and 20 °C). Connect it between port 6 of the Injection Valve and port 3 of the Trap Valve as indicated in Figures 2a
and 2b.
1. Set the temperature of the Enzymate BEH Pepsin Column to between 10–20 °C. Higher temperatures aid digestion and lower
temperatures minimize back-exchange.
2. Equilibrate the Enzymate BEH Pepsin Column using 0.1% formic acid in water, pH 2.5 for at least twenty minutes (20 column volumes) at
a flow rate of 100 μL/min before performing protein digestions.
1
2
3
4
6
5
2
3
4
6
5
1
Binary Solvent
Manager (BSM)
Auxiliary Solvent
Manager (ASM)
Waste
Waste
Sample
Trap Valve
Injection Valve
Tra
p
Enzymate BEH
Pepsin Column
ACQUITY UPLC
®
Column
To MS
1
2
3
4
6
5
2
3
4
6
5
ACQUITY UPLC
®
Column
1
Binary Solvent
Manager (BSM)
Auxiliary Solvent
Manager (ASM)
Waste
Waste
Sample
Trap Valve
Injection Valve
To MS
Tra
p
Enzymate BEH
Pepsin Column
Figure 2a. Protein digestion in trapping mode (sample already injected and is in loop).
Figure 2b. After proteins are digested on the Enzymate BEH Pepsin Column, the valve is switched and the peptic peptides are separated using reversed-phase UPLC.
The trap column used is a VanGuard 2.1 x 5 mm Pre-Column containing 1.7 μm ACQUITY UPLC BEH C
18
packing material (part number
). The analytical
reversed-phase separation occurs on an ACQUITY UPLC Column containing 1.7 μm ACQUITY UPLC BEH C
18
packing material (1.0 x 100 mm, part number
or 1.0 x 50 mm, part number