5
Diagnostics
Determining the Delay Volume of the Pump
Thermo Scientific
Accela Pump Hardware Manual
49
Determining the Delay Volume of the Pump
The gradient delay volume of your pump is an important variable if you are developing
gradient methods that you plan to transfer to other instruments. The gradient delay volume
of the Accela Pump consists of the wetted components that lie in the flow path between the
solvent proportioning valves of the pump and the outlet of the LDA.
Y
To determine the gradient delay volume of the pump
1. Set up your LC system:
a. Bypass the autosampler by connecting the pump directly to the detector with tubing
of known volume. The volume of the inlet tubing to the Accela PDA Detector is
0.32
μ
L/inch. (P/N 803260, inlet tubing, insulated, red PEEK,
1/16 in. OD
×
0.005 in. ID)
b. Prepare a spiked methanol solution as described in
“Testing the Pump
Proportioning”
on
page 46
.
c.
Fill one solvent reservoir bottle with 100% methanol.
d. Fill a second solvent reservoir bottle with the spiked methanol solution.
e.
Purge the solvent lines to replace the old solvents with the new solvents.
2. Create a data acquisition method.
Use the following detector settings and chromatographic conditions to run and monitor
the linear gradient listed in
Table 5
:
• Flow: 200
μ
L/min
• Pressure:
approximately 100 bar by restriction
• Eluents:
A: methanol
B: spiked methanol
• Detection: UV (254 nm)
3. Equilibrate the system until the baseline stabilizes. To equilibrate the system, depending
on the data system you are using, do one of the following:
• For Xcalibur, go to step
step 4
.
• For ChromQuest, go to step
step 5
.
Table 5.
Gradient program for testing gradient delay volume
Time (min)
%Methanol
%Spiked Methanol
0.00
100.0
0.0
0.01
100.0
100.0
