GE AKTApure - Page 497

Note: •
Always set the delay volume from the UV monitor to the point of collection of eluent. De-
pending on the system configuration, this will be Delay volume: Monitor to outlet valve
or Delay volume: Monitor to frac in the system settings.
•
If the pH valve is mounted, make sure to set the value for the delay volume of the pH valve
restrictor. The system automatically adds the volumes for the flow restrictor and the pH
flow cell if they are part of the flow path.
Experimental method for determination of delay
volumes
Delay volumes can be determined experimentally for Fraction collector F9-R, but not for Fraction col-
lector F9-C. Instructions for measuring the delay volume using the UV monitor U9-M or UV monitor
U9-L are provided below.
Two volumes are used to perform experimental delay volume measurements:
•
V1: the volume between the Injection valve and the UV monitor used
•
V2: the volume between the Injection valve and the fractionation collector tubing tip
The delay volume is then measured using the following procedure.
ActionStep
Check that a flow rate of 1 ml/min is delivered by the pump.
Note:
If the flow rate is not correct, check if air bubbles may have entered the pump. Make sure
to correct any problems before continuing.
1
Fill a small sample loop, for example 20 μl, with a 1% to 5% acetone solution.2
Set all components between the monitor and the fraction collector to by-pass mode.3
Fill the system with water. Run the pump at a flow rate of 1 ml/min and inject the acetone
solution as a sample.
4
Note the volume from the point of injection to the maximum of the peak in the chro-
matogram. This volume is volume measurement V1.
5
Reconfigure the system as follows to include the fraction collector in the flow path before
the monitor:
6
•
Disconnect the two tubing segments from the UV monitor flow cell and connect these
with a low-dead-volume connector, a 1/16'' female to a 1/16'' female union connector.
•
Mount the fractionation collector tubing tip on the top of the UV monitor flow cell.
Connect tubing to the bottom of the UV monitor flow cell and lead this tubing to waste.
It is preferable if the waste tubing gives some back pressure as the flow restrictor
now is placed before the UV flow cell.
Set the Frac size to a large volume, for example 100 ml, so that the Outlet valve is in the
Frac position during the entire run.
7
ÄKTA pure User Manual 29119969 AB 497
9 Reference information
9.13 Delay volumes