Protein Expression and Purification Series - Bio-Rad

CHAPTER 8
BIOLOGIC DUOFLOW
PROTOCOL
Protein Expression and Purification Series
Figure 8.5. The purge buttons for Pump A and Pump B on the DuoFlow workstation. When the purge buttons on the
workstation front are pushed, the workstation pumps will run at a default flow rate of 10 ml/min and the indicator light will flash
green. Many columns cannot withstand the pressure that this would generate and therefore, it is critical that the AVR7-3 valve has
been set to the P (Purge) setting to ensure that the fluid flow will exit to waste and not flow onto the column at this flow rate.
1.4 Manual Control of the Workstation Pumps
The workstation pump parameters are set from the Manual screen either by clicking in the appropriate field
and entering a value from the keyboard or by using the arrows (Figure 8.6).
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7.
Set the flow rate to 1.00 ml/min by typing 1.00 into the Flowrate box or using the up/down arrows.
Set the Inlet A composition to 100%.
Set the high pressure limit to 200 psi and the low pressure limit to 0 psi.
To start the pump, click the Start button. The green light for Pump A should be lit.
Allow Pump A to run for three minutes.
With Pump A running, change the Inlet B value to 100% (notice that Inlet A automatically changes to
0%) and press the Set button to initiate the change. The green light for Pump A should go off and the
green light for Pump B should turn on.
Allow Pump B to run for three minutes and then press Stop to stop the pump.
Figure 8.6. Manual pump control. The flow rate for an F10 pump head
system can be set between 0.01 to 10 ml/min. The maximum flow rate
is determined by the type of column that is being used. Some resins
cannot withstand the pressure that builds from a high flowrate and will be
crushed (such as soft agarose-based beads). Other resins can withstand
much higher pressures/flow rates without being damaged. The Inlet A
and Inlet B settings determine how much buffer is pulled from each port.
Typical chromatography nomenclature refers to the percentage of buffer
B that is being used. So if a 95% Buffer A and 5% Buffer B mixture was
required, this would be referred to as 5% Buffer B. The High and Low
limits are pressure limits that can be set to have the instrument turn off if
they are exceeded or drop below the given values, respectively. These are
used to protect the column from overpressuring and to stop the run if the
pressure drops to zero, which is usually indicative of running out of buffer
or having a large air bubble in the tubing.
170 Chapter 8: Purification Protocol for BioLogic DuoFlow System