Bio-Rad BioLogic Signal Import Module (SIM) User Manual

Page 185

SAMPLE LOADING

ADVANCED SYSTEM APPLICATIONS

9-4

9.2 REVERSE FLOW CHROMATOGRAPHY

Reverse flow chromatography is generally used in affinity chromatography experiments to prevent peak
broadening. In affinity chromatography most protein sticks to the top of the column and is eluted off through
the column. This results in band broadened due to diffusion. In reverse flow chromatography, molecules are
eluted directly off the top of the column and do not pass through the column. This results in sharper bands.
For this application, an AVR7-3 reverse flow valve, in addition to the AVR7-3 sample inject valve, is required.

Figure 9-3. Reverse Flow Affinity Chromatography with an AVR7-3 Valve

System Setup

1. Plumb an AVR7-3 reverse flow valve as shown in Figure 9-3. The column should be connected

between port 4 (column inlet) and port 6 (column outlet).

2. Plumb a short piece of tubing between ports 1 and 2 of the reverse flow valve.
3. Plumb port 5 of the reverse flow valve to port 4 of the upstream sample inject valve.
4. Plumb port 3 of the reverse flow valve to your detector.
5. In the device setup, configure the AVR7-3 valve as a user-define valve and name the valve "Change

Flow Valve". Name valve positions 1, 2 and 3 as "Forward Flow", "Reverse Flow" and "Purge',
respectively.

Writing a Flow Switching Protocol

The following three steps should be included as part of a reverse flow experiment.

1. Add a Change Valve step at the beginning of the protocol and place the Reverse Flow valve in the

Forward Flow position.

2. Just before the elution step add a Change Valve step to your protocol and place the Reverse Flow

valve in the Reverse Flow position.

AVR7-3 VALVE

FOR SAMPLE APPLICATION

AVR7-3 VALVE

AS "CHANGE FLOW" VALVE

INJECT

COLUMN

WORKSTATION
PUMP

SAMPLE
LOOP

SAMPLE

INJECT

LOAD

UV DETECTOR AND
FRACTION COLLECTOR

REVERSE

FLOW