Bio-Rad Media Sampler Pack - Manual (Page 7)

5
1. Packing begins with preparation of the tubing and the column. Clean and
rinse the column well with packing buffer before packing. The rinse is also
used to displace air from the column and its exit tubing. Make sure there is no
air trapped in the bottom flow cell. After removal of all the air from the bottom
flow cell and exit tubing, leave about 2 cm of liquid in the bottom of the
column. Close the outlet valve(s).
2. In a suitable container, mix the packing buffer with the media to form a 50%
slurry.
3. Pour or pump the slurry into the column.
4. Insert the movable piston and lower it to the surface of the liquid, making sure
no air is trapped under the piston.
5. Pressurize the inflatable seal to 2 bar (29 psi), so when lowering the piston
into the slurry, the buffer exits the top of the column through the inlet line. Be
sure the inlet line is completely filled with packing buffer to avoid air being
pumped back into the column.
6. Connect a pump and a pressure gauge, open all inlet and outlet valves, and
start packing at constant flow rate or pressure. Keep the flow rate or pressure
constant throughout the packing. Check the pressure at the column inlet. Do
not exceed the pressure limit of column or media.
7. Lower the piston as the bed compresses.
8. When the media bed no longer compresses, mark the bed height on the
column tube, close the outlet valve, and stop the pump. The bed will start to
expand in the column.
9. Lower the piston to within 1 cm of the surface of the media bed. Fully inflate
the seal (4 bar), start the pump, open the valves, and continue packing.
10. Repeat steps 8 and 9 until there is a maximum of 1 cm between media bed
surface and piston when the media bed is stable.
11. Close the bottom valve, stop the pump, disconnect the column inlet, and
lower the piston to the gel bed surface. The column is ready for testing of
column packing efficiency.
Section 7
Column Packing Evaluation
To check the packing efficiency and to monitor the column’s status over time,
packing efficiency and asymmetry should be tested immediately after packing and
at regular intervals to ensure reproducible performance. The values to be
determined for the packed column are the height equivalent theoretical plate
(HETP), number of theoretical plates (N), and the asymmetry factor (A
s
). The
method used involves applying a test sample of a low molecular weight substance
LIT486C.qxd 11/2/2004 3:12 PM Page 5