Bio-Rad Media Sampler Pack - Manual (Page 9)

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 medium.
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
Packing efficiency should be tested before use and re-evaluated, as necessary. The
values to be determined for the packed column are the height equivalent theoretical plate
(HETP) or number of theoretical plates (N), and the asymmetry (A
s
). The method involves
applying a test sample of a low molecular weight substance that has no interaction with
the media, e.g., 2 M NaCl. When using salt as the test substance, use a concentration of
2 M NaCl in water with 0.5 M NaCl in water as the elution buffer. Concentrated buffer
solutions, e.g., 10X buffer concentrate, can also be used. HETP varies depending on the
test conditions. Since HETP and A
s
are used as reference values, it is important to keep
conditions and equipment constant so results can be compared over time. Changes in
solute, solvent, elution buffer, sample volume, flow rate, flow path, temperature, etc.
influence results. For optimal results, ensure that the sample volume does not exceed
2.5% of the column volume, and maintain the flow rate between 75 and 150 cm/hr. The
column HETP and/or calculated plate number, N, can be used as acceptance criteria for
packed columns. If a UV absorbing substance is used as the test sample, use a UV
absorbance monitor set at 280 nm. If 2 M NaCl is the sample, use a conductivity
monitor; the elution buffer should be 500 mM NaCl. Apply the sample as close to the
column inlet as possible to avoid dilution in the tubing and piping leading up to the
column inlet. Calculate HETP, the number of theoretical plates (N), and A
s
as follows:
A
s
= b/a
HETP = L/N
N = 5.54(V
e
/W
½h
) 2
L = Bed height (cm)
N = Number of theoretical plates
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