Bio-Rad Bio-Scale™ Mini UNOsphere SUPrA™ Affinity Cartridges - Manual (Page 10)

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• Allow the slurry to settle until a 2–5 cm layer of clear supernatant packing
buffer is observed above the settled bed
• Lower the top piston into supernatant layer and engage the seal. Continue
to lower the top piston to remove any trapped air and to purge the top
piston assembly of air
• After ensuring that all of the air has been removed, begin flow packing at
approximately 300 cm/hr. This value may change depending on your
process requirements
o Columns equipped with speed-controlled dynamic axial compression
capabilities (such as the Bio-Rad EasyPack™ column) may be packed
using a combination of flow packing and axial compression, or simply by
using axial compression.
o Columns without axial compression capabilities may be flow-packed
without any precaution
• The optimal compression percentage for UNOsphere SUPrA media is 12%
4.3 Stall-Packing Closed Column Systems
When using process scale closed column systems that require stall packing,
consult the user manual of your specific column model.
Section 5
Column Packing Evaluation
After the completion of the packing operation, it is highly recommended and often
routine to verify the quality of the packing. This verification can also be done at
anytime throughout the lifetime of the packed bed to verify efficiency.
The verification consists of determining height equivalent to theoretical plate (HETP)
as well as the asymmetry factor (A
s
).
To determine HETP, equilibrate the column with 3–5 column volumes (CV) of
starting buffer or until a baseline conductivity (or UV) trace is achieved. To test the
effectiveness of column packing, inject a sample of a low molecular weight,
unretained compound (e.g., acetone or 1 M NaCl). If acetone is used as the test
marker (use an absorbance monitor set at 280 nm), the starting buffer must have a
salt concentration less than 100 mM. If 1 M NaCl is the test marker (use a
conductivity monitor), then the testing buffer salt concentration should be 100–200 mM.
The sample injection volume should be 1–3% of the total column volume. The
column testing should be performed at approximately 100 cm/hr.
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