Bio-Rad Media Sampler Pack - Manual (Page 5)

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7. Attach the column to your chromatography system, and purge the column with
starting buffer at linear velocities up to 1,200 cm/hr. If the bed compresses,
repeat steps 6 and 7.
Packing Large Columns
In large columns, UNOsphere should be packed using a 20–50% slurry at 1–2 bar
(constant pressure systems) or at 100–1,200 cm/hr (constant flow systems). It is
recommended to never use a packed column at a pressure or flow rate that is
>75% of the maximum pressure and flow rate achieved during its initial packing.
Given the industrial column hardware and packing skids, we recommend using
your standard operating procedures for column packing.
Section 5
Column Packing Evaluation
Once column packing is complete, equilibrate the column with up to 5 column
volumes (CV) of starting buffer. 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 between 100 and 200 mM. The sample volume should be
2–5% of the total column volume. The column testing should be operated using the
same linear velocity used to load and elute the sample.
To obtain comparable Height Equivalent to a Theoretical Plate (HETP) values
between columns, the same conditions must be applied. Minimum theoretical plate
values should be between 1,000 and 3,000 plates/m for linear velocities of
50–500 cm/hr.
HETP = L/N
N = 5.54(Ve/W
½h
) 2
L = Bed height (cm)
N = Number of theoretical plates
Ve = Peak elution volume or time
W
½h
= Peak width at peak’s half height in volume or time
Ve and W
½h
should always be in the same units.
Peaks should be symmetrical, and the asymmetry factor as close as possible to 1.
Values of 0.8 to 1.5 are acceptable. A change in the shape of the peak is usually
the first indication of deteriorating performance.
Peak asymmetry factor calculation:
As = b/a
a = Front section of peak width at 10% of peak height bisected by line denoting Ve.
b = Latter section of peak width at 10% of peak height bisected by line denoting Ve.
As = 0.8–1.8 is acceptable.
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