Bio-Rad UNOsphere™ Rapid S Media - Manual (Page 5)

3
Packing Large Columns
In large columns, UNOsphere rapid S should be packed using a 20–50% slurry,
ideally using a combination of axial and flow compression. The best overall
performance of UNOsphere rapid S will be obtained when a compression ratio of
17–18% is used. After achieving the desired compression it is recommended to
flow condition the column with fresh packing buffer for 3 column volumes of up flow
followed by 3 column volumes of down flow at 100 cm/hr. After flow conditioning it
is recommended to evaluate the column efficiency using your standard operating
procedures or the procedure described in section 5 below.
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 100–200 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 1,000–3,000 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 0.8–1.2 are acceptable.
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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