Bio-Rad Nuvia™ cPrime™ Media - Manual (Page 10)

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Section 5
Column Packing Evaluation
After column packing is complete, equilibrate the column with up
to 5 CV equilibration buffer. To test the efficiency of the column
packing operation, inject a sample of a low molecular weight,
unretained compound (for example, acetone or 1 M NaCl) to
determine height equivalent to a theoretical plate (HETP). If acetone
is used as the test marker (use a UV absorbance monitor set at
280 nm), the equilibration buffer must have a salt concentration
<100 mM. If 1 M NaCl is the test marker (use a conductivity
monitor), then the equilibration buffer salt concentration should be
100–200 mM. The sample volume should be 1–3% of the total
column volume. Column testing should be operated using the
same linear velocity used to load and/or elute the sample.
To obtain comparable HETP values among 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/W½h)2
L = Bed height (cm)
N = Number of theoretical plates
Ve = Peak elution volume or time
W½h = Peak width at peak half height in volume or time
Ve and W½h should always be in the same units
Peaks should be symmetrical and the asymmetry factor as close as
possible to 1. Values of 0.8–1.8 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