Bio-Rad Nuvia™ HR-S Media - Manual (Page 10)

6 Nuvia HR-S Instruction Manual
Section 5 : Column Packing
Evaluation
When column packing is complete, equilibrate the
column until baseline conductivity is stable. To test the
effectiveness of column packing, inject a sample of a low
molecular weight, unretained compound (for example,
acetone or 1 M NaCl). 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 height equivalent to a theoretical
plate (HETP) values among columns, the same conditions
must be applied.
N = Number of theoretical plates
N = 5.54(V
e
/W
½h
) 2
L = Bed height, cm
HETP = L/N
V
e
= Peak elution volume or time
W
½h
= Peak width at peak half height in volume or time
V
e
and W
½h
should always be in the same units
Peaks should be symmetrical and the asymmetry factor as close
as possible to 1.
Peak asymmetry factor calculation:
A
s
= b/a
a = Front section of peak width at 10% of peak height
bisected by line denoting V
e
b = Latter section of peak width at 10% of peak height
bisected by line denoting V
e
A
s
equal to 0.8–2.5 should be easily achieved under normal
operating conditions.