5.4
Performance evaluation of the column
Background
The efficiency of a column depends on how well it is packed. A poorly packed column
gives rise to uneven flow, resulting in zone broadening and reduced resolution. A column
efficiency test should be performed directly after packing and at regular intervals during
the working life of the packed bed. The test is also useful when the separation perfor-
mance is seen to deteriorate. Poor test results indicate that the media should be repacked
or replaced.
A widely used method for expressing the efficiency of a packed bed is in terms of the
height equivalent to a theoretical plate (HETP) and the peak asymmetry factor (A
s
). These
values can easily be determined by applying a sample of for example acetone or NaCl
solution to the column.
It is important that the column is properly equilibrated before the packing is
evaluated.
Note:
The sample volume should be approximately 1 to 2% of the total bed volume, and 1 to
2% v/v acetone can be used. Alternatively 0.8 M sodium chloride, or equivalent can be
applied to a 0.4 M NaCl elution buffer, depending on the media that is used. Use a liquid
velocity 30 to 40 cm/h for 34 µm and 50 µm media and 20 cm/h for 75 and 90 µm media.
Running the HETP test at a higher velocity or using a larger sample volume will affect
both HETP and A
S
values. The airtrap and any filters should be bypassed during the
sample application to avoid back-mixing.
HETP calculation
Calculate the height equivalent of a theoretical plate (HETP) and asymmetry factor (A
S
)
from the UV curve, or conductivity curve if sodium chloride is used as sample, as follows:
HETP = L/N
N=5.54 (V
R
/w
h
)
2
Meaning
Variable
Bed height
L
Plate number
N
Retention volume
V
R
Peak width at half peak height
w
h
V
R
and w
h
are in the same units
100
AxiChrom 50, 70 and 100 columns Operating instructions 28-9331-08 AG
5 Operation
5.4 Performance evaluation of the column
