Bio-Rad Media Sampler Pack - Manual (Page 8)

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that has no interaction with the media, e.g., 2 M NaCl. When using salt as the test
substance, use a concentration of 2 M NaCl in water with 0.5 M NaCl in water as
the elution buffer. Concentrated buffer solutions, e.g., 10x buffer concentrate, is
also used. HETP varies depending on the test conditions. Since it is used as a
reference value, it is important to keep conditions and equipment constant so
results can be compared over time. Changes in solute, solvent, elution buffer,
sample volume, flow rate, flow path, temperature, etc. influence results. For
optimal results, ensure that the sample volume does not exceed 2.5% of the col-
umn volume, and maintain the flow rate between 75 and 150 cm/hr. If an accept-
ance limit is defined in relation to column performance, the column HETP and/or
calculated plate number, N, can be used as acceptance criteria for column use.
If a UV absorbing substance is used as the test sample, use a UV absorbance
monitor set at 280 nm. If 2 M NaCl is the probe, use a conductivity monitor; the
elution buffer should be 500 mM NaCl. Apply the sample as close to the column
inlet as possible to avoid dilution in the tubing and piping leading up to the column
inlet. Calculate HETP and the number of theoretical plates (N) as follows:
HETP = L/N
N = 5.54(V
e
/W
½h
) 2
L = Bed height (cm)
N = Number of theoretical plates
V
e
= Peak elution volume or time
W
½h
= Peak width at peak’s half height in volume or time
V
e
and W
½h
should always be the same units
Fig. 3. A simulated chromatography profile from which HETP and A
s
values are calculated.
a b 10% of peak
h i h
W
h,
width at half height
Bed height = 20 cm
Column Volume = 20 L
Flow rate = 100 cm/h
Ve = 10 L
W
h
= 1 L
HETP = 0.036 cm
a = 1
b = 1.2
A
s
= 1.2
Bed height = 20 cm
Column volume = 20 L
Flow rate = 100 cm/h
V
e
= 10 L
W
½h
= 1 L
HETP = 0.036 cm
a = 1
b = 1.2
A
s
= 1.2
W
½h
width
at half height
10% of peak a b
V
e
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