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p. 9
The reduced plate height is calculated:
HETP/d
where d is the diameter of the bead. As a guideline, a value of <3 is normally
acceptable.
The peak should be symmetrical, and the asymmetry factor as close as
possible to 1 (values between 0.8–1.5 are usually acceptable). A change in
the shape of the peak is usually the first indication of bed deterioration due
to use.
Peak asymmetry factor calculation:
A
S
= b/a
where
a = 1st half peak width at 10% of peak height
b = 2nd half peak width at 10% of peak height.
Figure 2 shows a UV trace for acetone in a typical test chromatogram in
which the HETP and As values are calculated.
Fig 2. UV trace for acetone in a typical test chromatogram showing the HETP
AND A
S
value calculations.
Column: BPG 300
Media: Sepharose 6 Fast Flow
Bed height: 57.5 cm
Bed volume: 40.6 litres
Eluent: Distilled water
Sample: 1.05 litres (1% acetone)
Flow velocity: 19 cm/h
W
e
=18.7
W
h
=0.9
HETP =0.024 cm
a: 0.90
b: 0.85
A
s
: 0.94
UV
absorption
W
h
V
e
a
b
Volume