p. 9
The reduced plate heig ht is calculated:
HETP/d
where d is the diameter of th e bead. As a guidel ine, a value of <3 is norm ally
acceptable.
The peak should be symm etrical, and the as ymmetry factor a s close as
possible to 1 (values b etween 0.8–1.5 are usua lly acceptable) . A change in
the shape of the peak is us ually the first i ndication of bed d eterioration du e
to use.
Peak asymmetry facto r calculation:
A
S = b/a
where
a = 1st half peak width at 10 % of peak height
b = 2nd half peak width at 10% of p eak height.
Figure 2 shows a UV trace for ac etone in a typical tes t chromatogram in
which the HETP and As val ues are calculated .
Fig 2. UV trace for acetone in a typical t est chromatogra m showing the HETP
AND AS value calculatio ns.
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
We=18.7
Wh=0.9
HETP =0.024 cm
a: 0.90
b: 0.85
As: 0.94
UV
absorption
Wh
Ve
ab
Volume