GE 71-5000-15 AD - Manual (Page 8)

p. 8
The calculated plate number will vary depending on the test conditions
and it should therefore be used as a reference value only. It is also
important that conditions and equipment are kept constant so that results
are comparable. Changes in solute, solvent, eluent, sample volume, flow
rate, liquid pathway, temperature, etc., will influence the results.
For optimal results, the sample volume should be at max. 2.5% of the
column volume and the flow velocity between 15 and 30 cm/h.
If an acceptance limit is defined in relation to column performance, the
column plate number can be used as part of the acceptance criteria for
column use.
Method for measuring HETP and As
To avoid dilution of the sample, apply it as close to the column inlet as
possible.
Conditions
Sample volume: 2.5% of the bed volume
Sample conc.: 1.0% v/v acetone
Flow velocity: 15 cm/h
UV: 280 nm, 1 cm, 0.1 AU
Calculate HETP and A
S
from the UV curve (or conductivity curve if NaCl is
used as sample) as follows:
HETP = L/N
N = 5.54(V
e
/W
h
)2
where L = Bed height (cm)
N = number of theoretical plates
V
e
= Peak elution distance
W
h
= Peak width at half peak height
Ve and Wh are in the same units.
To facilitate comparison of column performance the concept of reduced
plate height is often used.