p. 8
The calculated plate numb er will vary dependi ng on the test conditi ons
and it should therefore be u sed as a reference valu e only. It is also
important that cond itions and equip ment are kept consta nt so that results
are comparable. Change s in solute, solvent , eluent, sample vo lume, flow
rate, liquid pathway, te mperature, etc., wi ll influence the re sults.
For optimal results, t he sample volume sh ould be at max. 2.5% of t he
column volume and the fl ow velocity betw een 15 and 30 cm/h.
If an acceptance limit is def ined in relatio n to column perfor mance, the
column plate number can b e used as part of th e acceptance criter ia for
column use.
Method for measuring HE TP and As
To avoid dilution of the sampl e, apply it as close to t he 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 AS f rom the UV curve (or c onductivity cu rve if NaCl is
used as sample) as foll ows:
HETP = L/N
N = 5.54(Ve/Wh)2
where L = Bed height (cm)
N = number of theoretical p lates
V
e = Peak elution distan ce
W
h = Peak width at half peak h eight
Ve and Wh are in the same units .
To facilitate comparison of co lumn perform ance the concept of red uced
plate height is often us ed.