Bio-Rad Bio-Scale™ Mini UNOsphere SUPrA™ Affinity Cartridges - Manual (Page 11)

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To obtain comparable HETP values between columns, the same conditions must
be applied. The number of theoretical plates is often expressed in terms of plates
per meter (N/m) to normalize for column bed height. Minimum theoretical plate
values should be approximately 4,000–7,000 plates/m. However, since protein
A-based separations are not plate-based, obtaining a particular number of
theoretical plates/meter is not required.
HETP = L/N
N = 5.54 (Ve/W
½h
) 2
L = Bed height (cm)
N = Number of theoretical plates
Ve = Peak elution volume or time
W
½h
= Peak width at peak’s half height in volume or time
Ve and W
½h
should always be in the same units.
Note: Peaks should be symmetrical without significant leading or trailing
shoulders. A split peak may indicate a cracked bed which requires repacking.
Peak asymmetry factor calculation:
See Figure 2
As = b/a
a = Front end of peak width at 10% of peak height bisected by line denoting Ve
b = Back end of peak width at 10% of peak height bisected by line denoting Ve
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