Bio-Rad Foresight™ Chromatography Columns, Prepacked - Manual (Page 33)

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c) Collect the supernatant from each well by
centrifugation (300 x g, 3 min) onto a 96-well
collection plate.
d) Withdraw 200 µl of each supernatant and
apply it onto a 96-well UV plate and measure
the absorbance at 280 nm in each designated
well. Calculate the protein concentration by
using a mass extinction coefficient of 1.4. This
concentration is equivalent to the equilibrium
liquid phase concentration (C).
e) Calculate the protein adsorbed phase
concentration using the following mass balance:
( )
0
0
ads
V
q C C
V
= −
Where q represents the absorbed phase
concentration in equilibrium with C, C
0
represents
the initial hIgG concentration, and C represents
the liquid phase concentration at equilibrium.
f) Obtain the two parameters in the Langmuir
equation by fitting the model to the isotherm
data using the solver-add in Microsoft Excel. This
process is done using as objective function the
sum of squared errors.
Results and Discussion
The human IgG adsorption isotherm for mixed mode
resin is shown in the figure. The corresponding fitted
Langmuir adsorption capacity and the isotherm equilibrium
constant are 50.7 mg/ml and 34.0 mg/ml respectively.
The adsorption capacity calculated is consistent with a
dynamic binding capacity of 43.1 to 53.8 mg/ml obtained
in column chromatography.