Prof. Dr. Cristina Dias-Cabral
Chemistry Department
Health Sciences Research Centre (CICS)
University of Beira Interior

Host: Alois Jungbauer

The greatest challenge of any chromatographic technique is predicting the adsorptive
behavior of the biomolecule onto the chromatography resin. Thermodynamic analyses of
biomolecule adsorption have helped to elucidate complex adsorption mechanisms in liquid
chromatography. Information about the thermodynamic quantities associated with liquid
chromatographic processes may be obtained from batch equilibrium experiments, by analyses
of data presented as van’t Hoff plots or from microcalorimetric measurements. Nevertheless,
the resolution with which variations in thermodynamic behavior, as a function of protein
loading and temperature, can be detected by batch equilibrium experiments is limited, and
the indirect method of van’t Hoff analysis may be complicated by the presence of multiple
sub-processes associated with adsorption. Furthermore the results may not be representative
of what occurs under overloaded conditions. Calorimetric methods, have proven to be a
reliable methodology to elucidate complex adsorption mechanisms in liquid chromatography,
as they measure the heat flows caused by interactions during the adsorption process.
Specially, flow microcalorimetry (FMC), as a dynamic means of heat signal measurement,
provides valuable insight about the driving forces and underlying mechanisms of adsorption,
both under linear and overloaded conditions. The heat signals produced from this technique
corresponds to adsorption and desorption events occurring in a small column filled with the
adsorbent. A flow microcalorimeter is operated in a manner that is analogous to the operation
mode of a chromatograph and hence the results are expected to be representative of what
happens in an actual chromatography column. This technique has been used, with good
results, to study the adsorption mechanism of biomolecules, such as proteins and plasmid
DNA, onto different supports.
The presentation will give an overview of the technique and examples of its application in the
study of the adsorption mechanism of different biomolecules (protein and plasmid DNA) onto
different chromatographic supports (ion exchange and hydrophobic interaction).