Thesis final - after defense-7 - Jacobs University

Chapter 3
work, the effects of larger proteins (> 50 KDa) on retention have been examined as a function
of the adsorbent type. The number of larger proteins in each fraction was divided by the total
number of proteins to normalize the data. The normalized values of the larger proteins from
the three ligands was presented in Figure 30, where the larger proteins were found to be
higher at low salt concentrations than at high salt concentrations. In other words, the larger
proteins were higher in the latter fractions in comparison to the earlier ones. This revealed that
larger proteins retained longer on the adsorbents. In case of larger proteins, the retention was
higher for Toyopearl-Butyl and Toyopearl-Hexyl in comparison to the other adsorbents. It
was reported before that if the pore space of the adsorbent is smaller than the dimensions of
the magnitude as the protein has, then it will result in less retention of protein (33). However,
this was not applicable on Toyopearl-Ether and Toyopearl-Phenyl adsorbents where larger
proteins has shown less retention, although they have high pore size (100 nm). The reason
could be the less hydrophobic nature of the two adsorbents which retained larger proteins for
a shorter time during chromatography. In a similar way, Source-Phenyl has given less
retention to larger proteins, although it has a high hydrophobic nature, but the pore size is
smaller (34 nm). These results declared that the pore size and hydrophobicity of the
adsorbents could be the combine contributing parameters affecting the retention of larger
proteins.
In case of smaller proteins it was reported once that it has no effect on retention (33).
However, in another report it was reported that smaller proteins exhibited a decrease in
retention due to the less contact surface area with adsorbents (68). This was confirmed in this
work when smaller proteins have exhibited an inverse relationship to the corresponding
retention behavior and most of the smaller proteins were eluted at the beginning of the
chromatography. The reason could be that smaller proteins have less non-polar surface area to
interact with the adsorbents.
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