Thesis final - after defense-7 - Jacobs University

Chapter 3
acidic pH and decreased at basic pH values of the mobile phase (43). To study the sole effect
of the pI, a neutral pH was maintained during chromatographic experiments. The neutral pH
is also a preferred choice in the downstream processing of proteins. The chromatographic
fractions were analyzed on 2-D gels and the normalized values of the acidic and basic proteins
were calculated. The normalized values of the basic proteins in each gel (fraction) were
presented versus corresponding elution position for different ligands (Figure 31). A higher
number of basic proteins were found at low salt concentrations at the end of the
chromatography which revealed the longer retention of basic proteins during chromatography.
The Sepharose-Phenyl and Toyopearl-Butyl have given longer retention to the basic proteins
in comparison to the other adsorbents. This revealed the partial negative charges on these
adsorbents. The negative charges on adsorbents may attract the positive charges on basic
proteins and resulted in ionic interactions in addition to HIC. The combination of the ionic
and hydrophobic interactions resulted in longer retention of the basic proteins during
chromatography. The partial negative charges on hydrophobic adsorbents were also reported
in literature, although the ration of negative charges varied among adsorbents (133). The
uncharged adsorbents were reported to be preferred for the true hydrophobic interactions,
otherwise at low salt concentrations; the interaction will be more electrostatic than
hydrophobic, which can affect the separation based on hydrophobic interactions (43).
However, in other reports the presence of charges on the adsorbents was favored in order to
maintain the native structures of the proteins (21, 102).
The neutral proteins pI (6-8) and acidic proteins (pI < 6) have been eluted in almost equal
distribution during the chromatographic separations. The reason could be the neutral pH of
the mobile phase, which allowed the neutral proteins to elute on the basis of hydrophobicity.
The analysis of 2-D gels revealed that the gels at the end have mostly neutral proteins, while
acidic and basic proteins are rarely available. The proteins with pI around 6, 7 and 8 have
been highly retained at neutral pH and the reason could be that the charge was near to zero
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