Thesis final - after defense-7 - Jacobs University

Chapter 1
The ions at the start of the series are called kosmotropes or anti-chaotropes and have been
reported to have stronger interactions with water molecules and promote hydrophobic
interactions (40, 41). The effects of the salts on protein retention can also be explained by the
number of water molecules released by the induction of different types of salts. The
selectivity of the different salts can also be predicted by the differences in their capability to
exclude water molecules from proteins and adsorbent surface (27).
The mobile phase pH is another factor affecting the protein adsorption during hydrophobic
interaction chromatography. At highly basic pH values (up to 9-10), a decrease in
hydrophobic interactions between proteins and adsorbent occur, due to the changes in the
hydrophilicity of proteins. In contrast, hydrophobic interactions increase by decreasing the pH
of the mobile phase. The proteins which are usually not able to bind at neutral pH will have
more chances of binding at acidic pH values (42). The basic proteins, such as lysozyme (pI =
10.7) has shown longer retention at basic pH values. In contrast, acidic proteins, such as
human serum albumin (pI = 5.2) has revealed less retention during chromatography at basic
pH values (43). In another report, the total number of releasing water molecules were found
higher, when the mobile phase pH was close to the pI of the protein and decreased when pH
was away from the pI (44). The effect of pH has been rarely reported and the reason could be
the instability of the proteins and adsorbents at elevated pH conditions. Another study also
stated that an optimal pH should be maintained in HIC to obtain high purity and yield at their
maximum extent (45-48). The temperature was also reported to have an effect in HIC. It has
been observed that increasing the temperature promoted protein retention and lowering the
temperature enhanced protein elution. At higher temperature, the proteins are usually
denatured and hydrophobic residues come on surface which resulted in high hydrophobic
interactions. Due to the higher chances of unfolding at elevated temperatures, the unstable
proteins should be separated at low temperatures. The proteins purified in the cold room may
not be reproducible at the room temperature (49, 50). All the reports for the effects of these
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