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Effect of temperature
Unlike most other LC techniques binding strength increases in HIC with temperature. The fact that bulk water occurs as loosely associated, temperature sensitive clusters in dynamic equilibrium with “free” water molecules probably explains this. Increasing the temperature shifts the equilibrium towards “free” water, hereby increasing the difference in entropy between bulk water and the shell of ordered water at the hydrophobic surface. The gain in entropy upon minimizing the exposure of hydrophobic surfaces thus increases with temperature, which strengthens the hydrophobic interaction.
Fig 6.1. The bulk water cluster sizes and turn over rates increase
with temperature, a fact that strengthens hydrophobic interaction.
From a practical point of view this means that the eluent, the sample and the chromatography system all should hold the same temperature or unexpected results may be experienced. In Figure 6.2 several column volumes of a sample at 4o C (presumably taken directly from the fridge!) were applied to the column at room temperature. This caused the target protein to stay unadsorbed and to appear in the flow-through fraction. After having raised the sample temperature to equal that of the chromatography system (i.e. room temperature) the target protein binds and elutes as expected.
Fig 6.2. The temperature of the sample must equal that of
the chromatography system or results may deviate from those expected.
The experiment in Figure 6.3 shows that temperature can be used to control the experiment in much the same way as with the salt concentration of the binding buffer.
Moving from room temperature (23o C) to cold room temperature (4o C) required an elevation of the ammonium sulfate concentration in the binding buffer from 1.25 M to 1.55 M to restore the chromatogram.
Fig 6.3. By adjusting the salt concentration of buffer A
the temperature effect can be fully compensated. |
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