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Hydrophobic Interaction Chromatography (HIC)

Importance of Hydrophobic interaction in biological systems

1. Folding of globular proteins (e.g formation of molten globule state)

2. Binding of many small moleculest to proteins during enzyme catalysis,


regulation and transport across many surface

3. Self association of phospholipids and other lipids to form membrane


bilayer and binding of integral membrane proteins.

4. In HIC, hydrophobic interaction used for binding of proteins to adsorbents


with hydrophobic ligands.

- Elution according to differences in strength of interaction between the


protein and the amphiphilic matrix is brought about by decreasing salt
concentration of the eluent or decrease of the solvent polarity.

Theory

-Water is a poor solvent for nonpolar solutes and dissolving a nonpolar substance
in water is thermodynamically unfavourable. Due to the hydrogen bonding
capability, water has a unique structure, causing a high surface tension.

-A non polar solute forces the water to form a cavity in which the solute fits. In
this process, many hydrogen bonds between the water molecules are broken but
new hydrogen bonds are formed among the water molecules, surrounding the
cavity, leading to a negative change in enthalpy.

- the increase in degree of order of solvent molecules near the solute explains
negative change in entrophy. In all, the net change in free energy is positive and
unfavourable.

-In hydrophobic interaction, the increase in entrophy originating from water


molecules leaving the more ordered structure around the nonassociated solutes
for the more unstructured bulk water is the main driving force. Positive
enthalphy is smaller than entropy term and does not influence spontaneous
association to a great extent.

- The strength of hydrophobic interactions should increase with an increase in


temperature, at least in the temperature range that is of interest for HIC.

Factors important for HIC

1) Type and concentration of salt used

2) Additives that change polarity of the solvent

Type and concentration of salt used


Non-ionic interactions enhanced by ‘salting-out’ ; hydrophobic interaction surface
attached to matrix

- The formation of a cavity in water with a salt that gives a high surface
tension needs a bigger input of energy than does the formation of a cavity
with ethe same surface area in water containing a salt giving lower
surface tension.

- The higher the salt concentration, the lesser the availability of water
molecules in solution, increasing the surface tension and hence the
stronger the interaction.

Additives that change polarity of solvent

- Ethylene glycol decrease interaction btw HIC gel and proteins by changing
the overall structure of water slightly towards a structure resembling an
organic solvent.

Interaction btw Protein and the HIC adsorbent

Strength of interaction depends on

1. Proportion of nonpolar surface area of a protein

2. Distribution of the nonpolar surface area of a protein

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