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The Separation Mechanism
Before going into details with the separation mechanism of HIC there is a need to define terms like hydrophilicity and hydrohobicity, and to explain the phenomenon called hydrophobic interaction.
Definitions
Hydrophilic means "loves water" and is assigned to molecules which readily interact with water through dipole-dipole interaction or by hydrogen bonding.
Hydrophobic means "hates water" and is assigned to molecules which do not interact with water.
There is no sharp boundary between hydrophilic and hydrophobic but rather a continuum of hydrophobicity (or hydrophilicity for that matter) as illustrated in Figure 1.1.
Fig 1.1. The ability to "love water" resides in the possibility of
interacting with water molecules trough
dipole-dipole interaction or by hydrogen bonding.
In HIC sample molecules (in most cases proteins) are made to interact with hydrophobic ligands grafted on a hydrophilic matrix.
There is no general answer to how a protein surface interacts with hydrophobic ligands.
The perhaps most widely accepted, however, explains the hydrophobic interaction as an entropy-driven mechanism (Ref. Porath et al. 1973, Nature 245: 465).
Hydrophobic interaction
Consider what happens when you prepare a salad dressing. You basically mix oil, water and herbs. When you shake this mixture a lot of small droplets of oil are formed and suspended in the water. If you have done your shaking well the suspension will last for some time, but will eventually go back to the state with two separate phases, one water phase and one oil phase.
If you follow this return to two separate phases closely you will notice that it starts with the joining of small droplets to form larger and larger ones.
Fig 1.2. Oil droplets dispersed in water will join to form larger and
larger droplets, which eventually form two continuous phases over time.
Water is an exceptionally good solvent for polar substances but not for non-polar ones like oil and fat. These solubility properties stem from water being a strong dipole which, moreover, can form hydrogen bonds with molecules containing HX groups (X = O; F; N or Cl).
In liquid water a majority of the water molecules occur in clusters due to hydrogen bonding between them selves. One water molecule can bind to four others (Fig 1.3). .Although the half-life of water clusters is very short, the net effect is a very strong cohesion between the water molecules reflected e.g. by its high boiling point.
Fig 1.3. The solubilising properties of water resides
in its ability to interact with dipoles and to
form hydrogen bonds with HX-containing molecules.
At an air-water interface, water molecules arrange themselves into a strong shell of highly ordered structure. The possibility to form hydrogen bonds is here no longer balanced but is dominated by the liquid side of the interface. This gives rise to the ordered structure and manifests itself in a strong surface tension.
Anything that influences the stability of the water shell will also affect the surface tension.
Fig 1.4. Surface tension at the air-water interface stems from a shell of
highly ordered water. In contrast, bulk water is organised in short-lived
clusters by hydrogen bonding. A rise in temperature will lead to smaller clusters
and a higher exchange rate of water molecules between them.
When a hydrophobic substance is immersed in water something analogous to the surface tension phenomenon happens.
The water molecules cannot "wet" the surface of the hydrophobic substance. Instead they will form a highly ordered shell, around the hydrophobic substance, caused by the lack of possibility to form hydrogen bonds in all directions.
Now, minimising the extent of this shell will lead to a decrease in the number of ordered water molecules which represents a thermodynamically more favourable situation by an increase in entropy (DS). Consequently hydrophobic surfaces combine to accomplish this.
Fig 1.5. Hydrophobic surfaces in water are surrounded by a shell of highly ordered water.
The system strives to minimize the total area of such shells in order to gain in entropy
and forces hydrophobic substances to merge.
This phenomenon is called hydrophobic interaction and depends on the behaviour of the water molecules rather than on direct attraction between the hydrophobic molecules.
Turning back to the oil droplets of the dressing, we now have an explanation for their tendency to combine.
Proteins carry both hydrophilic and hydrophobic areas on their surfaces and at high concentrations of certain salts they precipitate (salting out), the main cause is enforced hydrophobic interaction.
Fig 1.6. Salting out is (at least in part) a result of the
capability of certain salts to enhance hydrophobic interaction.
Adsorption
HIC media contain ligands that can combine with hydrophobic surfaces of proteins.
In pure water this hydrophobic effect is too weak to cause interaction neither between ligand and proteins, nor between the proteins themselves. However, certain salts enhance hydrophobic interactions and adding such salts brings about adsorption to HIC media.
Fig 1.7. HIC deals with the relation between water shells
around hydrophobic surfaces, bulk water clusters
and salts enhancing hydrophobic interaction.
The following salts strengthen hydrophobic interaction in the order indicated:
Na2SO4 > K2SO4 > (NH4)2SO4 > NaCl > NH4Cl > NaBr > NaSCN
Ammonium sulfate is the salt commonly used to control adsorption in HIC. The sample is applied and adsorbed at high salt concentrations (~ 1M).
To bring about selective desorption, the salt concentration is then lowered gradually and the sample components elute in order of hydrophobicity.
Fig 1.8. The adsorption of hydrophobic molecules
is a reversible reaction whose equilibrium
is controlled by the salt concentration.
Desorption curves
The adsorption reaction is a dynamic equilibrium between free and adsorbed molecules. It can be described in terms of a desorption curve obtained by plotting the relative amount of free sample molecules as a function of the salt concentration as shown in Figure 1.9.
Fig 1.9. The desorption curve reflects the distribution of the sample between
the mobile and the stationary phase. Within the partition zone this distribution
varies as a function of the salt concentration and the elution velocity varies accordingly.
(Desorption curves have little practical value and are used here only to demonstrate the working principles of IEX.)
In a column experiment all transport of a sample down the column is carried out by the mobile phase (the eluent) and acts only on the molecules present in the mobile phase.
When a sample travels down the column, its velocity is proportional to the part of sample molecules present in the mobile phase.
The desorption curve thus represents the velocity of a sample zone as a function on the salt concentration. The salt concentration interval corresponding to the desorption curve will be referred to as the partition zone.
The position of the desorption curve along the salt concentration axis is governed by the net hydrophobicity of the sample. An increase of the net hydrophobicity will shift it to the right and a decrease to the left (Fig 1.10).
Fig 1.10. The desorption curve is shifted to the right with increasing net hydrophobicity.
Within the partition zone the sample will move down the column by way of a continuous re-partitioning mechanism.
The transport of the molecules present in the mobile phase creates an uneven distribution of
“stationary” and “mobile” sample molecules in that the concentration profile in the mobile phase will always be slightly ahead of that in the stationary phase.
The partitioning mechanism strives to correct this, resulting in a mass transfer of sample molecules from the mobile phase to the stationary phase at the front of the sample zone and a mass transfer in the opposite direction at the rear end of the zone (Fig 1.11).
Fig 1.11. Mass transfer directions in a travelling sample zone. Blue arrows indicate net transport direction between mobile and stationary phases.
This positional discrepancy in concentration profiles causes broadening of the sample zone and is a consequence of the chromatographic process itself i.e. the continuous re-partitioning.
Although always present, the extent of this type of zone broadening depends on:
- Mass transfer rate.
- Mobile phase flow rate.
To minimise zone broadening, mass transfer must be given time enough for equilibration (Fig 1.12).
Fig 1.12. The flow rate must be balanced against the mass transfer rate or sample zones will broaden excessively due to incomplete mass transfer. Arrow length indicates flow rate.
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