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The separation mechanism
Reversed Phase Chromatography (RPC) utilises solubility properties of the sample in much the same way as the organic chemist does when he purifies a crude sample by partitioning it between two different liquid phases in a separation funnel (Fig 1.1).
Fig 1.1 Purification by partitioning the sample between two liquid phases.
The distribution is controlled by the difference in polar properties of the respective phases.
The distribution of sample components between the two phases will depend on their respective solubility characteristics and the polar properties of the phases.
The solubility "rule of thumb" says: "equal dissolves equal" i. e. non-polar substances dissolve preferably in non-polar solvents, while polar ones dissolve in polar solvents.
By varying the polar properties of one of the phases (the lower phase in Fig 1.1) one can change the distribution of the components to be purified as well as the contaminants.
A more efficient way to use the very same principle is to turn to column chromatography (figure 1.2) in which the funnel experiment is repeated a very large number of times per run.
Fig 1.2 Reversed Phase Chromatography utilises solubility differences between
the sample components by a continuous re-partitioning mechanism.
In the column the sample is continuously re-partitioned between a stationary non-polar phase (the beads in the column) and a mobile phase (the liquid pumped through the column).
The distribution between the stationary and the mobile phase is controlled by the polar properties of the mobile phase.
The more of a sample component that dissolves in (adsorbs to) the stationary phase, the more the zone will be retarded while carried down the column by the mobile phase or eluent.
The somewhat odd name reversed phase chromatography deserves an explanation.
The most commonly used solubility-dependent chromatography techniques are:
The name reversed phase chromatography was coined in relation to the older technique, normal phase chromatography.
Solubility properties
The ability of a solvent to dissolve a substance depends on its ability to interact with the substance.
Non-polar substances are held together by Van der Waals interactions (Fig 1.3) which polar solvents do not break, since dipole-dipole interaction between the solvent molecules is too strong.
Fig 1.3 Hydrocarbon chains attract each
other mainly by Van der Waals interactions.
Polar substances are held together by the attraction between dipoles (Fig 1.4). It takes polar solvents to break these bond in order to bring about solubilisation.
Fig 1.4 Dipoles interact in a head-to-tail manner
between the polar centres.
Hydrogen bonding is typical for water and makes water molecules appear in clusters rather than as "free" single molecules (Fig 1.5). The HX- group is also a dipole, which makes water an excellent solvent for polar substances especially those containing HX-groups ( X= O; N; F; Cl).
Fig 1.5. Hydrogen bonding occurs betweenmolecules containing HX-groups.
Each water molecule can bind four other water molecules.
This fact constitutes the very special properties of water
and makes it an excellent solvent for polar substances.
The difference in hydrophobicity forms the basis for separation in RPC.
Fig 1.6 Because of their solubility in water, polar substances are called hydrophilic.
Non-polar substances, on the other hand, are called hydrophobic because they do not
dissolve in water.
There is no distinct limit between hydrophilic and hydrophobic behaviour,
but rather a continuum.
Adsorption mechanisms
RPC was first applied to relatively small organic molecules which more or less dissolved in the hydrocarbon phase, in other words the distribution between the stationary and the mobile phases worked in close analogy to the separation funnel.
Fig 1.7 Organic molecules are "embraced" by the carbon chains of the stationary phase.
With peptides and proteins, however, the mechanism is a bit different. First of all both peptides and proteins carry a mix of hydrophilic and hydrophobic amino acids accessible for interaction with the RPC ligands. Moreover, they are rather large at least in comparison to the traditional organic target molecule. They therefore cannot be completely "embraced" by the hydrocarbon phase. Instead there is a high probability for multi-point attachment. Hydrophobic surfaces are known to combine by a mechanism called hydrophobic interaction (see below!). Together this leads to adsorption rather than to dissolution and the properties of the interaction for peptides and proteins deviates distinctly from that of the typical organic molecule.
Fig 1.8 Unlike the typical organic target molecule peptides and
proteins adsorb to the stationary phase often by multi-point attachment.
Hydrophobic interaction
Hydrophobic surfaces are surrounded with layers of highly organized water molecules, when exposed to aqueous solvents. A decrease in the amount of this organized water would lead to a thermodynamically more favourable situation by an increase in entropy (DS).
Hydrophobic surfaces therefore combine to minimise the total area exposed to the aqueous solvent and thereby the amount of organized water.
RPC media carry ligands consisting of hydrocarbon chains, which can combine with hydrophobic surfaces of peptides and proteins in this way. In fact, in RPC the hydrophobic interaction is strong enough to adsorb these molecules in pure water.
To bring about desorption, eluents consist of mixtures of water and organic solvents like acetonitrile.
 
Fig 1.9. In contrast to bulk water, hydrophobic surfaces are covered by a
shell of highly ordered water molecules. The carbon chains of the
stationary phase combine with the hydrophobic areas of peptide
and proteins to minimise this shell and so gain in entropy.
Fig 1.10. A decrease in the polar properties of the
mobile phase will weaken the hydrophobic interaction.
Fig 1.11. Protein tertiary and quaternary structures depend to a
large extent on hydrophobic interaction as a stabilising force.
RPC eluents are designed to weaken hydrophobic interactions
and are thus potential denaturants.
Peptides and proteins are made to be biologically active under "physiological" conditions i.e. contact with quite polar solvents.
Hydrophobic interaction is one of the important forces stabilizing the tertiary and quaternary structures of proteins and eluents that weaken hydrophobic interaction are also potentially denaturants. If a protein becomes partially unfolded at an eluting strength below or near that needed to desorb the protein, more hydrophobic areas may be uncovered resulting in increased co-operative binding. This in turn requires an even higher eluting strength for desorption leading to further unfolding and so on. Such a chain of events may lead to complete denaturation and/or irreversible adsorption.
Fig 1.12 Peptide secondary structure is stabilized mainly by
hydrogen bonding and is less sensitive to RPC eluents.
Protein tertiary structure depends on hydrophobic
interaction as one of the stabilizing forces
and is thus sensitive to RPC eluents.
Peptides contain a low (if any) degree of tertiary structure and are readily renaturated. They are therefore less likely to be harmed by RPC eluents.
Proteins, on the other hand, depend on their tertiary and quaternary structures for their biological function and are much more difficult to renaturate.
RPC of proteins is therefore a delicate balance between desorption and denaturation and care must be taken to satisfy this balance or the protein may be irreversibly destroyed.
Desorption curves
The adsorption reaction is a dynamic equilibrium between free and adsorbed molecules and is controlled by the content of an organic solvent in the eluent. 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 organic solvent concentration as shown in Figure 1.13.
(Desorption curves have little practical value and are used here only to demonstrate the working principles of RPC.)
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 portion of sample molecules present in the mobile phase.
The desorption curve thus represents the velocity of a sample zone as a function of the organic solvent concentration. The concentration interval corresponding to the desorption curve will be referred to as the partition zone.
Fig 1.13 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. |
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