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The Separation Mechanism
Ion exchange chromatography (IEX) separates molecules according to net charge.
Matrix-bound charged groups reversibly adsorb oppositely charged sample molecules like proteins, peptides and nucleotides (Fig 1.1).
Fig 1.1. Charged sample molecules adsorb to ion exchangers of the opposite sign. The interaction is a dynamic equilibrium that can be influenced by pH or salt concentration.
Desorption is brought about either by a change in the pH or by an increase in the salt concentration of the eluent.
Adsorption
Proteins carry charged amino acids on their surfaces and can thus be adsorbed to ion exchangers. Proteins with net negative charges (excess of negative charges) adsorb to anion exchangers, while those with net positive charges (excess of positive charges) adsorb to cation exchangers. The strength of the adsorption increases with increased net charge (Fig 1.2).
Fig 1.2. The larger the net charge, the stronger is the adsorption. The size of the arrow to the right represents the adsorption strength.
Charged amino acids contain either weak acid or amino groups, whose degree of dissociation depends on pH. Consequently the net charge will vary with pH in a way that is fairly specific for each individual protein (see Charge properties of proteins and peptides).
The main way of influencing selectivity in IEX is consequently by varying the running pH.
Desorption
Essentially two possibilities exist to desorb sample molecules from the ion exchanger:
- Reducing the net charge by changing pH.
- Adding a competing ion to "block" the charges on the ion exchanger.
Varying the pH is a powerful way of influencing the net charges of the sample molecules and is therefore normally used to control the selectivity (elution order and distance between eluted peaks).
Adding a competing ion will not influence the selectivity, but provide a means of desorbing the sample molecules in order of increasing net charges (Fig 1.3).
Fig 1.3. The higher the net charge, the higher the salt concentration required for desorption.
Most IEX experiments use a neutral monovalent salt such as NaCl as the desorbing agent, mainly because NaCl has little or no effect on the running pH.
The higher the net charge, the stronger the adsorption and the higher the salt concentration needed to desorb the sample molecule.
Desorption curves
The adsorption reaction is a dynamic equilibrium between free and adsorbed molecules and is controlled by adding a neutral salt. 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 1.4. (Desorption curves have little practical value and are used here only to demonstrate the working principles of IEX.)
Fig 1.4. 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.
In a column 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 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 charge of the sample. An increase of the net charge will shift the desorption curve to the right and a decrease will shift it to the left (Fig 1.5).
Fig 1.5. The desorption curve is shifted to the right with increasing net charge.
When a sample zone travels down the column under continuous re-partitioning, a certaindeviationfrom equilibrium is created. This, however, is unavoidablyassociated with broadeningof the sample zone.
To read more about zone broadening effects:
Basic principles in gel filtration, Section 5: Peak with... |
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