The principle behind Ion-Exchange Chromatography is that we are creating a competition between the target ions and other ions of the same charge to bind to the adsorbent matrix of the column, which has opposite charge. Hence, we are exploiting the fast that other than at isoelectric point, by changing the pH, we are able to obtain protein in ionic form, where it is positively charged or otherwise. The ionic protein would have to compete with the other ions present in the matrix to bind to the adsorbent beads. The separation of the proteins depends on the reversible adsorption of charged solute molecules to immobilized ion-exchange groups of opposite charge.
Steps in IEC:
- Equilibration
- Sample application and washing
- Elution
- Washing and re-equilibration
The process of equilibration is to ensure that all the adsorbent beads are in equilibration to the counter ions. Usually, for anion exchanger, Cl- ion is used while for cation exchanger, Na+ is used.
Strong exchanger means that the exchanger remains charged over a large pH range while weak exchanger means the exchanger remains charged over a small pH range.
Sample Application and Washing
Before the sample is inserted into the column, we should dissolve the protein sample into the starting buffer and adjust the pH. As IEC has the ability to concentrate the protein sample, the sample applied into the column can be of any volume, and also maximize the buffering capacity of the buffer.
Upon application of sample, the proteins that carry appropriate charge would displace the counter ions and bind reversibly. The protein that binds the strongest to the column is usually found closest to the top of the column. The column would then be washed with the starting buffer to wash out the protein samples or component that do not adsorb and they are recovered in a breakthrough peak. Sometimes, in the process of adsorption, protein bands can be observed.
Figure 4.14 above shows that the α decreases with increasing salt concentration. As the salt concentration is increasing, the protein band is becoming sharper. When salt is added, desorption occurs and proteins start to move down the column. However, the proteins are not moving as fast as the salt passing by. Hence, each part of the protein band experiences increase in salt concentration. At the same time, the edge of the band is sharpen due to the protein concentration effect.
Importance of pH in IEC
Actually, the pH of the micro-environment of the ion-exchanger differs slightly from the pH of the starting buffer added to the column. Hence, the effect of pH in Ion-Exchange Chromatography cannot be underestimated due to Donnan's effect. Due to Donnan's effect, the proton in the adsorbent matrix can be repelled or attracted. Hence, for anionic ion-exchanger, normally the pH is 1 unit higher while for cationic ion-exchanger, the pH is 1 unit lower. The lower of ionic strength of the buffer results in greater the difference in pH due to Donnan's effect. Hence, it would have great impact on the stability of the protein, especially enzyme, which is a function of pH. Enzyme protein usually prefers mild alkaline solution than mild acidic environment. For anionic ion-exchanger, denatured due to high pH is normally rare. Hence, in order to prevent the denaturation due to low pH in cationic-exchanger, it is wise to dilute the sample first before application into the column.
Elution
Desorption of the proteins can be done by:
- Changing the ionic strength
- Changing the pH
- Inclusion of new ionic species
Isocratic elution
Isocratic elution is done using solvent that has constant composition throughout the elution process. Such elution method can only be used on proteins with known chemical properties and the proteins are run in repeated trials.
Stepwise elution
Stepwise is the serial application of isocratic elution. Such a method might not work efficiently in protein separation because sometimes the ionic strength of the buffer used at any changes in the concentration might be too high, or too low, resulting in false separation. If the buffer used is too weak, it might result in sample being separated into broad peak while if the buffer used is too strong, it will result in the several different proteins elute at the same time.
Gradient elution
Gradient elution is by increasing the concentration of buffer added to the column gradually. At low salt concentration, protein that are weakly attached to the adsorbent would be desorbed and eluted out, while at higher salt concentration, the second protein would get eluted, and so on. The problem of having broad elution peak in isocratic elution is actually minimize in gradient elution as the elution power of the salt solution increases as the gradient increases.
Linear gradient is highly recommended. However, the difference lies in the fact that whether it is a short steep linear gradient or a long shallow gradient. There is no perfect choice of gradient. For short steep linear gradient, the peak produced would be sharp and faster separation but with the compromise of having short retention distance between the peaks. For long shallow linear gradient, maximum separation of the peak is achieved but the separation will be long and the peak broadening might result.
The gradient elution can be created by mixing the initial buffer and final buffer together initially. Gradually, the proportion of final buffer increased while the proportion of initial buffer decreases along the process of elution.
Washing and re-equilibration
The column should be washed as it might contain denature proteins as well as other components such as lipid. The presence of such components might affect the efficiency of the column in the future re-use, present as contaminant in the future run, or reduce adsorption.
Advantages of Using IEC
IEC is the most popular chromatography method used to separate protein, due to:
- High resolution
- High protein-binding capacity
- Versatility as there are various kinds of IEC available and the pH and buffer condition vary.
- Straight-forward as the separation is based on charge
- Ease of peformance
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