As seen in previous post, there are a number of different types of chromatography used in the purification of proteins:
- Gel permeation chromatography, which separates molecules by size
- Ion exchange chromatography, which separates molecules based on differences in their charge density
- Hydrophobic interaction chromatography, which separates molecules based on differences in hydrophobicity
- Affinity chromatography, which separates molecules based on differences in their affinity for a target ligand attached to the chromatography resin
GEL PERMEATION CHROMATOGRAPHY
Gel filtration chromatography, is also known as gel permeation and size exclusion chromatography. Gel filtration chromatography allows for the separation of proteins based on differences in their size. Larger molecules do not fit within the pores of the chromatography resin, and therefore pass completely through the column and are not retained. By contrast, smaller molecules can partition into the pores of the chromatography resin, and therefore elute at a later time.
An example of this type of resin is Sephadex (cross-linked dextran), that is porous, yet strong enough so they didn't collapse during operation, and hydrophilic, so that proteins would not irreversibly adsorb to them.
(a) non porous resin vs (b) porous resin
The retention volume for a molecule on a gel filtration resin is given by this equation:
In this case, Vi is the intra-particle pore volume and KD is the partition coefficient of the molecule, and is related to its molecular weight.
Given this, the distance between two peaks is then equal to:
Therefore, a gel filtration material with a maximum difference between KD2 and KD1 and a high pore volume, Vi, will give the maximum resolution and a maximal separation between the two molecules
In ion exchange chromatography, the protein interacts with the resin based on an electrostatic attraction between the protein and negatively or positively charged groups on the resin. The strength of this interaction depends on the charge of the protein, the charge of the resin, the dielectric constant of the mobile phase, and competition from other ions in solution. A protein will adsorb to the resin when they are of opposite charges. There are two types of ion exchange chromatography: anion exchange chromatography, which adsorbs negatively charged groups on the protein to positively charged groups on the resin, and cation exchange chromatography, which is simply the opposite of anion exchange.
Ion exchange chromatography is most useful as a polishing step to remove charged impurities such as DNA and host cell proteins.
HYDROPOHOBIC INTERACTION CHROMATOGRAPHY
In hydrophobic interaction chromatography, hydrophobic residues on the protein’s surface interact with hydrophobic ligands attached to a hydrophilic chromatographic resin. The type of hydrophobic ligand and its concentration on the resin are important parameters that control its separation properties
The type of hydrophobic ligand, for example the varying links of alkyl chains, in its concentration on the resin are important parameters of the resin that control its hydrophobicity and separation properties.
With hydrophobic interaction chromatography, the interaction of the protein with the resin is modulated by adding or removing anitichaotropic salts, such as ammonium sulfate, to the buffer. High salt concentrations promote adsorption to the hydrophobic groups in the column. Therefore, to elute adsorbed proteins for the column, the salt concentration is decreased. As the salt concentration is decreased, proteins of increasing hydrophobicity will desorb and elute from the column.
Hydrophobic interaction chromatography is often most useful as a polishing step to remove higher molecular weight impurities that may not be removed by other methods.
In affinity chromatography, the proteins interact with ligands attached to the chromatographic resin. These ligands are chosen for their specific and selective interaction with the target biologic. As the sample is loaded onto the column, the target biologic will bind to the ligand, while other impurities will not. We can then recover our protein with an appropriate elution buffer, usually of low pH.
When affinity chromatography is available, it is most useful as an initial purification step. Because of the ligand's high affinity for the target biologic, affinity chromatography can significantly increase the purity and concentration of the biologic in one step.
The number of molecules that will bind to the column is dependent on the concentration of the protein in the mobile phase, C sub M, the concentration of molecules already adsorbed to the surface, C sub s, and the concentration of adsorption sites on the resin itself, S. At equilibrium, the association constant K, is given by this equation.
We can define the binding capacity, Q, as the concentration of the total number of binding sites on the resin, C sub S, plus S Solving for C sub s gives us the following, which is known as the Langmuir adsorption isotherm.
So, the retention volume is giving by this equation:
Where A sub S is either the total area of adsorbent on the column, or the weight of adsorbent.