Chromatography is the science of separation. The discovery of chromatography is generally credited to Tswett, who, in 1903 described his work on using a chalk column to separate the pigments in green leaves. The term "chromatography" was coined by Tswett to describe the colored zones that moved down the column.

Basically, chromatography involves the flow of a mobile (liquid) phase over a stationary phase (which may be a solid or a liquid). As the mobile phase moves past the stationary phase, repeated adsorption and desorption of the solute occurs at a rate determined by its solubility.


Chromatographic separations can be carried out using a variety of supports, including  immobilized silica on glass plates (thin layer chromatography), volatile gases (gas chromatography), paper (paper chromatography), and liquids which may incorporate hydrophilic, insoluble molecules (liquid chromatography).




Ion-Exchange Chromatography

Proteins are made up of twenty common amino acids. Some of these amino acids possess side groups ("R" groups) which are either positively or negatively charged. A comparison of the overall number of positive and negative charges will give a clue as to the nature of the protein. If the protein has more positive charges than negative charges, it is said to be a basic protein. If the negative charges are greater than the positive charges, the protein is acidic. When the protein contains a predominance of ionic charges, it can be bound to a support that carries the opposite charge. A basic protein, which is positively charged, will bind to a support which is negatively charged. An acidic protein, which is negatively charged, will bind to a positive support. The use of ion-exchange chromatography, then, allows molecules to be separated based upon their charge. Families of molecules (acidics, basics and neutrals) can be easily separated by this technique. This is perhaps the most frequently used chromatographic technique used for protein purification.

Ion exchange resins contain charged groups.

These may be acidic in nature (in which case the resin is a cation exchanger) or basic (in which case it is an anion exchanger). 
Cation and anion exchangers may be broken down further into weak and strong exchangers (reflecting binding affinity). 

Type of exchanger                Functional group 
Weak cation exchanger         carboxymethyl 

Strong cation exchanger       sulfopropyl 

Weak anion exchanger        diethylaminoethyl 

Strong anion exchanger      quaternary amine 

Usually, samples are loaded under low ionic strength conditions and bound material is eluted using either a step or gradient elution of buffer with higher ionic strength. 
Generally speaking, a protein will bind to a cation exchange resin if the buffer pH is lower than the isoelectric point (pI) of the protein, and will bind to an anion exchange resin if the pH is higher than the pI. 

Knowledge of the pI of the protein is therefore helpful in designing a purification protocol using ion exchange resins (however, you can always simply try different resins to see which works best). 

Generally speaking, ion exchange columns are short and fat in dimensions.

Elution of proteins from ion exchange resins 

Proteins bound to ion exchange resins are bound via non-covalent ionic (salt-bridge) interactions. We can compete for these ionic binding sites on the resin with other ionic groups, namely, salts 
There are two general types of methods when eluting with a salt solution: 

1. Gradient elution and 2. Step elution 

A gradient elution refers to a smooth transition of salt concentration (from low to high) in the elution buffer. Weakly binding proteins elute first, and stronger binding proteins elute last (i.e. they require higher salt concentrations in the buffer to compete them off the column) 
A gradient salt concentration can be made using a gradient maker. In its simplest form, this consists of two containers (must be the same shape) connected by a siphon (or tube at the bottom). One container contains the low salt buffer, and the other contains high salt buffer. The buffer is withdrawn from the low salt container: 

This will produce a linear gradient from low to high salt concentrations over the total volume of the gradient 

If we know the concentration range of salt over which a protein of interest will elute we can simply elute with a buffer containing that concentration of salt. This is known as a step elution. 
Step elutions are generally faster to run, and elute the protein in a smaller overall volume than with gradient elutions. They generally work best when contaminants elute at a significantly different salt concentration than the protein of interest 



Hydrophobic Interaction Chromatography

Not all of the common amino acids found in proteins are charged molecules. There are some amino acids that contain hydrocarbon side-chains which are not charged and therefore cannot be purified by the same principles involved in ion-exchange chromatography. These hydrophobic ("water-hating") amino acids are usually buried away in the inside of the protein as it folds into it's biologically active conformation. However, there is usually some distribution of these hydrophobic residues on the surface of the molecule. Since most of the hydrophobic groups are not on the surface, the use of HIC allows a much greater selectivity than is observed for ion-exchange chromatography. These hydrophobic amino acids can bind on a support which contains immobilized hydrophobic groups. It should be noted that these HIC supports work by a "clustering" effect; no covalent or ionic bonds are formed or shared when these molecules associate.

Gel-Filtration Chromatography

This technique separates proteins based on size and shape. The support for gel-filtration chromatography are beads which contain holes, called "pores," of given sizes. Larger molecules, which can't penetrate the pores, move around the beads and migrate through the spaces which separate the beads faster than the smaller molecules, which may penetrate the pores. This is the only chromatographic technique which does not involve binding of the protein to a support.



This is a very flexible and powerful technique. It is the only technique which can potentially allow a one-step purification of the target molecule. In order to work, a specific ligand (a molecule which recognizes the target protein) must be immobilized on a support in such a way that allows it to bind to the target molecule. A classic example of this would be the use of an immobilized protein to capture it's receptor (the reverse would also work). This technique has the potential to be used for the purification of any protein, provided that a specific ligand is available. Ligand availability and the cost of the specialized media are usually prohibitive at large-scale.

Other Methods

While the methods above are typically chosen for use in a purification process, there are in fact many others that can be used. Each of these methods or techniques takes advantage of a specific part of the protein being purified. The commonality is that all of the techniques employed are based on the protein's structure.


               IT'S MAGIC!


Affinity chromatography involves the use of packing which has been chemically modified by attaching a compound with a specific affinity for the desired molecules, primarily biological compounds. The packing material used, called the affinity matrix, must be inert and easily modified. Agarose is the most common substance used, in spite of its cost. The ligands, or "affinity tails", that are inserted into the matrix can be genetically engineered to possess a specific affinity. In a process similar to ion exchange chromatography, the desired molecules adsorb to the ligands on the matrix until a solution of high salt concentration is passed through the column. This causes desorption of the molecules from the ligands, and they elute from the column. Fouling of the matrix can occur when a large number of impurities are present, therefore, this type of chromatography is usually implemented late in the process. (link)

There are a number of proteins and other biological macromolecules that complex with some other biological entity with a high degree of specificity. This fact is made use of in product recovery operations via the use of affinity chromatography. 

Suppose a certain biomolecule (a) is attached to a solid used to pack a chromatographic column. Now consider a molecule (b) in solution, which has a specific affinity for (a). It is but natural that (b) will want to get out of solution and bind to (a), right? It's this attraction of (b) for (a) which is defined as the partition coefficient 'K'. Now since 'K' for (b) is going to be much higher than that of any other proteins in solution, it will bind to the column while the rest of the complex solution will merely pass through the column with insignificant amounts of non-specific binding occurring.

What are some examples of molecules which may be used for this technique? 




With the advent of Monoclonal Antibody production, which allows the synthesis of a single type of antibody with a very high specific binding constant to its corresponding antigen (a particular protein or other molecule), the preparation of affinity columns has become not only routine, but commercial.

With such 'immunosorbent' column separation, some important practical and theoretical differences arise compared to more conventional forms of chromatographic resolution. 

They are as follows:
1. The dominant cost in the process is the antibody needed to make the immunosorbent column. Generally speaking, this is much more costly than the antigen-containing broth itself.
2. A small column of repeated, high capacity use is required. 
3. Elution of the adsorbed product requires
breaking the antigen-antibody complex. Now this means that denaturing conditions must be employed. Since the antibodies themselves are proteins too, loss of some antibody binding affinity typically occurs, resulting in gradual loss of column capacity. 

4. A first cycle on a new column gives poorer recovery than successive operations, apparently due to some irreversible binding. 

5. A major economic goal in designing any affinity chromatography setup is determination of optimal elution buffer wash volumes and concentrations.

Now let us look at a specific example where affinity chromatography is used, namely, in the purification of human leukocyte interferon made in E. coli. 

Proteins synthesized in genetically engineered organisms and intended for injection into animals must be stringently purified. Pyrogens from E. coli, including the outer envelope lipopolysaccharide (LPS) must be removed or inactivated. Hence product recovery operations such as affinity chromatography are an important step in the manufacturing process. 

Given below is a schematic representation of the typical steps involved in processing human leukocyte interferon produced by recombinant DNA techniques. This will give you an idea of where exactly affinity
chromatography is usually involved in the realm of bioprocessing. 


Another Use of Affinity Columns:

You have the Antigen and are seeking the purified antibody: