32.9: Types Of Column Chromatography

The stability and compatibility of column material with samples are crucial for efficient purification in chromatographic techniques. Various operating parameters such as pH, temperature, or solvent affect the packing of the column material, thereby determining the purification efficiency. The choice of column material also plays an essential role in deciding the operating parameters and can be modified based on the proteins that need to be purified.

Gel Filtration Chromatography

When the protein's chemical nature is unknown, gel filtration chromatography is used for purification based on size. Matrices such as agarose, polyacrylamide, dextran derivatives, or silica beads are used for low-pressure systems, whereas polymeric resins are used for higher flow rates. The varying degree of cross-linking of these polymeric materials determines the pore size of the matrix, which decides the size range of proteins that can be separated.

Ion Exchange Chromatography

Ion-exchange chromatography can purify proteins based on their net charge at a particular pH determined by their isoelectric point or pI. The net charge on matrix resin is used to purify oppositely charged proteins by replacing the cations or anions from the matrix resin with the charged proteins. The resins are categorized based on the type of ion that is exchanged — positively charged proteins replace cations from cation-exchange resins, and vice versa. Strong ion exchangers with stability over a wide range of buffer pH include quaternary ammonium, sulfonate, and sulfopropyl resins. In contrast, weak ion exchangers that are effective only in a narrow pH range include diethyl aminoethyl (DEAE) or carboxymethyl (CM) resins.

Affinity Chromatography

Affinity chromatography involves immobilizing protein-specific antibodies or enzyme-specific substrates to the column material, usually agarose beads. For example, a column with the immobilized antibody of choice is prepared to separate specific antigens. When the mixed antigen sample is passed through the column, the target antigens bind to the immobilized antibodies and separate from the mixture. To separate recombinant proteins tagged with enzymes such as beta-galactosidase, the corresponding substrate Isopropyl-β-D-thiogalactopyranoside (IPTG) is immobilized on the column to purify the enzyme.

High-pressure liquid chromatography (HPLC) and gas chromatography (GC) are the most commonly used column chromatography techniques. In HPLC, the solvent flows at high pressure through a tightly packed column that improves efficiency. Gas chromatography analyzes volatile compounds by converting them to their gaseous forms upon heating. The volatilized sample is pushed through the column by inert gasses such as nitrogen and helium.

Gel filtration, ion-exchange, and affinity chromatography are the common types of column chromatography used to extract pure compounds from a mixture, such as a protein suspension.

Gel filtration chromatography uses an inert matrix of polymeric gel beads to create specific pore sizes.

In the case of a protein suspension, the proteins larger than the pore size elute first, while the smaller ones are trapped in the gel beads. These trapped proteins can then be eluted with multiple solvent washes.

In contrast, an ion-exchange column is packed with matrix beads coated with weak acids or bases that give them a net positive or negative charge at a certain pH.

This helps separate proteins based on their surface charge by forming ionic bonds with the oppositely charged proteins.

Lastly, affinity chromatography uses a matrix tagged with ligands, such as antibodies, to pull out a target protein from a mixture based on specific binding interactions.

In affinity and ion-exchange chromatography, the target protein is finally eluted by adding an elution solvent that weakens the column-protein interactions due to a change in conditions such as the pH, ionic strength, or introduction of a competitive molecule.