Column Chromatography 2z1je

Column Chromatography

Ion Exchange Chromatography • Ion exchange chromatography is commonly used to separate charged biological molecules such as proteins, peptides, amino acids, or nucleotides. • The amino acids that make up proteins are zwitterionic compounds that contain both positively and negatively charged chemical groups. • Depending on the pH of their environment, proteins may carry a net positive charge, a net negative charge, or no charge. The pH at which a molecule has no net charge is called its isoelectric point, or pI.

• In a buffer with a pH GREATER than the pI of the protein of interest, the protein will carry a net negative charge; therefore, a positively charged ANION EXCHANGE resin is chosen to capture this protein. • In a buffer with a pH LOWER than the pI of the protein of interest, the protein will carry a positive net charge; thus a negativelycharged CATION EXCHANGE resin is chosen.

Ion exchange chromatography • After loading an impure protein sample onto an ion exchange chromatography column, the column is washed to remove undesired proteins and other impurities, and then the protein(s) of interest is eluted using either a salt gradient or a change in pH. • To elute proteins from an anion exchange resin, a decreasing pH gradient is chosen, while an increasing pH gradient is chosen for elution from cation exchangers.

Affinity Chromatography • Affinity chromatography is a separation method based on a specific binding interaction between an immobilized ligand and its binding partner. • Affinity chromatography offers high selectivity, resolution, and capacity in most protein purification schemes. It has the advantage of utilizing a protein's biological structure or function for purification. As a result, purifications that would otherwise be time consuming and complicated, can often be easily achieved with affinity chromatography. • A commonly used metaphor to illustrate affinity binding is the lock and key analogy. A unique structure present on the surface of a protein is the key that will only bind to the corresponding lock, a specific ligand on a chromatographic .

ELUTION • pH elution A change oh pH alters the degree of ionization of charged groups on the ligand and/or bound protein. This change may affect the binding sites directly, reducing their affinity , or cause indirect changes in affinity by alterations in conformation

• Chaotropic eluents Chaotropic agents such as guanidine hydrochloride or urea can be used to alter the structure of proteins, but these eluents may cause permanent or temporary damage to the ligand. • Competitive elution Selective eluents are used to separate substances on group specific or when the binding affinity of the ligand/target protein interaction is relative high. The eluting agents competes either for binding to the target of protein or for binding to the ligand. The concentration of competing compound should be similar to the concentration of coupled ligand.

Size exclusion chromatography • There are great diversity of proteins in the body and they all vary in size. One common purification method that separate proteins based on their size is gel filtration or size exclusion chromatography.

• The setup consists of long column that contains special gel beads. These beads are insoluble but porous. They typically consist of a hydrabed polymer such as dextran. • SEC only works if there is relatively large differences size protein in the mixture that we want to separate it.

Size exclusion chromatography A porous column acts as a “molecular sieve”, in which smaller molecules get stuck in pores and take longer to travel through the column , but the larger molecules quickly to the bottom.

Size exclusion chromatography • The small proteins enter the porous beads while the larger proteins cannot fit into the internal volume of the beads • therefore the larger protein reach the bottom first while the others emerge last.