Question

How would SEC (size-exclusive chromatography) enhance (or not) for protein purification and why? What advantages or disadvantages? What could be done in the future for greater purification?

SEC means size-exclusive chromatography

Answer 1

Size-exclusion chromatography is a method in which molecules in solution are separated by their size. It is usually applied to large molecules or macromolecular complexes such as proteins. SEC works by trapping smaller molecules in the pores of the adsorbent. This process is usually performed within a column, which typically consists of a hollow tube tightly packed with micron-scale polymer beads containing pores of different sizes. When solution travels down the column some particles enter into the pores. Larger particles cannot enter into pores. Large molecules pass by the pores because those molecules are too large to enter the pores. Larger molecules therefore flow through the column more quickly than smaller molecules.

Hence, the smaller the molecule the longer the retention time. The larger the molecules, the faster the elution.

Advantages-

It is feasible to determine the approximate molecular weight of a compound.

Good separation of large molecules from the small molecules with a minimal volume of eluate.

Various solutions can be applied without interfering with the filtration process.

Biological activity of the particles can be preserved during the process of separation.

This technique is can be combined with others that further separate molecules by other characteristics, such as charge and affinity.

There are short and well-defined separation times and narrow bands for this technique, which lead to good sensitivity.

No sample loss because solutes do not interact with the stationary phase.

Future of purification-

Phage-display technologies is generating a large repertoire of ligands specific for protein. ligands will be increasingly used in future applications for large-scale recombinant protein purification. Existing chromatographic processes are also constantly improving and processes such as back-flush ion exchange and displacement chromatography are continuously in development.

Companies have developed mixed-mode stationary phases which offer unique selectivity and higher capacity.

Large bead hydrogel-based technologies such as inside-out ligand attachment (IOLA), developed by LigoChem (NY, USA) are also very good.

Rapidly emerging systems like HPTFF and membrane chromatography for protein purification and provide alternatives to traditional chromatographic systems.