Chapter 5 Size-exclusion chromatography

Size-exclusion chromatography (SEC), also called “gel-permeation chromatography,” “molecular-sieve chromatography,” or “gel filtration,” separates molecules according to their sizes. Smaller molecules are retarded on a column, whereas larger ones are eluted more rapidly. As the retention time can be directly correlated with the sizes of molecules, this method is particularly useful for the determination of molecular weight. SEC is generally applicable to the separation of molecules in the range of 0.5–1000 kDa, but larger proteins or other giant molecules can also be separated. SEC can also physically separate folded macromolecules from the unfolded ones, particularly in the case of slow equilibria. The major applications of SEC include the determination of molecular weight and molecular-weight distribution of natural and synthetic polymers. Separation strongly depends on many factors such as column packing, column dimensions, flow rate, sample volume, and mobile-phase composition. Capillary SEC provides a separation strategy for microscale purification. Capillary SEC columns are best suited for direct coupling to electrospray ionization–mass spectrometry (ESI–MS) because they have comparable flow rates that can be delivered to the ESI source without splitting.