Lecture 25 summary

One of the most important applications of chromatography in biochemistry is size-exclusion chromatography: Size-exclusion chromatography is also called “gel filtration” or “molecular sieve” chromatography. Harris (p.504) refers to this type of chromatography as "molecular exclusion" chromatography. Size-exclusion chromatography separates molecules according to size and shape (although we will not consider the dependence on shape since size is normally a much more important factor in determining retention time).

Concepts and applications in size-exclusion chromatography

The retention volume of an analyte species is the volume of mobile phase that has passed through the column when the peak of elution occurs. Void volume (V₀) is the volume of the mobile phase in the column outside the stationary phase (Harris, p.504). Size-exclusion chromatography is widely used to estimate the native molecular mass of proteins. Ideally, there is a linear relationship between the logarithm of a protein's molecular mass and its retention volume.

See the example in Harris, p.505-506, where data from size-exclusion chromatography are given. First, proteins of known molecular mass* were chromatographed on a size-exclusion column to establish a calibration curve. The latter is then used to obtain an estimated mass for an unknown protein whose retention volume is determined on the same column.

(*and roughly globular shape)