there are thousands of different macromolecules in each cell, purification of a
specific one from all the others requires powerful separation techniques, such
as chromatography and electrophoresis. Both of these approaches take advantage
of physical and chemical properties that differ between the individual
A. Gel electrophoresis –In
gel electrophoresis, the macromolecules areplaced in a solid matrix, called a
gel, which is under a liquid buffer. An electric field is applied to this
system, and since biological macromolecules carry ionic charges, they will be
attracted towards one pole of the electric field and repelled by the opposite.
Thus, macromolecules characteristically migrate in either direction in the
field. The migration speed is determined by the charge-to-mass ratio of the
In a flat gel, also called a
horizontal or submarine gel, electrophoresis system, an agarose gel lies
horizontally below the electrophoresis buffer. This technique is mainly used to
separate large nucleic acids (DNA and RNA).
Ø A vertical electrophoresis system holds a polyacrylamide gel in the vertical position, and is mainly used to separate proteins or small-sized nucleic acids.
B. Chromatography –Chromatography
is a family of methods used toseparate macromolecules through their relative
affinity to a stationary phase (generally, solid chromatography beads) and a
mobile phase (generally, an aqueous buffer). The chromatography beads are
loaded into a tube, called a chromatography column, and buffer is dripped, or
pumped, through the column to carry the macromolecules along. The
macromolecules separate on their affinity for the mobile front. Some
chromatography beads separate by charge (ion exchange chromatography), by
interaction chromatography), or by a specific property of that protein (affinity
chromatography). Macromolecules can also be separated by size otherwise known
as size exclusion or gel filtration chromatography.
To overcome this limitation, high
performance (or high pressure) chromatography (HPLC) uses high-pressure pumps
and metal-jacketed columns to operate at high pressures and speed up the
A fraction collector collects the
released mobile phase (eluent) of a chromatography column. It automatically
measures a programmed volume (sometimes by the number of drops of liquid) into
a line of test tubes or microcentrifuge tubes.