Bio-Rad Bio-Gel A Agarose Gel User Manual
Page 5
of interest. In fractionation, molecules of varying
molecular weights are separated within the gel matrix.
With this method, the molecules of interest should fall
within the fractionation range of the gel. Common
applications include the fractionation and molecular
weight determination of proteins, nucleic acid
separations, plasmid purification, and polysaccharide
fractionation.
Resolution depends on the particle size, pore size,
flow rate, column length and diameter, and sample
volume. Generally, the highest resolution is obtained with
low flow rates (2–10 cm/hr), long narrow columns, small
particle size gels, small sample volumes (1–5% of the
total bed volume), a 2-fold difference in molecular weight
and a sample viscosity that is the same as the eluant. For
desalting, the sample volume can be as much as 30–40%
of the total bed volume, and shorter wider columns may
be used.
5
Section 3
Principles of Gel Filtration
Section 3.1 Gel Filtration Basics and Sample
Preparation
Gel filtration or size exclusion chromatography (SEC)
separates molecules based on their size. The gel media
consists of spherical beads containing pores of a specific
size distribution. Separation occurs when molecules of
different sizes are included or excluded from the pores
within the matrix. Small molecules diffuse into the pores
and their flow through the column is retarded, while large
molecules do not enter the pores and are eluted in the
column's void volume. Consequently, molecules separate
based on their size as they pass through the column and
are eluted in order of decreasing molecular weight.
Operating conditions and gel selection depend on the
application and the desired resolution. Two methods used
in size exclusion chromatography are group separation,
including desalting and buffer exchange, and frac-
tionation. In desalting, the molecule of interest is eluted in
the void volume, while smaller molecules are retained in
the gel pores. To obtain the desired separation, the gel
should have an exclusion limit smaller than the molecule
4
4006139A.qxd 6/23/98 8:11 AM Page 4