Biomedical Engineering Reference
In-Depth Information
The foundation of this technique is the penetration of molecules
into the cavities of a macroporous support particle. These particles
are mostly made from hydrophilic gels of dextran, agarose, poly-
acrylamide, or chemical derivatives of them, and the interactions
between the macromolecules of the chromatographic support and
the biomolecules are minimized. At given diameters of the cavities
of the chromatographic matrix, molecules with a hydrodynamic
diameter similar to or smaller than the pore diameter diffuse into
the matrix, whereas molecules with larger diameters are excluded.
Because in a rough approximation the diameter of a protein is pro-
portional to its molar mass, gel filtration is used for separation by
molecular weight (in a more detailed view, separation according to
molar mass is only possible if all molecules have the same shape,
e.g., spherical or ellipsoid).
GPC can be applied in two distinct ways:
Group separations
: One group of molecules of a sample is ex-
cluded from the gel, whereas the other group penetrates the pores
of the matrix. Examples: desalting; buffer exchange.
Fractionation according tomolecular size
(“molar mass”): The
components of a sample are separated according to their molecular
size caused be their ability to penetrate the macoporous beads. Ex-
amples: Analytical or preparative fractionation of macromolecules,
separation of oligomers from monomers.
Most of the supports are chemically stable at pH between 2
and 12 and are not influenced by salt. Especially the gels for low-
pressure chromatography are sensitive to organic solvents at higher
concentrations. Especially gels with high exclusion limit also col-
lapse irreversibly at higher pressure. Supports for medium and
high-pressure chromatography show the same chemical stability,
but they have much smaller particle diameters and are stable at
pressures of some hundred bar. The mechanical instability of the
soft swollen gels forbids the use of magnetic stirrers or fast moving
blade agitators.
Swelling of dry GPC beads takes up to several hours and may
be accelerated by heating (cf. Table 3.2). Take care that sufficient
solvent is available during swelling (as a rule of thumb twice the
expected volume).
To avoid irreversible compression of the gel, respect manu-
facturer's specifications of maximal pressure during filling of the
column and during run. (For selected gels, this value is given in
Table 3.2.) The hydrostatic pressure
∆
h during filling and run is
illustratedinFig.3.3.
If pumps, e.g., peristaltic pumps, are used for the chromato-
graphic run, occurring changes in viscosity result in an unexpected
increase of pressure; therefore, select flow rates significantly lower
than the maximal rate given for a distinct gel.
To compare the separation performance of two columns con-
taining the same media, the flow rate of the solvent (mobile phase)
