Biomedical Engineering Reference
In-Depth Information
by chromatography of defined homopolymers such as polysaccha-
rides or polystyrenes, or with a special set of protein standards.
Since a gel filtration matrix separates according to steric (hydrody-
namic) parameters, and an ellipsoid molecule has a larger hydro-
dynamic radius than a spherical one with the same molar mass,
both the molecules therefore show different retention behavior. If
the protein of interest is significantly larger than accompanying
substances, a matrix with a cutoff between them is recommended
because the target appears with the void volume as the first little
diluted fraction. So, for desalting of protein solutions or for buffer
exchange, mostly Sephadex G-25 medium is well suited.
Additional chemical and mechanical stability of supports should
betakenintoconsideration,aswellaspossibleinteractionsbetween
protein and matrix macromolecules.
3.3.2 Filling of a Gel Filtration Column
The GPC column, which is intended for fractionation in labora-
tory scale, should have a ratio of bed diameter d to bed height H
(Fig. 3.3B) of 1:50 to 1:200.
Slurrytheswollen,settledgelinaboutahalfofitsvolumeof
buffer and degas under vacuum for 10 min. Connect the column
outlet with tubing ending in a vessel at the level of the upper border
of the column. Then mount the filling reservoir to the vertical
column as shown in Fig. 3.3A. Fill the column up to one-fourth to
one-third of its height with buffer and pour the gel slurry without
bubbles in one portion into column and reservoir. Regulate the
recommended hydrostatic pressure
∆
h (cf. Fig. 3.3; Table 3.2) by
lowering the outlet.
When the gel has settled, fill the column with buffer and care-
fully insert the upper adaptor. Move the adaptor down to the surface
of the packed bed and push it further 1 - 3 mm into the GPC gel.
Avoid air bubbles and let the outlets open to avoid compression of
the gel.
If columns without adaptors are used, stabilize the surface of
the gel by a sheet of (glass fiber) filter paper. The surface of the bed
should be without craters.
The GPC columns with bubbles or clefts within the bed have to
be emptied and packed again prior use.
Wash the completely packed column with buffer (eluent) until
a constant flow rate is obtained. The eluent volume should be at
least fivefold that of the bed volume.
3.3.3 Sample Application
and Chromatographic Separation (Elution)
For fractionation the sample volume should be not more than 1/20
to 1/10 of bed volume and should have the shape of a cylinder.
