Bio-Rad Bio-Gel P Polyacrylamide Gel - Manual (Page 9)

sible to filter it, the sample should be centrifuged until it is clear.
Figure 7 shows the hypothetical effects of various chromatographic
conditions.
13
tional buffer to wash the sample into the bed. Replace the super-
natant buffer and attach column to reservoir.
11. Collect fractions for analysis, or monitor with continuous flow
equipment such as UV/Vis, conductivity, and refractive index
monitors.
Section 4
Sample
Gel filtration is largely independent of sample concentration. The
volume of the sample relative to the bed volume is far more impor-
tant. For analytical purposes the sample should not be larger than
1-5% of the bed volume, whereas for desalting the sample can be as
large as 30-35% of the bed volume. The viscosity of the sample may
limit the concentration of sample which can be used. Viscous samples
may be diluted to decrease the viscosity. It may be possible to achieve
better results by applying viscous samples at a lower flow rate. The
sample should be clear, and completely dissolved in running buffer,
without particles or solid contaminants. Filtration of samples will
increase column life. If, due to the nature of the sample, it is not pos-
12
Fig. 7. Aberations in gel
chromatography elution
profiles (hypothetical).
a.
Satisfactory separation.
b. Sample volume too large or
bed too short. c. Eluant flow rate
too high or gel particle size too
large.
d. Poor sample
application, nonuniform cross-
sectional bed resistance, or large
dead space volume.
e. Sample
viscosity too high.
a
b
c
d
e
LIT174B 10/13/98 9:26 AM Page 12