Biomedical Engineering Reference
In-Depth Information
3.7.5 Ultrafiltration
Solutions of macromolecules may be concentrated by means of
polymer membranes of defined pore size. Applying a pressure or
centrifugal force, small molecules pass the pores, whereas large
molecules retain. The nominal cutoff of an ultrafiltration mem-
brane (MWCO) helps you to select a membrane: Molecules smaller
than the MWCO will pass the membrane, whereas larger molecules
are held back. This separation is not sharp and depends on pro-
tein conformation and solvent composition. Complete retention is
achieved when using a membrane with a MWCO 1/3 to 1/5 of the
molar mass of the macromolecule of interest. Figure 3.6 illustrates
the separation of proteins by ultrafiltration.
Recovery of proteins from ultrafiltration depends not only on
the size, but also on the solute composition and the type of mem-
brane, since unspecific adsorption of the protein to the membrane
cannot be excluded. Furthermore, the chemical resistance of the
membrane to buffer components and sanitation ingredients should
be taken into consideration.
Unspecific adsorption of proteins may be decreased if the mem-
brane is preincubated with 5% Tween 20 (w/v) for 1 h and subse-
quently washed with ddH
2
O.
Desalting is also possible by ultrafiltration. For this purpose, the
sample is concentrated to about 10% of its volume, reconstituted to
itsoriginalvolumewithwaterorasecondbuffer,andultrafiltration
is repeated twice in the same way. Proteins are less likely to be
denatured, because ultrafiltration is a mechanical separation which
does not need harsh chemicals for separation.
Fig. 3.6.
Percentage of retention of proteins by ultrafiltration membranes. (Data according to
Millipore/Amicon application note)
