Page 264 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioTechnology

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Encyclopedia of Physical Science and Technology EN014J-683 July 30, 2001 20:3

672 Separation and Purification of Biochemicals

price, their application will most likely be limited to the bility to scale up the separation by modifying the column’s
isolation of high-value biologicals. length without changing the separation distance.
A very simple way to achieve a reduction in the intra-
particular mass transfer is the use of micropellicular, i.e., 1. Membranes
nonporous particles. However, due to the low capacity
Filter membranes are ubiquitous in bioseparation and
of such particles (90% of the adsorptive surface is usually
mainly used to roughly separate molecules according to
found inside a particle), such nonporous particles are more
differences in size. However, affinity, ion exchange, hy-
suitable for analytical than for preparative applications.
drophobic interaction or reversed-phase ligands may be
Another way to increase capacity without relying on the
coupled to such membranes (“affinity filters”) to increase
intraparticular surface area, which is more suitable to
their selectivity for the target molecule by several orders of
preparative applications, is represented by the so-called
magnitude. Different membrane materials have been used
tentacle gels. Gigaporous stationary (“perfusion”) parti-
for such purposes, including polyamide, regenerated cel-
cles and hyperdiffusive (“gel in a shell”) particles can also
lulose, polystyrene, and various copolymers. Another ad-
be envisaged for preparative separations.
vantage of membranes is their low flow resistance (back-
The pores of the hyperdiffusive particles are filled with
pressure). Since the separation efficiency (plate height)
a gel, which considerably improves the efficiency of the
shows hardly any dependency on the flow rate, separations
mass transfer by diffusion in such gels. Just as the hyper-
can be carried out within seconds at very high flow rates,
diffusive particles, the gigaporous particles were devel-
unless the adsorption kinetics themselves pose a limit.
oped in the 1980s to allow the use of higher flow rates
Membrane chromatography is often performed using
and improve the productivity of bioseparation. They con-
devices derived from conventional filtration units, al-
tain large through-pores (“convective pores”) of several
though some filter holders exist, which are compatible
hundred micrometers in diameter, which reach across the
particles, together with relatively small and shallow pores to typical chromatographic systems or flow injection an-
alyzers. In principle, five types of membrane units can be
(“diffusive pores”), which line the walls of the gigapores
distinguished:
and serve mainly to increase the adsorptive surface. When
these particles are packed into a column, the mobile phase
Hollow fiber membranes (large dead volume causes
flows around the particles as in conventional columns but
zone broadening)
alsothroughtheparticle.Thelattereffectreducestheprob-
Radial flow systems
lem of intraparticular mass transfer. However, theoretical
Dead end filter systems with single (or stack of)
considerations have shown that the full advantage of con-
membrane(s)
vective mass transfer through the particle is only obtained
Compact porous disks
at relatively high mobile phase flow rates.
Porous sheets loaded with specific binding particles

(not strictly speaking membrane chromatography)
B. Continuous Stationary Phases
Membranes inserted in such a housing function as very
Continuous stationary phases are single porous entities
short and wide chromatographic columns. The uniform
(“filters,”“sponges”), which are transfused by the mobile
distribution of the mobile phase over the relatively large
phase. The walls of the pores through which the mobile
cross-sectional area can be a problem, along with the col-
phase flows are themselves lined with the interactive sur-
lection of the target biomolecules without back-mixing
face; the mass transfer resistance of the system is thus re-
andpeakdistortion.Thescaleupofmembranechromatog-
duced to a minimum. Continuous stationary phases have
raphy is usually straightforward, as ample experience ex-
been realized in the form of flat sheets (membrane/disk
ists in the scaling up of filtration units, and stacking of
chromatography), but also in the form of porous rods (con-
membranes of different functionalities can bring interest-
tinuous bed column chromatography). The van Deemter
ing possibilities in mixed-mode separations.
curve of the various continuous bed-type stationary phases
is relatively “flat” even at elevated flow rates, see Fig. 4.
2. Monoliths
In other words, the efficiency of the macroporous mate-
rial does not decrease by increasing flow rates and such Chromatographic membranes were developed from typ-
materials can therefore be used for fast bioseparation. The ical filter membranes, which shows in their materials
scale-up of continuous beds remains a problem, since the and also in the physical characteristics. Many have, for
in situ polymerization cannot be handled at elevated col- example, relatively broad pore size distributions. Lately
umn scale. Some tube-like columns have been designed the advantages of chromatographic membranes (low back-
for radial chromatography, thus benefiting from the possi- pressure, superior mass transfer properties) have served