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248 Galaev and Mattiasson
in biotechnology is cryogels (from the Greek o (kryos) meaning frost or
ice). Cryogels are produced by polymerization in a partially frozen state when
the ice crystals perform as a porogen. After completing the polymerization and
melting ice crystals, a system of interconnected pores is formed (1).
Cryogels have a unique combination of properties:
• Pores of 10–100 μm in size allow even large (at molecular scale) objects like
microbial or mammalian cells to pass easily through the cryogel without being
trapped.
• Hydrophilic nature of the polymers, which form pore walls, minimizes the nonspecific
interactions with the pore walls.
• High polymer concentration in the pore walls and hence a good mechanical
stability of the cryogels.
The large pore size and the interconnected morphology of pores allow
unhindered mass transport of solutes of practically any size. The cryogel
columns have porosities exceeding 80–90%. High porosity and the interconnected
morphology of the pores result in a very small flow resistance of cryogel
columns. The columns can be operated at flow rates of about 750–2000 cm/h,
at hydrostatic pressure approximately 0.01 MPa (2). Due to the convective
flow of the mobile phase through the interconnected pores, the mass transfer
resistance is practically negligible, and the height equivalent to a theoretical
plate (HETP) is practically independent either of flow rate or of the size of the
marker (from acetone to Escherichia coli cells) (3).
Mechanically, the cryogel adsorbent is very stable. The continuous matrix
could easily be removed from the column, dried at 60°C and kept in a dry
state. The dry matrix has a slightly smaller diameter than the swollen one
and could be easily inserted inside the empty chromatographic column. After
re-hydration in the running buffer which takes usually less than a minute, the
cryogel column is ready for operation. The elasticity of the cryogel ensures the
tight connection of cryogel monoliths with the column walls and the absence
of by-pass of liquid in between the cryogel monolith and the column walls (4).
Commercially available pre-activated cryogel matrices are produced by
Protista Biotechnology AB as 0.25-, 2- or 5-ml monolithic columns. The
monolithic columns are made of cross-linked polyacrylamide or polydimethylacrylamide
(polyDMAAm) and contain 20–30 μmole epoxy groups/ml column
volume (CV).
The presence of epoxy groups allows easy coupling of a variety of ligands to
monolithic cryogel columns, for example, ion-exchange ligands (5), Immobilized
Metal Affinity Chromatography (IMAC) ligands (2,6), protein A and
antibodies (7,8). The produced monolithic chromatographic columns have
been used for the direct capture of histidine-tagged proteins from crude cell
homogenate (2) and from cell fermentation broth (6), for specific isolation