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






               662                                                                     Separation and Purification of Biochemicals


               time, however, the number of coordination sites available  them water-soluble, while their tore cavity is rather apo-
               for protein binding by the stationary phase is reduced.  lar. The separation mechanism is based on the partitioning
                 IMAC has been used in the chromatographic isolation  of the enantiomers between the outer and the inner sur-
               of proteins and nucleotides, but also to investigate the  face according to their polarity, and their inclusion in the
               surface topography of protein histidine residues. The  tore cavity. The chiral retention mechanism hence mixes
               three-dimensional protein structure is not strongly af-  ionic interaction, hydrogen bonding, dipole interactions,
               fected by the binding to the chromatographic surface;  steric, and π-π interactions as well as inclusion complex-
               therefore the biological activity of the product is usually  ation. CD phases are used in the polar organic mode or
               wellpreserved.Traditionally,softagarosebeadswereused  in the reversed-phase mode. Mobile phases for the polar
               for protein purification by IMAC. Since then a number of  organic mode are mainly acetonitrile and methanol, made
               rigid supports and chromatographic membranes have be-  polar through the addition of anhydrous acids and bases,
               come available. The mobile phase in IMAC is a buffered  whereas the mobile phases for reversed-phase chromatog-
               salt solution and the strength of the metal–protein interac-  raphy contain water. The pH, the buffer concentration and
               tion is modulated by the type and concentration of the salt.  type, as well as the organic modifier concentration and
               In preparative IMAC, the separation is often achieved by  type will affect the separation. Immobilized macrocyclic
               differential elution with stepwise changes in the salt con-  antibiotics (glycopeptides) have also been used as chiral
               centration. The pH also influences the retention behavior  selectors, very much in the same manner as cyclodex-
               of proteins and elution in IMAC may most conveniently  trins. Immobilized α 1 -acid glycoprotein (AGP) and hu-
               be achieved by lowering the mobile phase pH to 6 so that  man serum albumin (HSA) are known to act as chiral se-
               the histidine residues are protonated. An agent competing  lectors, which act via ionic binding, hydrogen bonding and
               for the metal sites, e.g., glycine, histidine, or imidazole, or  hydrophobic interaction. These ligands are mostly used in
               an organic modifier may also be used for the elution of the  the reversed-phase mode.
               protein. Complexation and hence removal of the chelated
               metal ions by EDTA presents another means for protein
                                                                 D. Separation by Size
               elution.
                                                                 In size-exclusion chromatography (SEC), also called gel
                                                                 filtration (GF) or gel permeation chromatography (GPC),
                 2. Chiral Chromatography
                                                                 the sample molecules are separated according to their hy-
               Chiral or enantioselective chromatography (chiral-HPLC)  drodynamic diameter (i.e., ultimately according to their
               may be considered another subdivision of affinity chro-  size and mass) and not as the result of an interaction-
               matography, which deals with small biochemicals rather  mediated process. The sample is passed though a column
               than with biopolymers such as proteins. Enantiomers are  packed with an inert porous material having appropriate
               nonoverlayable mirror images of one another, mostly  pore size distribution and volume. In analytical SEC, the
               molecules containing asymmetrical carbon atoms. Two  sample size should be no more than 3% of the column vol-
               enantiomers have the same physicochemical properties  ume (c.v.); however, in preparative work the sample may
               regarding charge, size, and solubility, but may differ con-  occupy up to 15% of the column volume. Since no inter-
               siderably in their biochemistry and activity. Even though  action between the solute and the stationary phase occurs,
               stereoselective chemical syntheses are often possible, the  all the sample components are eluted within one column
               (large-scale) separation of drug enantiomers in order to  volume, resulting in a quite low resolving power of com-
               produce an enantiopure drug remains a common chal-  plex mixtures by this method. In biochemical applications,
               lenge in the pharmaceutical industry. In some cases the  SEC is hence mostly used for the rapid and convenient sep-
               scale of these separations makes even the utilization of a  aration of sample components having substantially differ-
               simulated moving bed (see below) feasible. Another area,  ent molecular masses, such as in the desalting of a protein
               whichrequiresenantiopuresubstances,isthestudyofdrug  solution (or buffer exchange). Preparative SEC is often
               metabolism.                                       used as a polishing step following other chromatographic
                 Chiral-HPLC started in the 1980s, and the main appli-  separations.
               cations have been the separation of sugars, amino acids  In SEC, separation occurs due to differences in the ac-
               (small peptides), and their derivatives. Different types of  cessibility of the intraparticular void volume by the sample
               natural and modified cyclodextrins (CD) have been im-  componentsofdifferentmoleculardimensions.Molecules
               mobilized as enantioselective ligands onto conventional  larger than the upper exclusion limit cannot enter the intra-
               silica particles. Cyclodextrins are rings of glucose units,  particular void space and elute first, whereas sufficiently
               with a toroidal three-dimensional (3D) structure. Their  small molecules have access to all the pores, and therefore
               hydrophilic (abundant hydroxyl groups) surface makes  elute last, see Fig. 8.
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