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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.