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Oligo nucleotides Nucleic acid-binding enzymes
Denatured DNA Proteins binding to DNA
“All mentioned phases are available with ligands immobilized on cross-linked agarose beads (with particle diameters >30 ixm) suited for “fast protein liquid chromatography” (FPLC). Some are available also based on silica or highly cross-linked polymers suited for use under high pressure (HPLC).
Retention and Selectivity
salts (NaCl up to 1 M) or chaotropic agents to the eluent. Such agents reduce the hydrogen-bonding as well as the hydrophobic interactions. Typically used chaotropic agents are urea, thiourea, sodium thiocyanate, and guanidine hydrochloride, applied in concentrations between 1 and even 6 M. (In higher concentrations there is a risk of denaturation of labile proteins.)
Affinity chromatography is primarily used for the enrichment of the target analytes and for their specific isolation from all other substances, which are not retained, in a sample. Separation between similarly retained substances using this method is not entirely practicable. AFC is operated most frequently in a yes/no mode, which implies strong specific retention (and enrichment) of the target compound, followed by prompt desorption and elution using as little eluent volume as possible. Such separations do not rely on high plate numbers and high peak capacities, as offered by small particles in HPLC. Less rigid support materials, with larger particle sizes and with lower pressure limits, based on agarose beads are frequently used for the preparation of AFC phases used in so-called fast-protein liquid chromatography (FPLC).
B. Chiral-Stationary Phases
Chiral stationary phases (CSPs) are the main tool for separating enantiomers by HPLC. A particular chiral phase is effective for only a certain kind (family) of chiral analytes, because there has to be a high degree of structural correspondence between the chiral selector (chiral stationary phase in this instance) and the selectand (chiral analyte to be separated). With chiral stationary phases, the chiral selector is either immobilized on a support material as a chiral surface ligand, or it is coated in the form of a chiral polymer. Several mechanisms of chiral recognition are described in the following. From these different mechanisms, a list containing the main groups of stationary phases can be given (Table 10).
An alternative strategy for chiral separation involves chiral selectors that are not immobilized onto a solid support, but are added to the mobile phase, in which they act through a secondary equilibrium. This will be discussed in Sec. V. The molecular mechanisms of steric recognition are very similar in both strategies.
Discrimination (recognition) of enantiomers is possible if the Gibbs free energies of the interaction with a chiral selector are different for the two enantiomeric selectands. Enantio-meric recognition needs an ensemble of interactions between at least three points of the selector and three points of one of the retained enantiomeric selectands (analytes) 161,621. A schematic drawing illustrating selector-selectand interactions is given in Fig. 9. Interactions in this context can be attractive as well as repulsive, and steric hindrance (for adopting an optimum interaction position) is often a main factor. Generally, all types of attractive inter-actions between selector and selectand can be used for chiral recognition (i.e., dipole-dipole, charge-transfer, electrostatic, dispersive, or hydrophobic interactions). The strength and relative importance of these various interactions are influenced by the eluent composition. Nonpolar organic eluents enhance dipole-dipole and charge-transfer interactions, whereas aqueous eluents induce hydrophobic interactions. The main types of interactions needed to achieve stereorecognition thus determine the type of mobile phase to be employed. For the mobile phase, a distinction can be made between a normal-phase and a reversed-phase mode chiral chromatography. One must remember that, although the interactions between selector and analyte may be dominated by one type of interaction, chiral recognition always needs a set of interactions.
Various classification schemes have been proposed for chiral separations. To distinguish between mechanisms, the following may be useful.
1. Chiral recognition by the formation of 1:1 complexes between a surface-immobilized
selector and a selectand in a broad sense, covering the following types of complexes.
a. Host-guest complexation resulting in inclusion complexes between chiral analytes and chiral host molecules. Cyclic oligosaccharides (cyclodextrins) [63,641, chiral
Table 10 Commercially Available Types of Chiral Stationary Phases for HPLC
Selector Mobile, phase Support material
Chiral host phases Aqueous (organic) Chemically immobilized onto