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nbsp; It is clear that there is yet another limitation of which to be aware when exploring the effect of solvent composition on retention and selectivity. It is important to examine the effect of solvent composition over a range of temperatures, to ensure that the true effect of solvent composition on selectivity is disclosed. If the distribution system is evaluated at, or close to that temperature where the separation ratio remains constant and independent of solvent composition, then the potential advantages that can be gained from an optimized solvent mixture will never be disclosed. Any evaluation of either a particular stationary phase, or solvent mixture, for the separation of closely eluting solutes must be carried out over a range of temperatures.       Optimum

is distinctly more selective than the b material. It has been employed in the separation of a very wide range of compound classes, and from very small to very large molecules. Yet another stationary phase has been synthesized by substituting the cyclodextrin hydroxyl groups with pure the 'S' hydroxypropyl groups followed by permethylation. As a result, the size selectivity of the material is reduced but more polar (hydrophilic) groups are introduced. The b material has a greater chiral selectivity than the a or g phases. This material provides a good general purpose column. It is clear that there are many possibilities for derivatizing the cyclodextrins to provide unique interactive character; there are a large number commercially available and many more are likely to be synthesized in the future.   A number of examples of some specifically derivatized cyclodextrin stationary phases have been reported. In one example, the positions 2 and 6 are alkylated (pentylated) thus

or mixtures of both) so that polar selectivity remains dominant in the stationary phase. Unfortunately, all substances are not simply polar or dispersive or ionic but can exhibit various combinations of all three interactive characteristics. It follows, that the stationary phase will also need to have the appropriate mixture of interactive properties to maximize selectivity and in contrast the interactive character of the mobile phase will need to oppositely balanced to ensure maximum selectivity still resides in the stationary phase. Under some circumstances the selectivity of the mobile phase can also be exploited to interact more strongly with the neighboring substance and thus elute it more quickly. It is clear, that some considerable effort must be made to identify the bests phase system for the isolation of a substance from a hitherto unknown mixture. This work can be carried out on an analytical column and its success will determine the maximum load that can be used,

in competing for the benzene against the dispersive interactions of the n-hexadecane. Chiral Stationary Phases There are basically five general types of chiral stationary phase in common use in LC. The first is the protein based stationary phase. These stationary phases usually take the form of natural proteins bonded to a silica matrix. As they are proteins, they contain a large number of chiral centers and are known to interact strongly with small analytes exhibiting strong chiral selectivity. There are specific interactive sites that provide chiral selectivity, but there are many more sites that only contribute to general retention. These other sites can be deactivated by mobile phase additives (e.g. octylamine) which reduces the overall retention and increases the chiral selectivity. The second type consists of relatively small molecular weight chiral substances bonded to silica 9 Pirkle (37). Each bonded group has a limited number of chiral centers available but, due

small molecules might be separated on the derivatized a-cyclodextrin whereas, in contrast, larger molecules might be better separated on the derivatized g-cyclodextrin. It follows, that the derivatized b-cyclodextrin might be selected for separating the isomers of solute molecules of intermediate size. In contrast, if the 3-position hydroxyl group is derivatized all the cyclodextrins completely loose their size selectivity. Derivatizing the 6-hydroxyl position has little or no effect on chiral selectivity but does appear to enhance the loading capacity of the stationary phase. This position is also used to anchor the cyclodextrins to the surface of silica gel in the preparation of LC stationary phases. After permethylation the a-, b- or g- cyclodextrins must be dissolved directly in appropriate polysiloxanes mixtures, and the coated on the walls of the capillary column (glass or fused quartz) using the techniques already described. The underivatized cyclodextrins, and those that have

character, but it is interesting to note that the propane diols are eluted between the C10 and C11 hydrocarbons. This indicates that the C3 chain alcohol is retained by polar forces to an almost equivalent extent as the C11 hydrocarbon retained by dispersion forces only. This implies a fairly strong overall polar character to the stationary phase. It is also seen that the m and p xylenes are well separated showing good spatial selectivity and the separation of diol enantiomers good chiral selectivity. Courtesy of Supelco The a-DEX™ column was 30 m long, 0.25 mm I.D., carrying a film of stationary phase 0.25 mm thick. The temperature was 90˚C and the helium flow velocity was 30 cm/s.   Figure 41 Chromatogram of a Test Mixture for a Permethylated a-Cyclodextrin Stationary Phase As the two enantiomers are strongly polar it would also appear that the greater retention of the (–)-1,2-propane diol was due to polar interactions between the OH