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presently available for the GC separations. When the a, b, or g cyclodextrins are derivatized, the hydroxyl group on the 2-position reacts first. Nevertheless, the derivative is still size selective and interaction will be determined by the size and neighboring functional groups on the interacting molecule.   To give an example, very 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 the 2 and 6 positions, the hydroxyl group in the 3 position can then be trifluoroacetylated which produces a very different type of stationary phase. which has a wide field of application. It has been reported that the derivatized g-cyclodextrin 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

There is considerable evidence supporting the concept of solute inclusion in the cyclodextrin cavity during chromatographic development including a number of NMR studies (23). Cyclodextrins derivatize selectively and the reaction sites are distributed about  a mean. The 2-OH and 6-OH groups are the most reactive while the 3-OH group is the least reactive. Armstrong et al.  (24) obtained a plasma desorption mass spectrum for a mixture of O-(S)-2-hydroxypropyl-derivatized b-cyclodextrin which is shown in figure 40. The number above each peak denotes the number of substituted hydroxy propyl groups per cyclodextrin moiety. It is seen that there is a (more or less) symmetrical distribution of substituents about a mean of 6 hydroxyl groups reacted per cyclodextrin structure. The minimum appears to be about 2 and the maximum  about 12 substituents per moiety. This distribution resulting from substitution reaction, shows that the substituted cyclodextrin phases are

Courtesy of Supelco   Figure 27. A Molecular Model of Cyclodextrin   The thermal stability of the mixed stationary phase can be improved by incorporating a phenylpolysiloxane into the coating material. Phenylpolysiloxane also significantly improves the stability of the coating to oxidation, particularly at elevated temperatures (as cyclodextrin is basically a sugar, is will be very susceptible to oxidation at high temperatures). Some methysiloxane, however, must still be present to render the cyclodextrin soluble in the polymer matrix. Chiral selectivity can be further augmented by bonding other chirally active groups onto the secondary hydroxyl groups of the cyclodextrin (see again figure 27). Unfortunately, much of the chemistry used to derivatize these cyclodextrin compounds is considered proprietary and so synthetic

The reagents reacted quantitatively with primary and secondary amino functional groups, under mild conditions (55˚C for 10 min.), in the presence of triethylamine, to produce the corresponding fluorescent thiourea derivatives. The purity of the reagents were ascertained by separation on a cyclodextrin column and the results are shown in figure 50. The separation was carried out on a derivatized cyclodextrin column (ES-PhCD) 15 cm long and 6 mm I.D., packed with 5 mm particles. Chromatogram A shows the elution of the (R)-(–)-NBD-PyNCS isomer, B, the elution of the (R)-(+)-NBD-PyNCS isomer and C, the separation of the racemic mixture. The mobile phase was a mixture of acetonitrile/methanol/water : 3/3/4 v/v/v. Chromatogram D shows the elution of the (R)-(–)-DBD-PyNCS

derivatives of the cyclodextrins have been synthesized to provide specific types of interaction to increase their chiral selectivity. X-ray data has indicated that the b and g structures are quite rigid whereas the a structure appears to be somewhat flexible. Thus solute molecules, if spatially suitable, can be included in cyclodextrin cavity and interact by dispersive, polar of ionic forces with any neighboring groups to which they are appropriately close. The inclusion of a solute by the cyclodextrin structure is depicted in figure 37. Thus, if there is spatial differences between a pair of isomers, then this can introduce some interactive selectivity. This mechanism will be exhibited as another type of entropic contribution to the standard free energy of distribution which will also induce an attending enthalpic contribution. The concept, depicted in figure 37, is a grossly over simplified impression of the inclusion phenomena. Courtesy of ASTEC Inc. Figure 38.