those most commonly used for separating labile proteins. Stationary Phase Limitation by Chiral Selectivity. The extent to which an enantiomer can interact with the stationary phase depends on how close it can approach the molecules of the stationary phase. If the stationary phase is also chiral in nature, it is likely that one enantiomer in the sample will fit closely to the stationary phase surface whereas the other will be stearically excluded and thus have less stationary phase with which to interact. The first chiral separations in GC were reported by Gil-Av et al. as in 1966 (7), but, surprisingly, the use of GC for the separation of enantiomers has only recently been investigated and developed into a practical system. The use of chiral stationary phases in GC has been dogged by entantiomeric instability arising from the racemization of both the chiral stationary phase and the chiral solutes at elevated temperatures. In addition, at the elevated

controlled in a number of ways. Firstly, the stationary phase loading on the column can be varied to adjust the retention as required. A specific stationary phase loading may be selected, to either improve the resolution, or to reduce the analysis time, or in some instances, to increase the sample load. Sometimes, the stationary phase loading is reduced so the column is more amenable to specific compounds (e.g. to prevent proteins from being denatured). Secondly, the stationary phase can contain molecules of a special shape that can only make close contact with molecules having a complementary shape. Other molecules can not interact so closely with the stationary phase and consequently, the stationary phase available to them will be restricted. This approach is exploited in chiral chromatography where the stationary phase is made to consist largely of a specific enantiomer that confers chiral selectivity to the distribution system Thirdly, the stationary

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 to their small size, there can be a large number of groups

nbsp; Vancomycin is a very stable chiral stationary phase, has a relatively high sample capacity, and can be covalently bonded by multiple linkages to the silica gel surface. It can be used with mobile phases with a high water content, as a reversed phase, or with a high solvent content, as a largely polar stationary phase. For example, when used as a reversed phase THF–water mixtures are very effective mobile phases. Conversely, when used as a polar stationary phase, n-hexane–ethanol mixtures are often employed. Vancomycin has a number of ionizing groups and thus can be used over a range of different pH values (pH 4.0 to 7.0) and exhibit a wide range of retention characteristics and chiral selectivities. Ammonium nitrate, triethyl-ammonium acetate and sodium citrate buffers have all been used satisfactorily with this stationary phase. The effect of the chosen buffer has little or no effect on chiral selectivity only on pH control. An example of

particular difficult types of separation. In gas chromatography (GC), chiral selectivity is controlled by choice of stationary phase and operating temperature. From a practical point of view, chiral selectivity is achieved by introducing spatially oriented groups into the stationary phase molecules and, as a consequence, an additional entropic component to the standard energy of distribution. Physically, by choosing the right structure, a closer interaction of one solute enantiomer with the stationary phase, relative to that of the other can be achieved. This favored closer contact will result in stronger interactions between the chemical groups of that particular isomer and the neighboring groups on the stationary phase. As a consequence, both the entropic enthalpic contributions to the standard energy will be increased. Because the enthalpic contribution to the standard energy is temperature dependent, and the magnitude of the standard enthalpy of the two enantiomers will differ

the different chiral stationary phases available will differ considerably. Moreover, as the nature of many of the synthetic procedures involved can be very complex, the properties of the products have the potential for varying significantly from batch-to-batch. It follows, that test mixtures are needed to quality control the columns and also to demonstrate the nature of the stationary phase. The results from such test mixtures should not only reveal the general chromatographic properties of the stationary phase but also confirm its capacity for separating enantiomeric pairs. The latter point is important, as most application samples contain many compounds other than those of a chiral nature and all, or most, will require to be resolved. An example of a chromatogram of a test mixture used by Supelco to demonstrate the chromatographic characteristics of their a-DEX column is shown in figure 28. The stationary phase is claimed to have a strong shape selectivity for positional isomers (e.g