Inclusion chromatography, separations

Schurig, V., Grosenick, H., and Green, B. S. (1993) Preparative enantiomer separation of the anesthetic enflurane by gas inclusion chromatography. Angew. Chem. Int. Ed. 32, 1662-1663. 

Schurig, V. and Grosenick, H. (1994) Preparative enantiomer separation of enflurane and isoflurane by inclusion chromatography. J. Chromatogr. A 660 617-625. 

In gel inclusion chromatography (GIC), the insoluble, swelling cyclodextrin polymers are utilized (24-26). For routine analytical purposes this method is too slow and time consuming, but some highly effective preparative separations including enantiomeric resolutions have been published. This approach seems to be very promising for semi-micro or laboratory scale preparative separations. 

The study of the chromatographic behavior of natural indole alkaloids on cyclodextrin polymers was different, and unexpectedly high retentions were observed in mildly acidic buffer solutions at room temperature, which permitted their separation by inclusion chromatography (25) (Table IV). Figure 7 shows the separation of two Vinca-alkaloids of very similar structure, the (+)-vincamine and (+)-apovincamine. 

Figure 8 shows the analytical base-line separation of quebrachamine antipodes by inclusion chromatography on B-cyclodextrin polymers. 

Separations of [njhelicene racemates have also been attempted using n-acids, such as 2-(2,4,5,7)-tetranitrofluorenylidene-9-aminooxypropionic acid (TAPA, 112, see above) its butyric acid analogue TAB A 113 and binaphthyl-2,2-diyl-hydrogenphosphate (BPA, 115 Other employed methods were inclusion chromatography on triacetyl cellulose or helical polymers like (-l-)-poly(triphenyl-methyl-methacrylate [(-l-)-PTrMA, 776]... 

The helical compounds 198a and 199 were separated into the enantiomers by inclusion chromatography (medium-pressure column chromatography on cellulose triacetate). 200 was enantiomerically enriched (—)-200 was only weakly enrich-... 

A different fit of the two enantiomers into the asymmetric cavities—the key-lock principle—of these polymers effects separation of the antipodes. For optimal enantioselectivity the secondary structure of the chiral spatially fixed matrix is decisive. This type of separation is usually called inclusion chromatography. 

Analytically, the inclusion phenomenon has been used in chromatography both for the separation of ions and molecules, in Hquid and gas phase (1,79,170,171). Peralkylated cyclodextrins enjoy high popularity as the active component of hplc and gc stationary phases efficient in the optical separation of chiral compounds (57,172). Chromatographic isotope separations have also been shown to occur with the help of Werner clathrates and crown complexes (79,173). 

Immobilization. The abiUty of cyclodextrins to form inclusion complexes selectively with a wide variety of guest molecules or ions is well known (1,2) (see INCLUSION COMPOUNDS). Cyclodextrins immobilized on appropriate supports are used in high performance Hquid chromatography (hplc) to separate optical isomers. Immobilization of cyclodextrin on a soHd support offers several advantages over use as a mobile-phase modifier. For example, as a mobile-phase additive, P-cyclodextrin has a relatively low solubiUty. The cost of y- or a-cyclodextrin is high. Furthermore, when employed in thin-layer chromatography (tic) and hplc, cyclodextrin mobile phases usually produce relatively poor efficiencies. 

Chiral Chromatography. Chiral chromatography is used for the analysis of enantiomers, most useful for separations of pharmaceuticals and biochemical compounds (see Biopolymers, analytical techniques). There are several types of chiral stationary phases those that use attractive interactions, metal ligands, inclusion complexes, and protein complexes. The separation of optical isomers has important ramifications, especially in biochemistry and pharmaceutical chemistry, where one form of a compound may be bioactive and the other inactive, inhibitory, or toxic. 

In relation to separation of nucleotides, Hoffman61 found that adenine nucleotides interacted most strongly with cycloheptaamylose, presumably by inclusion of the base within the cavity of cyclodextrin. When epichlorohydrin-cross-linked cycloheptaamylose gel was used as a stationary phase for nucleic acid chromatography, adenine-containing compounds were retarded most strongly. 

In a contribution dealing with two related compound classes, space could be saved by treating them together in domains where they display close similarities. However, the only spheres where this applies to sulphones and sulphoxides are elemental sulphur determination and chromatography. The former is too unspecific to be considered for inclusion in this chapter. Chromatographic behaviour is determined by the whole molecule, but the widespread use of chromatographic methods does justify its treatment. At the risk of a very little duplication it has been deemed more suitable to provide separate accounts of the two compound classes. 

Hydrophobic interaction chromatography (HIC) can be considered to be a variant of reversed phase chromatography, in which the polarity of the mobile phase is modulated by adjusting the concentration of a salt such as ammonium sulfate. The analyte, which is initially adsorbed to a hydrophobic phase, desorbs as the ionic strength is decreased. One application demonstrating extraordinary selectivity was the separation of isoforms of a monoclonal antibody differing only in the inclusion of a particular aspartic acid residue in the normal, cyclic, or iso forms.27 The uses and limitations of hydrophobic interaction chromatography in process-scale purifications are discussed in Chapter 3. 

Chiral separations result from the formation of transient diastereomeric complexes between stationary phases, analytes, and mobile phases. Therefore, a column is the heart of chiral chromatography as in other forms of chromatography. Most chiral stationary phases designed for normal phase HPLC are also suitable for packed column SFC with the exception of protein-based chiral stationary phases. It was estimated that over 200 chiral stationary phases are commercially available [72]. Typical chiral stationary phases used in SFC include Pirkle-type, polysaccharide-based, inclusion-type, and cross-linked polymer-based phases. 

The three isomers of cresol are not as readily separated by HPLC, although recent techniques have been developed to accomplish this task. Reversed-phase chromatography columns have been used for the analysis of cresols with limited success. Recently, a new reversed-phase support has been developed that allows complete separation of the three cresol isomers (Bassler and Hartwick 1989). Inclusion complexes of the cresols with p-cyclodextrin cleanly separate the three isomers on commercially available columns (Yoshikawa et al. 1986). Detection limits down to 1 ppm can be obtained by this method. 

Other combinations are, of course, possible, depending on the particular separation problem. Combining gel filtration or liquid liquid partition with liquid chromatography (LC) is one solution. Inclusion of chromatography on polymeric supports can also provide additional means of solving a difficult separation. 

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