Stereoisomers complexation chromatograph

Although it is well understood that molecules must be able to enter the cavity of the cyclodextrin molecule for complexation to occur, and therefore, under chromatographic conditions, for retention to result, the differential binding of two stereoisomers within the cyclodextrin that allows for their differential retention is not always apparent. An understanding of this can be obtained through the use of three dimensional computer graphic imaging of the crystal structure of the inclusion complex. 

A central question in phosphotransferases and nucleotidyltransferases is the structure of the metal-nucleotide complex which is the true substrate for the enzyme. It is unlikely that all of the possible Mg-ATP complexes could serve as substrates for a given enzyme, but until recently there has been no way to determine which isomer is active. The difficulty is the coordination exchange equilibrium, which is rapidly set up and dynamically maintained in solutions of Mg-ATP. To avoid this problem, metal-nucleotide complexes have been synthesized using coordination exchange-inert metals such as Cr(III) and Co(IIl) in place of Mg(II) [7,60], The resulting complexes are structurally stable and can be separated by chromatographic methods into their coordination isomers and stereoisomers. The isomers can then be investigated as substrates or inhibitors of specific enzymes. 

One additional factor that may add complexity in pyrolysate analysis is the presence of several diastereoisomers for the same compound. Many polymers contain in their backbone asymmetric atoms (usually carbons). A single asymmetric carbon generates two enantiomers, and these are not separated using regular chromatographic columns [17]. The number of stereoisomers of a compound with n chiral centers in the molecule... 

The fifth alkaloid, (-l-)-epialloelaeocarpiline (16), m.p. 136—137°C, is also a conjugated dienone (u.v. spectrum), and is yet another stereoisomer of elaeo-carpiline (n.m.r. spectrum). Dehydrogenation gives a complex mixture of four products from which 7S,8R,9R-(-l-)-isoelaeocarpine (enantiomer of 4) may be isolated, and when (-f-)-epialloelaeocarpiline is chromatographed on silica-gel t.l.c. plates, isomerisation occurs in part to give some (+)-epi-isoelaeocarpiline... 

Coordination exchange-inert metal nucleotide complexes have been synthesized, their structural and stereoisomers have been separated by chromatographic and enzymic methods, and their structures have been determined by X-ray crystallography and correlated to their circular dichroism and P NMR spectra. The pure isomers have been tested as substrates for enzymes in place of MgATP or MgADP, and from the results the structures of the enzyme-bound and active isomers have been deduced. The most widely used complexes of this type have been Cr(III)-aquo complexes and Co(III)-ammine complexes such as those shown below. 

The use of mass spectrometry in the structural analysis of carbohydrates, first reported in 1958 (114), was developed in detail by Kochetkov and Chizhov (115). They showed that, under electron impact, the acetylated and methyl ether derivatives of monosaccharides provided a wealth of structural information through analysis of typical fragmentation pathways of the initial molecular ion. This has proved of enormous utility in the structural elucidation of polysaccharides and complex oligosaccharides sequential permethylation, hydrolysis, reduction to the alditol, and acetylation, affords mixtures of peracetylated, partially methylated alditol acetates that can be separated and analyzed by use of a gas chromatograph coupled directly to a mass spectrometer (25). The mass spectra of stereoisomers are normally identical, while the gas chromatographic retention times readily permit differentiation of stereoisomers. 

Aspects of chemical methods used in the structural elucidation of polysaccharides and complex carbohydrates have been reviewed. In a critical examination of the use of g.l.c.-m.s. in the identification of TMS ethers of monosaccharides, a standardized method, which uses a medium resolution mass spectrometer and short chromatographic columns, has been proposed. TMS Ethers of monosaccharides have been characterized by g.l.c.-chemical ionization m.s. with ammonia as reagent gas. Molecular weights were determined, and fragment ions were produced in a quantity high enough to differentiate between stereoisomers (epimers and anomers). Disaccharides have been determined by permethylation followed by g.l.c. The method has been used in the detection of carbohydrate intolerance secondary to intestinal disaccharidase deficiency. 

Analysis becomes much more complex when stereoisomers are quantified separately (Figure 56.1). Enantiomers cause identical detector responses in NPD, FID, or MS. Therefore, chiral separation systems are required to overcome tiiese detector limitations. Despite enormous progress in separation media and detector systems within the last two decades, the number of reports on chiral analysis of nerve agents valuable for toxicokinetic studies is still very small. Chiral separations make use of special chromatographic columns modified with chiral ligands. 

Chromatographic separation was carried out on silica gel plates and cellulose plates impregnated with sodium borate (B). The experiments revealed that borate complexes of AA and DHAA could be separated from each other. TLC was also attempted using direct (D), reverse-phase (RP), and reverse-phase sodium borate (RP-B) TLC plates with and without metaphosphoric acid (MPA). A good separation of the three AA isomers and DHAA isomers was achieved on D and RP cellulose plates. Using an RP-silica-MPA plate, the three A A isomers as well as the three DHAA isomers were fairly separated from their own group members. The separation of six stereoisomers, three AA, and three DHAA was achieved using D-cellulose-MPA plate. 

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