Separation and Identification of Isomers

Separating chiral isomers requires chiral counterions. Cations are frequently resolved by using anions t/-tartrate, antimony r/-tartrate, and a-bromocamphor-7r -sulfonate anionic complexes are resolved by the bases brucine or strychnine or by resolved cationic complexes such as [Rh(en)3]. A novel strategy using I- and i-phenylalanine to resolve Ni(II) complexes that form chiral helical chains has been reported. For compounds that racemize at appreciable rates, adding a chiral counterion may shift the equilibrium, even if it does not precipitate one form interactions between the ions in solution may be sufficient to stabilize one form over the other.  

FIGURE 9J23 The Cotton Effect in ORD and CD. Idealized optical rotatory dispersion (ORD) and circular dichroism (CD) curves at an absorption peak, with a positive Cotton effect. 

Circular dichroism (CD), is caused by a difference in the absorption of right- and left-circularly polarized light, defined by the equation 

CD spectra are not always easily interpreted, because there may be overlapping bands of different signs. Interpretation requires determination of the overall symmetry around the metal ion and assignment of absorption spectra to specific transitions between energy levels (discussed in Chapter 11) in order to assign specific CD peaks to the appropriate transitions. 

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BuserHR. 1975. Polychlorinated dibenzo-p-dioxins Separation and identification of isomers by gas chromatography-mass spectrometry. J Chromatogr 114 95-108. 

SFC-NMR was used for the separation and identification of a mixture of five vitamin A acetate isomers using supercritical CO2 as the eluent [74], An advantage pointed out in this report is the lack of a solvent resonance, eliminating the need for solvent suppression and allowing unrestricted observation of the entire... 

R) (S)-3,6,6-Trimethyl-1-heptene. (R)(S)-3,6,6-Trimethyl-l-hept-ene has been hydroformylated in exactly the same conditions adopted for the hydroformylation of its isomer 2,2,5-trimethyl-3-heptene. The separation and identification of the reaction products have been made using the technique previously described. The results are reported in Table I. 

MH Saleh, B Tan. Separation and identification of cis/trans carotenoid isomers. J Agric Food Chem 39 1438-1443, 1991. 

Ecknig, W., Rotzsche, H., Kriegsmann, H. Separation and identification of hexa-methylcyclotetrasiloxane isomers by a combination of analytical gas chromatography and infra-red spectroscopy. Vortr. Symp. Gas-Chromatog. 4, Leuna, Ger. 

Additional analytical methods are appropriate when a more complete characterization of the CLA isomers in biological samples is required. Most often, a combination of GC and silver ion-HPLC is used and permits excellent separation and identification of positional and geometrical isomers of CLA (see Adlof, 2003, and Kramer et al., 2004, for detailed reviews of this approach). In addition, the use of gas chromatography-mass spectrometry (GC-MS) has become increasingly popular and represents a very powerful technique for identification of the position of double bonds in fatty acids (see Dobson, 2003), and the orientation of those bonds in CLA isomers (Michaud et al., 2003). 

S. Strohschein, C. Rentel,T. Lacker, E. Bayer, and K. Albert, Separation and identification of tocotrienol isomers by FIPLC-MS and HPLC-NMR coupling, Anal. Chem. 71 (1999), 1780-1785. 

Ephedrine and ist isomer pseudoephedrine are alkaloids in Ephedra species. Brochmann-Hans-sen and Baerheim Svendsen reported the first gas chromatographic separation of these two alkaloids in a study on the separation and identification of 11 sympathomimetic amines on a 1.15 % SE-30 packed column at 104°C. However, ephedrine and pseudoephedrine could not be separated as such, but were separated as their oxazolidine derivatives after treatment with acetone. A typical chromatogram is given in Tale 13.1. 

There have been many publications on the use of HPLC-DAD. As one set of examples that highlights both the growth and now-current wide application, is the analysis of the larger PAHs. This class of compounds has many, many isomers that have very similar structures and retention times. The collection of spectra by the DAD allows each HPLC peak to be monitored and compared to standard or reference spectra. PAH mixtures are usually very complex, so the separation and identification of these samples by using HPLC-DAD highlights the powerful capability possible with this combination. 

Sehat, N., Yurawecz, M.P., Roach, J.A.G., Mossoba, MM., Kramer, JX.G., and Ku, Y. (1998) Silver-Ion High-Perfonnance Liquid Chromatographic Separation and Identification of Conjugated Linoleic Acid Isomers. Lipids 33,217-221. 

In the last few years, 200 m capillary columns have become available (CP SELECT FOR FAMES, Varian Inc.). Even though this column is of intermediate polarity (Carbowax type column), the increased length does provide some improved separations of the 18 1 isomer region (96 P. Mockel and G. Jahreis private communication). However, using such a column wiU require higher head pressures, and the joint between the two 100 m columns is prone to leak (P. Mockel and G. Jahreis, personal communication). Details on the separations and identifications of FAME in all the different regions remain to be worked out. 

Sehat, N., M.R Yurawecz, J.A.G. Roach, M.M. Mossoba, J.K.G. Kramer, and Y. Ku. Silver-Ion High-Performance Liquid Chromatographic Separation and Identification of Conjugated LinoleicAcid Isomers. j j 33 217-221 (1998). 

Silver-ion High-Performance Liquid Ghromatographic Separation and Identification of Conjugated Linoleic Acid Isomers. i i 33 217—221 (1998). 

The separation conditions mentioned above for CZE with dynamically coated capillaries UV detection and formic acid were also compatible with the operative of electrospray ionization in CE-MS system and CE-MS-MS analyses.It was shown that an essential advantage of CE-MS over LC-MS was the possibility of separation and identification of three desmethyl isomers of MC-LR differing in the Adda, Masp, and Mdha residues. 

Juaneda, P., and Sebedio, J.-L. (1999) Combined Silver-Ion and Reversed-Phase High-Performance Liquid Chromatography for the Separation and Identification of C-20 Metabolites of Conjugated Linoleic Acid Isomers in Rat Liver Lipids, 7. Chromatogr. B 724, 213-219. 

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