Enantiomer analysis, separation technique

Enantiomeric separations have become increasingly important, especially in the pharmaceutical and agricultural industries as optical isomers often possess different biological properties. The analysis and preparation of a pure enantiomer usually involves its resolution from the antipode. Among all the chiral separation techniques, HPLC has proven to be the most convenient, reproducible and widely applicable method. Most of the HPLC methods employ a chiral selector as the chiral stationary phase (CSP). 

CE has been established as a very efficient technique for the separation of drug enantiomers. Chiral separations in CE are also based on the formation of diastereomeric complexes between the enantiomers and a chiral selector. The main advantages of the technique are its high efficiency, short analysis times, versatility due to the great variety of chiral selectors that can be added to the BGE, short equilibration times required when changing the chiral selector and low consumption of selector. °... 

Sensors for the detection of enantiomers are of great interest, as so far the on-line monitoring of production processes and medical diagnostics using standard chemical analytical methods is not possible. Quite often only one enantiomer of a chiral compound is actually a bioactive therapeutic. Therefore a proper analysis of the final product is essential. Currently, this involves separation techniques like liquid chromatography, GC and capillary electrophoresis, and determination of enantiomeric purity with circular dichro-ism and specific rotation. These are all off-line procedures and therefore no real-time analysis can be performed. Sensing devices for the distinction of different enantiomers would be a much cheaper, faster and easier-to-use alternative for this task, amenable to automation. 

Instead by solvent extraction [207], aroma compounds from aqueous media, e.g. fruit juices, can even be separated and enriched by techniques of solid phase micro extraction (SPME), preferably from the headspace [208] , corresponding devices can often be directly connected to GC systems. These techniques provide the complete spec-tmm of the individual compounds of an aroma. As it will normally not be possible and even not necessary to analyse all components of the complex mixture, the separation of its main compounds may demand a multi-dimensional (MD) gas chromatographic system [209[ as displayed in Fig. 6.14 [210[. Examples for the multi-ele-ment/multi-compound isotope analysis by such systems will be given later (6.2.2.4.4, [211[) they can even integrate the identification of the compounds by molecular mass spectrometry and a simultaneous determination of the enantiomer ratios of isomers [210, 211 [. The importance of enantiomer analysis as a tool for authenticity assessment is extensively treated in chapter 6.2.3. 

As Louis Pasteur proposed in his time, enantiomers can be separated as diastereomeric derivatives after a chemical reaction with a chiral selector using fractional recrystallization (see Chapter 5). However, separation techniques such as chromatography and electrophoresis are now recognized for fhe separation of enantiomers. Even if chromatography is considered as fhe method of choice for chiral analytical purposes (see Chapter 7), capillary electrophoresis (CE) has recently gained importance in fhe field of stereoselective analysis. 

HPLC. " Different perfluorinated carboxylic acids have been examined as the ion-pairing agents (pH 2.5. 5) in the analyses of enriched yeast and vegetable extracts Alkylsulfonic acids have been successfully used in speciation analysis carried out on nuts, onion leaves and yeast " " Owing to their different biological activities, optical enantiomers of Se-amino acids were analyzed by several chiral separation techniques - ... 

Enantioselectivity was introduced especially for use in analysis of pharmaceuticals, where it was found that some pharmaceutical products have a chiral center and only one of the enantiomers exhibits the required pharmacological and pharmacokinetic behavior. The term was introduced first in relation to separation techniques,278 281 and later sensor technology.282... 

The most important advantages of capillary electrophoresis (CE) are extremely high peak efficiency, small sample size, minute amounts of chiral selectors and buffers, low costs and less environmental problems. A capillary format leads exclusively to all these advantages. At the same time, the capillary format is a clear disadvantage from the preparative point of view. Thus, CE is an excellent technique for enantiomer analysis, but not for their preparative separation. Therefore, at this point the technique is not discussed further. 

In 1960, a patent was granted for the separation of D,L-prollne on a lactose column (56). Davankov s laboratory was the first to report separation of amino acid Isomers on polymeric resins derlvatlzed with optically active amino acids (57). However, separation of amino acid enantiomers by these techniques has been hampered by long separation times (ca. 10 hr) and the difficulty In synthesizing supports of sufficient quality for modern HPLC (spherical particles, small size, uniform chemical modification). Separation of amino acid Isomers on a column consisting of silica bonded with L-amlno acids and complexed with copper (II) has been reported by Gubltz and Jellenz (42). Short analysis times for separation of mixtures of single D,L-amlno acids were reported (ca. 30 min), but complex mixtures have not been separated. 

Just as modem liquid chromatography (LC) is the dominant separative technique in the analysis of pharmaceuticals, so, in the evolution of methodologies for the chiral analysis of pharmaceuticals, LC has emerged as the pre-eminent technique. As already intimated, discrimination between enantiomers requires the presence of a chiral selector. In the determination of enantiomers by LC... 

Analyses of amphetamines, like most other drugs, are most commonly accomplished using GC-MS, which remains the gold standard for confirmation analysis. Concern about the (R)-enantiomer has also led to chiral separation techniques being used for definitive analysis of methamphetamine to establish whether or not the drug was an over-the-counter form or a controlled form of the drug. 

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. 

The efficiency of many CSPs increases dramatically when liquid eluents are replaced with sub- or supercritical fluids. During a comparison of LC and SFC performed with a Chiralcel OD CSP, Lynam and Nicolas reported that the number of theoretical plates obtained was three to five times higher in SFC than in LC [26]. The separation of metoprolol enantiomers by LC and SFC on a Chiralcel OD CSP is illustrated in Fig. 12-2. Although impressive selectivity is achieved by both techniques, resolution is higher in SFC (R = 12.7) than in LC (R = 4.8), and the higher flowrate in SFC reduces the analysis time. The increased efficiency of SFC also improves peak symmetry. 

CSPs and chiral mobile phase additives have also been used in the separation of amino acid enantiomers. Another technique that should be mentioned is an analysis system employing column-switching. D-and L- amino acids are first isolated as the racemic mixture by reverse-phase HPLC. The isolated fractions are introduced to a second column (a CSP or a mobile phase containing a chiral selector) for separation of enantiomers. Long et al. (2001) applied this technique to the determination of D- and L-Asp in cell culture medium, within cells and in rat blood. 

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