Chiral compounds electrophoresis

S Fanali, G Caponecchi, Z Aturki. Enantiomeric resolution by capillary zone electrophoresis use of pepsin for separation of chiral compounds of pharmaceutical interest. J Microcolumn Sep 9 9—14, 1997. 

This unit describes those methods that can differentiate between enantiomers found in foods that contribute to their taste and aroma. These compounds are volatile odorants that are most easily analyzed using enantioselective high resolution-gas chromatography (HRGC). Other methods exist for the separation and analysis of chiral compounds, which include optical methods, liquid and planar chromatography, and electrophoresis, but for food volatiles, gas chromatography has evolved to the point where it is now the cornerstone for the most comprehensive analysis of volatile compounds. 

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. 

The two forms of capillary array electrophoresis are emerging as powerful methods for the determination of enantiomeric purity of chiral compounds in a truly high-through-put manner. Various modifications are possible, for example, detection systems based on UV/Vis, MS, or electrical conductivity. Moreover, chiral selectors in the CE electrolyte are not even necessary if the mixture of enantiomers is first converted into diastereo-mers, for example, using chiral fluorescent-active derivatization agents [51,57]. 

Fig. 1 Flow diagram indicating starting point and optimization procedure for enantiomeric separation of basic, acidic, and neutral compounds. [Adopted with kind permission from M. M. Rogan and K. D. Altria, Introduction to the theory and applications of chiral capillary electrophoresis, Beckman primer Vol. IV, p. 22, Part No. 726388.]...

Marina, M.L. Crego, A.L. Capillary electrophoresis A good alternative for the separation of chiral compounds of environmental interest. J. Liq. Chromatogr. Relat. Technol. 1997, 20, 1337-1365. 

In this context, there has been a considerable development of enantioselective synthetic methodologies, which have now reached a high degree of diversity and complexity. Simultaneously, this trend has created an intensive demand for stereoselective separation techniques and analytical assays for precise determination of the enantiomeric purity of chiral compounds. The development of chiral stationary phases (CSPs) or chiral selectors for gas chromatography (GC), hquid chromatography (LC) and capillary electrophoresis (CE) rapidly opened a new dimension in the area of separation technologies. 

Electrophoretic methods are widely used alternatives for the analytical determination of the enantiomeric purity of chiral compounds [194]. Due to the high elTi-ciency of capillary electrophoresis, separations can be achieved even when very low selectivities are observed. At a preparative scale, these methods are well established for the purification of proteins and cells [195] but there is very little published on enantioselective separations. Only recently, some interest in chiral preparative applications has been manifested. Separation of the enantiomers ofterbu-taline [196] and piperoxan [197] have been reported by classical gel electrophoresis using sulfated cyclodextrin as a chiral additive, while the separation of the enantiomers of methadone could be successfully achieved by using free-fluid isotachophoresis [198] and by applying a process called interval-flow electrophoresis [199]. 

K Verleysen, P Sandra. Separation of chiral compounds by capillary electrophoresis. Electrophoresis 19 2798-2833, 1998. 

In capillary electrophoresis (CE), CDs and their ionic and neutral derivatives have been successfully used as additives in the carrier system for the separation of structural isomers and structurally related compounds [53]. The commonly used neutral CDs are the native a-, /3- and y-CDs and the dimethyl, trimethyl, hydroxyethyl and hydroxypropyl forms [54]. The charged CDs are carboxymethyl, sulfobutyl ether, sulfated and amino CDs. The methyl derivatives of the CD are effective in separating chiral compounds, enantiomers of terbutaline, ephedrine and carnitine. The neutral derivatives of hydroxyalkylated /3-CD and the mixture... 

The enantiomeric purity of the chiral compounds was determined by capillary electrophoresis. 

Figure 6 Chiral capillary electrophoresis analysis of tramadol and its metabolites in urine. (A) Separation of reference compounds, (B) analysis of a urine sample collected 6-8h after oral administration of fOOmg tramadol. The internal standard (IS) was a chiral analog that is also resolved. Experimental conditions 50/57 cm fused silica capillary, 50 iim, 50 mmol I sodium borate buffer, pH 10.1, 30 mg ml carboxymethyl-/i-cyclodextrin, 20 kV, UV detection at 214nm. (Adapted with permission from Kurth Band Blaschke G (1999) Achiral and chiral determination of tramadol and its metabolites in urine by capillary electrophoresis. Electrophoresis 20 555-563 Wiley-VCH.)...

An important task that can be solved by electrophoresis is the separation of enantiomers of chiral compounds. It is performed by means of their different interactions with a chiral pseudostationary phase - another chiral substance, called chiral selector, which is present in the separation system. The enantiomers of a chiral analyte have the same physicochemical properties in the achiral environment, so their mobilities are also the same and caimot be separated when the separation system is achiral. However, when a chiral substance, which is able to interact (to form complexes) with the analyte, is added to the system, the constants of complexity are generally different and the mobilities of the complexes with both enantiomers may also be different. This enables their separation. 

Synchronous cyclic capillary electrophoresis (SCCE) was proposed by Jorgenson s group as a technique which allows overcoming the dispersion problems in FCCE caused by the parabolic counterflow profile [31], High resolution is achieved in SCCE by driving the samples in a virtually closed loop until desired resolutions are achieved. This technique was applied for isotopic and chiral separations. In the third cycle, the chiral compound (a-hydroxybenzyl) methyltrimethylammonium with a selectivity as low as 1.0078 was almost baseline separated in 3.5 h [40]. As mentioned by the authors, similar separations with a multicycle LC system as described in [41] would also be possible but would have taken 43 h. 

The different methodologies developed for the precise determination of the stereoisomeric composition of chiral compounds and chiral separation, such as gas and liquid chromatography on chiral stationary phases, capillary electrophoresis, and nuclear magnetic resonance (NMR) spectroscopy the methodologies for the determination of... 

Enantioresolution in capillary electrophoresis (CE) is typically achieved with the help of chiral additives dissolved in the background electrolyte. A number of low as well as high molecular weight compounds such as proteins, antibiotics, crown ethers, and cyclodextrins have already been tested and optimized. Since the mechanism of retention and resolution remains ambiguous, the selection of an additive best suited for the specific separation relies on the one-at-a-time testing of each individual compound, a tedious process at best. Obviously, the use of a mixed library of chiral additives combined with an efficient deconvolution strategy has the potential to accelerate this selection. 

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