Reversed-phase chiral

Among the most successful of the liquid chromatographic reversed-phase chiral stationary phases have been the cyclodextrin-based phases, introduced by Armstrong and commercially available through Advanced... 

Franco et al. [45] described an HPLC method for simultaneous determination of the R-( ) and (S)-(+)-enantiomers of vigabatrin in human serum after precolumn derivatization with 2,4,6-trinitrobenzene sulfonic acid (TNBSA) and detection at 340 nm. Separation was achieved on a reversed phase chiral column (Chiralcel-ODR, 25 cm x 4.6 mm) using 0.05 M potassium hexafluorophosphate (pH4.5) acetonitrile ethanol (50 40 10) as a mobile phase at a flow rate of 0.9 ml/min. The calibration graphs for each enantiomer were linear over the concentration range of 0.5-40 fig/ml with a limit of quantification of 0.5 fig/ml. No interferences were found from commonly coadministered antiepileptic drugs. 

Pu G.-Q., Yamamoto M., Takeuchi Y., Yamazawa H. and Ando T. (1999) Resolution of epoxydienes by reversed-phase chiral HPLC and its application to stereochemistry assignment of mulberry looper sex pheromone. J. Chem. Ecol. 25, 1151-1162. 

Karlsson, A. and Charron, C. Reversed phase chiral ion-pair chromatography at a column temperature below 0°C using three generations of Hypercarb as solid phase. J. Chromatogr. A. 1996, 732, 245-253. 

See also Ion Exchange Principles. Liquid Chromatography Normal Phase Reversed Phase Chiral. 

See alsa Chromatography Overview Principles. Electrophoresis Affinity Techniques. Enzymes Immobilized Enzymes. Immunoassays Ovenriew. Liquid Chromatography Overview Principles Reversed Phase Chiral Multidimensional. 

Peng L, Jayapalan S, Chankvetadze B, Farkas T. Reversed phase chiral HPLC and LC/ MS analysis with tris(chloromethylphenylcarbamate) derivatives of cellulose and amylose as chiral stationary phases. J Chromatogr A 2010 1217 6942-55. 

Reversed phase chiral separations are desired simply for efficiency in generating results from laboratories whose instrumentation is routinely configured to run in reversed and not normal phase modes.Normal phase conditions are less attractive to the analytical chemist for this reason and deter laboratory efficiency. Typical commercial chiral LC columns found on pharmaceutical reversed phase LC chiral method development screens are listed in Table 8. Table 11 shows suggested chromatographic conditions employed in reversed phase chiral screening. 

Holzheuer, W. B., Wong, M. M. and Webster, G. K. Reversed Phase Chiral Method Development Screening for Compounds of Pharmaceutical Interest. Curr. Pharm. Anal 5 346-357, 2009. 

Nonpolar organic mobile phases, such as hexane with ethanol or 2-propanol as typical polar modifiers, are most commonly used with these types of phases. Under these conditions, retention seems to foUow normal phase-type behavior (eg, increased mobile phase polarity produces decreased retention). The normal mobile-phase components only weakly interact with the stationary phase and are easily displaced by the chiral analytes thereby promoting enantiospecific interactions. Some of the Pirkle-types of phases have also been used, to a lesser extent, in the reversed phase mode. 

Chiral separations have become of significant importance because the optical isomer of an active component can be considered an impurity. Optical isomers can have potentially different therapeutic or toxicological activities. The pharmaceutical Hterature is trying to address the issues pertaining to these compounds (155). Frequendy separations can be accompHshed by glc, hplc, or ce. For example, separation of R(+) and 5 (—) pindolol was accompHshed on a reversed-phase ceUulose-based chiral column with duorescence emission (156). The limits of detection were 1.2 ng/mL of R(+) and 4.3 ng/mL of 3 (—) pindolol in semm, and 21 and 76 ng/mL in urine, respectively. 

Cyclodextrin stationary phases utilize cyclodextrins bound to a soHd support in such a way that the cyclodextrin is free to interact with solutes in solution. These bonded phases consist of cyclodextrin molecules linked to siUca gel by specific nonhydrolytic silane linkages (5,6). This stable cyclodextrin bonded phase is sold commercially under the trade name Cyclobond (Advanced Separation Technologies, Whippany, New Jersey). The vast majority of all reported hplc separations on CD-bonded phases utilize this media which was also the first chiral stationary phase (csp) developed for use in the reversed-phase mode. 

When analytes lack the selectivity in the new polar organic mode or reversed-phase mode, typical normal phase (hexane with ethanol or isopropanol) can also be tested. Normally, 20 % ethanol will give a reasonable retention time for most analytes on vancomycin and teicoplanin, while 40 % ethanol is more appropriate for ristocetin A CSP. The hexane/alcohol composition is favored on many occasions (preparative scale, for example) and offers better selectivity for some less polar compounds. Those compounds with a carbonyl group in the a or (3 position to the chiral center have an excellent chance to be resolved in this mode. The simplified method development protocols are illustrated in Fig. 2-6. The optimization will be discussed in detail later in this chapter. 

A general phenomenon observed with chiral stationary phases having hydrophobic pockets is that a decrease of flow rate results in an increase in resolution. This change has significant impact mostly in reversed-phase mode (see Fig. 2-10). 

This is because the increased turbulence from higher flow rates decreases the possibility for inclusion complexation, a necessary event for chiral recognition in reversed phase. Some effect has also been observed in the new polar organic mode when (capacity factor) is small (< 1). Flow rate has no effect on selectivity in the typic normal-phase system, even at flow rates up to 3 inL miir (see Fig. 2-11). 

Comparisons of LC and SFC have also been performed on naphthylethylcar-bamoylated-(3-cyclodextrin CSPs. These multimodal CSPs can be used in conjunction with normal phase, reversed phase, and polar organic eluents. Discrete sets of chiral compounds tend to be resolved in each of the three mobile phase modes in LC. As demonstrated by Williams et al., separations obtained in each of the different mobile phase modes in LC could be replicated with a simple CO,-methanol eluent in SFC [54]. Separation of tropicamide enantiomers on a Cyclobond I SN CSP with a modified CO, eluent is illustrated in Fig. 12-4. An aqueous-organic mobile phase was required for enantioresolution of the same compound on the Cyclobond I SN CSP in LC. In this case, SFC offered a means of simplifying method development for the derivatized cyclodextrin CSPs. Higher resolution was also achieved in SFC. 

This relatively new class of CSPs incorporates glycopeptides attached covalently to silica gel. These CSPs can be used in the normal phase, reversed phase, and polar organic modes in LC [62]. Various functional groups on the macrocyclic antibiotic molecule provide opportunities for tt-tt complexation, hydrogen bonding, and steric interactions between the analyte and the chiral selector. Association of the analyte... 

Many racemic mixtures can be separated by ordinary reverse phase columns by adding a suitable chiral reagent to the mobile phase. If the material is adsorbed strongly on the stationary phase then selectivity will reside in the stationary phase, if the reagent is predominantly in the mobile phase then the chiral selectivity will remain in the mobile phase. Examples of some suitable additives are camphor sulphonic acid (10) and quinine (11). Chiral selectivity can also be achieved by bonding chirally selective compounds to silica in much the same way as a reverse phase. A example of this type of chiral stationary phase is afforded by the cyclodextrins. 

There are two basic procedures that have been successfully used for the separation of isomers. The first is to add a chiral agent to the mobile phase such that it is adsorbed, for example, on the surface of a reverse phase, producing a chirally active surface. This approach has been discussed on page (38) in chapter 2. The alternative is to employ a stationary phase that has been produced with chiral groups bonded to the surface. 

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