Chiral separations optimization

Temperature can also be used to optimize enantioselectivity in SFC. The selectivity of most CSPs increases as temperature decreases. For this reason, most chiral separations in SFC are performed at ambient or subambient temperatures [50, 74]. Subambient temperatures are particularly useful for compounds having low conformational stability [75]. Stringham and Blackwell explored the concept of entropically driven separations [76]. As temperature increased, enantioselectivity decreased until the enantiomers co-eluted at the isoelution temperature. Further increases in temperature resulted in reversal of elution order of the enantiomers. The temperature limitations of the CSP should be considered before working at elevated temperatures. 

Rawjee, Y. Y and Vigh, Gy., Efficiency optimization in capillary electrophoretic chiral separations using dynamic mobility matching, Anal. Chem., 66,3777,1994. 

In achiral-chiral LC-LC, the mobile phases used with the achiral and chiral columns must be miscible with one another. Since the enantiomeric separation is usually the most difficult to optimize, it is usually the separation that dictates the mode of operation of the total analysis. Thus, it makes sense that a chiral column that operates in the normal phase mode would require an achiral column that also works in the normal phase mode. Polar organic mode chiral separations are universal in that they can be paired with an achiral column that operates in either the reverse phase or normal phase mode. The choice of the achiral column is always determined after selecting the chiral column and the mode of operation. As with traditional liquid chromatography, different achiral columns will give different selectivity. 

Beesley, T.E., Lee, J.T., and Wang, A.X., Method development and optimization of enantiomeric separations using macrocyclic glycopeptide chiral stationary phases, in Chiral Separation Techniques, Second completely revised and updated edition, Subramanian, G., Ed., Wiley-VCH Weinheim, 2001, 25. 

Analysis using a CMPA is usually resolved on a nonchiral column. A transient diastereomeric complex is formed between the enantiomer and the chiral component in the mobile phase, similar to the complexes formed with chiral stationary phases. A review by Liu and Liu (2002) cites several papers where addition of CPMAs has been used in analyzing amphetamine-related compounds. Some CPMAs include amino acid enantiomers, metal ions, proteins, and cyclodextrins. Advantages of this method of analysis include the use of less expensive columns and more flexibility in the optimization of chiral separation (Misl anova and Hutta, 2003). 

Proteins have also been successfully used for chiral separations in CE. One of the characteristics of proteins is their isoprotic point, pi. The protein will mainly be charged positively if pHpI. Therefore, the pH of the BGE is a very important parameter for the optimization of the separations. As with charged-CD derivatives, it is possible to separate both charged and uncharged chiral species with... 

SG Penn, ET Bergstrom, DM Goodall, JS Loran. Capillary electrophoresis with chiral selectors. Optimization of separation and determination of thermodynamic parameters for binding of ticonazole enantiomers to cyclodextrins. Anal Chem 66 2866-2873, 1994. 

There are numerous reports of the use of temperature to enhance chiral separations. In some cases, the optimal separation is achieved at elevated temperatures and in others at sub-ambient temperatures [46]. Tian et al. [47] noted that for the separation of chiral pesticides, most gave better separation factors at a low temperature. The exception was pyriproxyfen, which gave a larger separation factor at higher temperatures. Sun et al. [48] studied the chiral separation of clenbuterol... 

There is no systematic way to predict the impact of temperature on chiral separations. It is advisable for anyone attempting to optimize a chiral separation to explore temperatures over the range available with their instrumentation to determine the best conditions. 

Sponsler, S. andBiederman, M. 1997. Optimization of chiral separations using capillary gas chromatography. Am. Lab. 24C-H. 

Resolution is optimized by adjusting the buffer pH and the amount of organic modifiers. The most commonly used buffers are perchlorate, acetate, and phosphate. The protocol of the selection and optimization of the mobile phase for the enantiomeric resolution of drugs on polysaccharide-based CSPs in reversed-phase mode is presented in Scheme 2. Table 4 correlates the effects of separation conditions for neutral, acidic, and basic drugs on polysaccharide-based CSPs. From Table 4, it may be concluded that a simple mixture of water and an organic modifier will produce chiral separation of a neutral molecule because there is no... 

Temperature and pressure are rarely optimized in HPLC, but these parameters are very important in SFC, hence can alter retention, selectivity, and resolution. Toribio et al. [149] presented the chiral separation of ketoconazole and its precursors on Chiralpak AD and Chiralcel OD CSPs. The authors also reported that alcohol modifiers provided better separation than acetonitrile. Further, Wilson [143] studied the effects of composition, pressure, temperature, and flow rate of the mobile phase on the chiral resolution of ibuprofen on a Chiralpak AD CSP. It was observed that temperature affords the greatest change in resolution, followed by pressure and composition. An increase in methanol concentration, pressure, and temperature has resulted in poor chiral resolution. At first chiral resolution increased with an increase of flow rate (up to 1.5 mL/min) but then started to decrease. Contrary to this, Biermann et al. [135] described the... 

Karlsson and Hermansson [30] used chemometrics for optimization of chiral separation of omeprazole and one of its metabolites on immobilized al-acid glycoprotein. Plasma was centrifuged at 2500 rpm and a portion (20-50 ji ) was injected into a 5-/rm Chiral-AGP column (10 cm x 4 mm) with al-acid glycoprotein immobilized to silica as a chiral stationary phase and acetonitrile-phosphate buffer of pH 5.7-7.2 as mobile phase (1 ml/min). Detection of omeprazole and its main metabolite, hydroxy-lated omeprazole, was performed at 302 nm. A statistical model was developed for the optimization of the operational parameters. The experimental data were evaluated with multivariate analyses column temperature and acetonitrile concentration were the most important variables for the enantioseparations. Complete enantiomeric separation for omeprazole and hydoxylated omeprazole was obtained within 15 min. 

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