36


May/June 2013 Table 3: Peak area and retention time data for 9 replicate chiral separations (n = 9) (Reprinted from ref [[33]] with permission). Mean peak area


Mean corrected peak area


Relative migration time


Corrected Peak Areas Altria [[33]


Enantiomer 1 1199781


103827 Migration time (min) 11.55 (1.5 % RSD) 0.962 (0.04 % RSD) ] demonstrated by the separation of


a racemic mixture of picumeterol that it is important to correct the peak areas of the separate enantiomers for their migration velocity difference through the detection window. Early migrating analytes move faster through the detection window than later migrating analytes and thus show up as smaller peaks. Therefore the peak areas are corrected by either dividing by the migration time or by the migration velocity (Table 3).


Injection


For good precision in chiral CE, the same attention to detail is needed as for other CE techniques [[31]


]. That is, a constant


temperature on the samples and the BGE. As short injection time as possible above 3 s should be used. This is because in some CE instruments the variability in hydrodynamic injection volume is reduced by stringent control of the injection time x injection pressure. For this mechanism to function properly, at least 3 s injection time is needed. The vials should be levelled to prevent siphoning. Excess of sample sticking on the outside of the capillary after injection can be removed by dipping the capillary inlet after injection into a vial filled with water or BGE. The precision can be further increased by injection of a BGE or water plug after the injection of the sample [[31]


]. Around two


millimetres of the polyimide coating should be removed from the capillary inlet whenever possible, and of course straight capillary ends [[32]


]. An enantiomeric purity


determination often requires detection as low as 0.05 % of the enantiomeric impurity. In order to obtain these levels of sensitivity, it is often needed to use sample stacking. The simplest approach is to dissolve/dilute the sample in a less conducting solvent compared to the BGE. When the voltage is applied, the low-conductivity sample zone will have a higher electric field strength than the BGE. Consequently the analytes migrate faster in


the injection solution zone until they reach the boundary of the BGE zone. In the more conductive BGE the field strength is lower and the analytes stack, i.e. slow down and concentrate, which reduces the initial band width. However, many other sample stacking techniques are also available, see e.g. [[34]


]. Concluding Remarks


Capillary electrophoresis has developed into a highly efficient separation technique and is currently applied in several different areas of analysis such as chiral separations. Further implementation of CE and chiral CE in industry depends on sharing and training of good CE working practices and access to the industrial expert network, as well as on continuous development of instrumentation [[1]


].


Capillary electrophoresis made a significant contribution to the Human Genome Project and is emerging as an import analytical tool in the area of metabolomics and other ‘-omics’. In these areas the objective is to simultaneously analyse a large number of hydrophilic compounds such as amino acids, peptides and other analytes that might be present as enantiomers. Chiral CE shows high separation efficiency and is suitable for hydrophilic analytes and might thus be the preferred technique. A key factor for success is CE-MS. Unfortunately, the combination of CE and MS has not yet been fully established to be used routinely in the laboratory. When hyphenated CE-MS/MS becomes an analytical tool as ordinary as LC MS/MS is today, it will certainly make CE an even more attractive separation technique in several areas. In chiral CE, it would be preferable to apply the partial filling technique to avoid the selector to reach the MS instrument [[35]


]


and thus minimise time-consuming cleaning of the MS interface.


Automation and miniaturisation of analytical systems have been and still are general trends in analytical chemistry. The use of a multi-capillary CE system has proven to


Enantiomer 2 1246293


103841 12.00 (1.5 % RSD)


Peak area ratio 1.04


1.00 References


[1] C.E. Sänger – van de Griend, R.E. Majors, LC-GC North America 30 (2012) 954


[2] General Chapter 2.2.47 Capillary Electrophoresis, European Pharmacopoeia


[3] General Information <1053> Capillary Electrophoresis, United States Pharmacopeia


[4] V. Piette, M. Lammerhofer, W. Lindner, J. Crommen, J Chromatogr A 987 (2003) 421


[5] Y. Carlsson , M Hedeland, U. Bondesson , C. Pettersson, Chromatogr. A 922 (2001) 303


[6] C. Petterson, C. Gioeli, Chirality 5 (1993) 241


[7] S.A.C. Wren, R.C. Rowe, J. Chromatogr. 603 (1992) 235


[8] S.A.C. Wren, J. Chromatogr. 636 (1993) 57


[9] B. Chankvetadze, Capillary Electrophoresis in chiral analysis, John Wiley & Sons, 1997, ISBN 0 471 97415 3


[10] C.E. Sänger – van de Griend, K. Gröningsson, D. Westerlund, Chromatographia 42 (1996) 263


[11] C.E. Sänger – van de Griend, K. Gröningsson, J. Pharm. Biomed. Anal. 14 (1996) 295


[12] C.E. Sänger – van de Griend, H. Wahlström, K. Gröningsson, M. Widahl- Näsman, J Pharm. Biomed. Anal. 15 (1997) 1051


[13] 2335 Ropivacaine Hydrochloride Monohydrate, European Pharmacopoeia


[14] Monograph Ropivacaine Hydrochloride Monohydrate, United States Pharmacopeia


[15] A. Amini, T. Rundlöf, M.B. Grön-Rydberg, T. Arvidsson, J. Sep. Sci. 27 (2004) 1102


[16] G. Gübitz, M.G. Schmid, Cyclodextrin mediated chiral separations, in Chiral Separations by Capillary Electrophoresis, Ed. A. Van Eeckhaut, Y. Michotte, CRC Press, 2009, ISBN 978-1-4200-6933-4


considerably improve the sample throughput [e.g. [36][37]


]. Interesting studies applying


electrophoretic microfluidic devices for fast chiral separations have been published [[38] and more studies will be published in the future.


, [39] ]


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60