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1、11.1IntroductionThe separation of enantiomers is a very important topic to the pharmaceutical indus-try. It is well recognized that the biological activities and bioavailabilities of enan-tiomers often differ 1. To further complicate matters,the pharmacokinetic profile of the racemate is often not j

2、ust the sum of the profiles of the individual enantiomers.In many cases,one enantiomer has the desired pharmacological activity,whereas the other enantiomer may be responsible for undesirable side-effects. What often gets lost however is the fact that,in some cases,one enantiomer may be inert and,in

3、 many cases,both enantiomers may have therapeutic value,though not for the same disease state. It is also possible for one enantiomer to mediate the harmful effects of the other enantiomer. For instance,in the case of indacrinone,one enantiomer is a diuretic but causes uric acid retention,whereas th

4、e other enantiomer causes uric acid elimination. Thus,administration of a mixture of enantiomers,although not neces-sarily racemic,may have therapeutic value.Despite tremendous advances in stereospecific synthesis,chiral separations will continue to be important because of possible racemization alon

5、g the synthetic path-way 2,during storage or in vivo (e.g.,ibuprofen 3. While analytical methods are necessary and have become almost routine,economical methods for preparative and semipreparative scale-chiral separation remains largely unexplored. Yet,preparative chiral separations may be particula

6、rly important in an R&D setting where only small amounts of material may be required to initiate screening prior to developing a potentially more costly stereospecific synthetic strategy. In addition,the pharmaceu-tical industry has a critical need for methods which produce pure enantiomers for

7、reference materials.During the past two decades,significant progress has been made in chromato-graphic chiral separation technology. H owever,the bioavailability of drug sub-stances dictates that the compounds be water-soluble,and many are ionized at phys-iological pH. The pK a s of many drugs (see

8、Table 11-1 are well outside the safe operating range for silica-based media,and almost all high-performance liquid chro-matography (HPLC chiral stationary phases currently available commercially are on silica substrates. In addition,most preparative liquid chromatography (LC11Electrophoretically Dri

9、ven PreparativeChiral Separations using CyclodextrinsApryll M. StalcupChiral Separation Techniques:A Practical Approach,Second,completely revised and updated editionEdited by G. SubramanianCopyright ©2001 Wiley-VCH Verlag GmbHISBNs:3-527-29875-4 (Hardcover; 3-527-60036-1 (Electronic28811Electro

10、phoretically Driven Preparative Chiral Separations using Cyclodextrins Fig. 11-1. Effect of the addition of methanol on the enantiomeric separation of terbutaline using 2 %sulfated cyclodextrin in 25 m M phosphate buffer (pH 3.methods. An analogous relationship may be seen in the use of chiral mobil

11、e phase additives in thin-layer chromatography (TLC for screening potential chiral selectors for immobilization in chiral stationary phases for HPLC.In considering the applicability of preparative classical electrophoretic methods to chiral separations,it should be noted that practitioners in the ar

12、t of classical elec-trophoresis have been particularly inventive in designing novel separation strategies.For instance,pH,ionic strength and density gradients have all been used. Isoelectric focusing and isotachophoresis are well-established separation modes in classical electrophoresis and are also

13、 being implemented in CE separations 7,8. These trends are also reflected in the preparative electrophoretic approaches discussed here.11.2Classical Electrophoretic Chiral Separations:Batch ProcessesClassical gel electrophoresis has been used extensively for protein and nucleic acid purification and

14、 characterization 9,10,but has not been used routinely for small molecule separations,other than for polypeptides. A comparison between TLC and electrophoresis reveals that while detection is usually accomplished off-line in both electrophoretic and TLC methods,the analyte remains localized in the T

15、LC bed and the mobile phase is immediately removed subsequent to chromatographic develop-ment. In contrast,in gel electrophoresis,the gel matrix serves primarily as an anti-11.2Classical Electrophoretic Chiral Separation:Batch Processes 289convective medium and is usually designed to minimize intera

16、ctions with the solute, excluding molecular sieving effects. In addition,the presence of bulk liquid in the post-run gel no doubt contributes to the solute diffusivity problem,thereby reducing efficiency and complicating detection. Hence,separation of small molecules by clas-sical gel electrophoresi

17、s is generally not done. H owever,solute diffusion may be reduced through complexation with bulky additives.Righetti and co-workers 11 were one of the first to demonstrate the utility of classical isoelectric focusing for the chiral separation of small molecules in a slab gel configuration. In their

18、 system,dansylated amino acids were resolved enan-tiomerically through complexation with -cyclodextrin. Preferential complexation between the cyclodextrin and the derivatized amino acid induced as much as a 0.1pH unit difference in the pK b s of the dansyl group.Stalcup et al. 12 also demonstrated t

19、hat chiral analytes complexed with a bulky chiral additive (e.g. sulfated cyclodextrin,MW 20002500 Da,depending on degree of substitution,DS,with reasonably large binding constants (103 m 1 13could be resolved enantiomerically using classical gel electrophoresis. Initial work used a tube agarose gel

20、 containing sulfated cyclodextrin as the chiral additive.Mechanical support and cooling for the gel was provided by a condenser from an organic synthetic glassware kit (Fig. 11-2. Although 10 mg of racemate were loaded onto the gel and significant enantiomeric enrichment was obtained,recovery of the

21、 analyte required extrusion and slicing of the gel with subsequent extraction of the individual slices followed by chiral analysis of the extracts,a fairly labor-intensive process.Fig. 11-2.Schematic for preparative gel electrophoresis using a condenser for mechanical support and cooling.29011Electr

22、ophoretically Driven Preparative Chiral Separations using Cyclodextrins Fig. 11-3. Mini-prep continuous elution electrophoretic cell.Fig. 11-4.UV trace of piperoxan enantiomers eluting from mini-prepelectrophoresis cell.Stalcup and co-workers 14 adapted this method to a continuous elution mini-prep

23、electrophoresis apparatus shown in Fig. 11-3. In this apparatus,the end of the electrophoretic gel is continuously washed with elution buffer. The eluent can then be monitored using an HPLC detector (Fig. 11-4 and sent to a fraction collector where the purified enantiomers,as well as the chiral addi

24、tive,may be recovered. In this system,the gel configuration was approximately 100 mm ×7 mm,and was air-cooled. The number of theoretical plates obtained for 0.5 mg of piperoxan with this gel was approximately 200. A larger,water-cooled gel was able to handle 15 mg of 11.2Classical Electrophoret

25、ic Chiral Separation:Batch Processes 291 Fig. 11-5. electrophoresis apparatus.the binding constants,mediated by the dimensions of the chamber as well as the spe-cific conductance and linear velocity of the buffer.Despite the use of density and pH gradients,cooling and performance in micro-gravitatio

26、nal environments (e.g. the space shuttle 18,convection and heat dissipa-tion contributed to flow stream instability which was parasitic to the desired separa-tions and limited the utility of this approach.Recent innovations 19 have circumvented the heat dissipation and sample stream distortion inher

27、ent in most of the previous designs. In one apparatus,devel-oped by R&S Technologies,Inc. (Wakefield,RI,USA,Teflon capillary tubes are aligned close to each other in the electrophoretic chamber. Coolant is pumped through the Teflon capillary tubes during the electrophoretic run while the elec-tr

28、ophoretic separation is accomplished in the interstitial volume between the Teflon tubes.Continuous free flow electrophoresis has been used for the separation of biopoly-mers (e.g. ovalbumin and lysozyme 20 as well as smaller inorganic species (e.g.Co III(sepulchrate3+and Co III(CN63- 21. Sample pro

29、cessing rates of 15 mg h1were reported for a mixture of Amaranth (MW:804 and Patent Blue VF (MW: 1159 22.Three basic approaches have been used for chiral separations by continuous free flow electrophoresis. Thormann and co-workers 23 used 2-hydroxypropyl-b-cyclodextrin as an additive for the enantio

30、meric enrichment of methadone in an Octopus continuous free flow electrophoresis apparatus. In this work,both zone and isotachophoretic electrophoresis was used. Processing rates were on the order of 1020 mg h1,which represents a significant improvement in sample throughput relative to CE or the ear

31、lier gel work. The authors realized higher enantiomeric purities with interrupted buffer flow than with continuous buffer flow,and sugges-ted the potential of multistage continuous free flow for achieving even higher puri-ties.Glukhovskiy and Vigh 24 also used 2-hydroxypropyl-cyclodextrin as an addi

32、-tive,but their strategy involved isoelectric focusing. These authors developed the theoretical framework and effectively demonstrated the synergism between CE and continuous free flow electrophoresis. In this work,also using an Octopus continuous free flow apparatus,they were able to establish a pH

33、 gradient between 3.5 to 3.6 across the electrophoretic chamber by using polydisperse ampholytes or Biers ser-ine-propionic acid binary buffers in the buffer stream. As in Righettis earlier work, complexation with the cyclodextrin additive induced sufficient differences in the pI of various dansylat

34、ed amino acid enantiomers that complete enantioresolution was obtained. Although production rates were somewhat lower (1.3 mg h1 than achieved by Thormann and co-workers,the enantiomeric purity was significantly higher.In a different approach,Stalcup and co-workers 25 used sulfated -cyclodextrin for

35、 the enantioseparation of piperoxan in work directly derived from earlier CE and classical gel results. Their results were obtained using a continuous free flow appa-ratus developed by R&S Technologies,Inc. Processing rates on the order of 4.5 mg h1were reported. than in a chromatographic system

36、 because there is no solid sorbent that is subject to degradation or that needs to be pre-equilibrated with the mobile phase. Fig. 11-6.Histograms showing the distribution of piperoxan enantiomers in the absence (aand pre-sence (b of sulfated cyclodextrin in continuous free flow electrophoresis.A b

37、s o r b a n c e A b s o r b a n c eVial numberVial number(a(bHistogram for CFFE Run 3 05/07/99Histogram for CFFE Run 3 05/07/9911.4 Conclusions 297 11.4 Conclusions Clearly, chiral separations, particularly preparative, present such a challenging problem that no single technology can provide complet

38、e satisfaction. Much of the activity in chiral separations by CE may be attributed to the advantages of CE relative to liquid chromatography (e.g., efficiency, rapid method development, ease of changing chiral selector, etc. However, it must be noted that the true countercurrent processes possible i

39、n CE also allow selectivity to be manipulated to a much greater extent and with greater ease than is generally feasible in liquid chromatography using immobilized chiral selectors. In principle, any of the chiral entities enantiomerically resolved by CE should be amenable to preparative electrophore

40、tic methods. An important consideration for the ultimate economic viability of any preparative electrophoretic approach is the potential recovery of the chiral additive. Because the electrophoretic separation depends only upon the stability of the chiral additive itself and not the combined stabilit

41、y of an immobilized chiral selector, a spacer and an underlying substrate, as in the case of cyclodextrins immobilized on a silica chromatographic support, preparative electrophoretic separations have the potential to be more robust than analogous chromatographic methods. Although still in its infan

42、cy, preparative chiral electrophoresis represents an important technological advance in chiral separations, and has the potential to complement preparative chiral chromatographic methods as well as chiral CE complements chiral analytical chromatography. Acknowledgments The author would like to ackno

43、wledge R& S Technologies, Inc. (Wakefield, RI, USA for the loan of the continuous free flow electrophoresis system, and Cerestar, Inc. for the donation of the sulfated cyclodextrin. The author would also like to thank Drs. Chris Welch and Prabha Painuly for helpful discussions. References 1 W.D.

44、 Hooer and M.S. Qing. Clin Pharmacol. Ther., 48 (1990 633. 2 D. W. Armstrong, L. F. He, T. Yu, J. T. Lee and Y. S. Liu. Tet. Assym., 10 (1999 37. 3 W. J. Wechter, D. G. Loughhead, R.J. Reischer, G. J. Van Giessen and D. G. Kaiser. Biochem. Biophys. Res. Comm., 61 (1974 833. 4 T. J. Ward. Anal. Chem.

45、, 66 (1994 632A. 5 H. Nishi and S. Terabe. J. Chromatogr. A, 694 (1995 245. 6 S. R. Gratz and A. M. Stalcup. Anal. Chem., 70 (1998 5166. 7 X. W. Yao and F. E. Regnier. J. Chromatogr., 632 (1993 185. 8 E. Kenndler. Chromatographia, 30 (1990 713. 298 11 Electrophoretically Driven Preparative Chiral Separations using