Capillary electrophoresis (CE) was used to optimize the

buffer pH, ionic strength and sulfated cyclodextrin

concentrations for enantiomeric separation of piperoxan.

These enantioseparation conditions were then applied to a

classical gel electrophoresis system. Binding constants of

the sulfated beta-cyclodextrin–piperoxan couple were

approximated using CE and the effects of organic solvents

on the system were also investigated

Gratz, Samuel R.

Stalcup, Apryll

Sutton, Richard M. C.

English

Capillary electrophoresis (CE) was used to optimize the
buffer pH, ionic strength and sulfated cyclodextrin
concentrations for enantiomeric separation of piperoxan.
These enantioseparation conditions were then applied to a
classical gel electrophoresis system. Binding constants of
the sulfated beta-cyclodextrin–piperoxan couple were
approximated using CE and the effects of organic solvents
on the system were also investigated

Irish Universities

Use of capillary electrophoresis as a method development tool for classical gel electrophoresis

Capillary electrophoresis (CE) was used to optimize the\ud
buffer pH, ionic strength and sulfated cyclodextrin\ud
concentrations for enantiomeric separation of piperoxan.\ud
These enantioseparation conditions were then applied to a\ud
classical gel electrophoresis system. Binding constants of\ud
the sulfated beta-cyclodextrin–piperoxan couple were\ud
approximated using CE and the effects of organic solvents\ud
on the system were also investigated

Name not available

 OOHN+Use of capillary electrophoresis as a methoddevelopment tool for classical gel electrophoresisRichard M. C. Sutton, Samuel R. Gratz and Apryll M. StalcupDepartment of Chemistry, P.O. Box 210172, University of Cincinnati, Cincinnati, OH 45221-0172,USACapillary electrophoresis (CE) was used to optimize thebuffer pH, ionic strength and sulfated cyclodextrinconcentrations for enantiomeric separation of piperoxan.These enantioseparation conditions were then applied to aclassical gel electrophoresis system. Binding constants ofthe sulfated b-cyclodextrin–piperoxan couple wereapproximated using CE and the effects of organic solventson the system were also investigated.Keywords: Capillary electrophoresis; piperoxan;enantiomers; gel electrophoresis; chiral separationIn recent years, capillary electrophoresis (CE) has been used toperform a number of separations, previously attempted usinghigh-performance liquid chromatography (HPLC).1 One of thereasons for the technique’s gain in popularity is due to itsextremely high separation efficiency and high resolutioncapabilities. A number of advantages are immediately apparentwith CE in comparison with HPLC, including simplicity, rapidmethod development and optimization, fast analysis times, avariety of separation modes and low cost. Other advantages ofusing CE are that relatively expensive reagents can be used,since they require minimal quantities of reagent and separationmedia. Therefore, large volumes of organic waste, associatedwith HPLC methods, are avoided.An area in which CE has become extremely important is thepharmaceutical industry. In the case of chiral drugs, the analytespossess identical physico-chemical properties in an achiralenvironment. The development of CE has enabled enantiomericseparations to be performed with relative ease in comparisonwith HPLC. There is no longer a requirement to investigate theseparating capabilities of relatively expensive chiral columns,since most chiral separations can be achieved by the simpleaddition of a chiral additive to the run buffer.2 Enantiomericseparations occur if two conditions are met. The bindingconstants of the chiral additive and the enantiomer couples aredifferent, and there is a difference in mobility of the free andcomplexed states.There are a number of chiral separation mechanismsavailable using CE. These include inclusion complexation,metal–chiral ligand complexation, micellar solubilization, af-finity interactions and ion-pairing interactions.2,3 Of particularinterest in this study is the use of sulfated b-cyclodextrin (SCD)as a chiral additive. The first sulfonated cyclodextrin wasintroduced for this purpose by Tait et al.4 Since then, directlysulfated cyclodextrins have been effectively demonstrated forthe enantiomeric separation of a wide number of chiraldrugs.5At present, analytical separations are readily achieved usingCE, but it is difficult to scale up separations to yield preparativeamounts. Stalcup et al.,6 however, recently demonstrated that itis possible to perform enantiomeric separations using a largerscale electrophoresis system operating under similar conditionsto those used in CE. Enantiomerically pure chiral drugs can beobtained in quantities which may be suitable for preliminarypharmacokinetic and pharmacodynamic studies in a researchand development environment.7 In addition, the application of agel system with continuous buffer elution from one end, enablesanalytes to be collected in a similar fashion to preparativeHPLC.A chiral compound which has exhibited good enantiosepara-tion using SCD as a chiral additive5 and which is suitable forthis study is piperoxan (Fig. 1). Piperoxan is an adrenergicblocker which is used to block or inhibit the release or activityof norepinephrine in the human body. This action dilates bloodvessels (thus lowering blood pressure) and slows the heart rate.Piperoxan is used to treat hypertension and is also used as adiagnostic aid for pheochromocytoma.8This report details the investigation of various buffer systemsused with a SCD chiral additive for the enantioseparation of d,l-piperoxan. After determination of suitable separation conditionsusing CE, baseline enantioseparation performed using a clas-sical gel electrophoresis system enables usable quantities ofenantiomerically pure piperoxan to be obtained. Enantiomericpurity of piperoxan fractions can be determined using CE. Inorder to further evaluate the system, determination of bindingconstants for the piperoxan–SCD couple and the effect oforganic modifiers on the CE separation are described.ExperimentalInstrumentationCapillary electrophoresis experiments were performed using aBio-Rad BioFocus 2000 system (Bio-Rad, Hercules, CA,USA). Fused silica capillary (25 mm id, 17 cm total length, 12.4cm to the detector) coated with polyacrylamide was alsoobtained from Bio-Rad. Detection of piperoxan was achieved atthe anodic end of the capillary at a wavelength of 270 nm. Allexperiments were performed between 23.0 and 28.0 kV andthermostatted at 15 °C. Hydrodynamic injections of 4 mmpiperoxan were made at 5 psi for 0.4 s (2 psi s21). The neutralmarker was 20 mm nitromethane.Classical gel electrophoresis investigations were performedusing a ‘Mini Prep Cell’ (Bio-Rad; (Fig. 2). The power supplyfor the system was a Bio-Rad PowerPac 3000. In the Mini PrepCell, the elution buffer, which flushed the bottom of theelectrophoretic bed ( ≈ 100 3 7 mm), was delivered by a Bio-Rad Econo peristaltic pump to a Shimadzu (New York, USA)SPD-6A UV variable-wavelength detector, interfaced to aShimadzu Chromatopac CR-501 data station. The eluate wasfractionated by an Isco Retriever II fraction collector.Viscosity measurements were made using a Cannon–Ubbelohde type viscometer. The relative viscosity, hr, wascalculated using the equation, hr ≈ (t/to), where to is the effluxtime of the solvent (water) and t is the efflux time of the buffersolution.9Fig. 1 Piperoxan.Analyst, July 1998, Vol. 123 (1477–1480) 1477ReagentsSulfated b-cyclodextrin (nominal 13 sulfates/cyclodextrin) wasdonated by Cerestar, Inc. (Hammond, IN, USA). The d,l-piperoxan hydrochloride was obtained from Sigma ChemicalCo. (St. Louis, MO, USA). The agarose (medium EEO,electrophoretic grade) and all other buffer components {4-mor-pholineethanesulfonic acid (MES), 2-[tris(hydroxymethyl)-methylamino]-1-ethanesulfonic acid (TES), glycine, citric acid,formic acid, acetonitrile, methanol and N,NA-dimethylforma-mide} were obtained from Fisher Scientific (St. Louis, MO,USA).MethodsThe buffer solutions for CE prepared from MES, TES andglycine were 100 mm each containing 2% SCD. The concentra-tion of citric acid used for investigations with changing SCDconcentration was kept constant (150 mm). For other CEinvestigations, citric acid was used at concentrations required toadjust the respective SCD concentration to pH 3. The conditions used for CE were applied to the classical gelelectrophoresis system. The gel was prepared by heating 2%agarose solution, prepared from buffer containing 1% SCDadjusted to pH 3 using ≈ 54 mm citric acid. The gel, heatedusing a microwave oven, was reheated and cooled three times toremove air bubbles. Finally, the hot gel solution was carefullypoured into the gel chamber. The gel bed was ≈ 100 3 7 mmdiameter. Run times using the Mini Prep Cell were about 4–5 hwith an applied potential of 120 V and current of ≈ 10 mA.Results and discussionCE served two functions in the investigations. Primarily,optimization of the buffer pH, ionic strength and sulfatedcyclodextrin concentrations was performed using the CEsystem. Secondly, fractions collected from the Mini Prep Cellidentified from the UV trace as containing piperoxan weresubjected to chiral CE analysis to determine the enantiomericcomposition of the fractions.The highly sulfated b-cyclodextrin (SCD) used in theinvestigations was designated as having 13 sulfate groups percyclodextrin, and thus negatively charged at pH 3. Themechanism of enantioseparation is therefore thought to occur bya process of electrostatic attraction and by inclusion ofpiperoxan in the hydrophobic cavity of the cyclodextrin.Earlier work6 with SCD as a chiral additive in CE used aphosphate buffer system. It was believed that a buffer systemwith a lower mobility than phosphate could enable the system tobe run at higher voltages and enhance the separation. Inaddition, it was felt that an organic buffer might be easier toremove from the fractions collected from the Mini Prep Cell,which would facilitate recovery of both the analytes andadditive.In preliminary investigations, MES, TES and glycine werechosen as buffer components since they are zwitterions at thepHs of interest and should therefore have relatively lowermobilities than positively or negatively charged compounds ofthe same size. The pHs of the aforementioned buffer solutionswere between 4 and 6 after addition to SCD at variousconcentrations. Although separation of piperoxan enantiomerswas achieved using the prepared buffers in a CE system with acoated capillary, the pH of the buffers was considered too highfor use in the gel electrophoresis system, where the EEO couldbe significant at pH > 3. A buffer system prepared using citricacid appeared to be suitable for the enantioseparation ofpiperoxan, and displayed better resolution than the other buffersystems investigated (Table 1).A study of mobility of the piperoxan enantiomers wasperformed, in which the concentration of citric acid was keptconstant (150 mm), but the concentration of SCD was increased.The buffer pH was adjusted with sodium hydroxide to pH 3. Atthis high concentration of citric acid, all of the buffer solutionshad the same citric acid concentration since this was theconcentration required to adjust the 3% SCD solution to pH 3.As expected, the apparent mobility of the piperoxan enantio-mers increased with increasing SCD concentration over therange investigated. This may be explained by the higherconcentration of SCDs, forcing the piperoxan to spend a largeramount of time in the complexed form, and thus migratetowards the detector. Current was found to increase withincreasing SCD concentration. Plot of mobility versus SCDconcentration for the individual enantiomers were essentiallyparallel. Thus, little or no benefit was obtained going to higherSCD concentrations.The measured viscosities in the range of 0.75–3.00% SCDwere essentially constant (hr ≈ 1.10 ± 0.01). It is probable thatthe high concentration of citric acid ensured that viscositydifferences between solutions was minimal.One aim was to determine binding constants of the piperoxanenantiomers with SCD and these were obtained from anequation for binding constants for lectin sugar systems10adapted by Tanaka et al.11 for use with cyclodextrins:  1 1 1 1m m m m m m-=-· +-f SCD f SCD fSCD]( ) [K(1)where m is the apparent mobility of the analyte, mf is theapparent mobility of the analyte with no SCD present, mSCD isthe apparent mobility of the analyte complexed with SCD and Kis the binding constant. The apparent electrophoretic mobilitieswere determined using the equation:12  m meph eo- =lLVt(2)where meph and meo are the electrophoretic and electroosmoticmobilities, l is the length of capillary to the detector, L is thetotal capillary length, V is the operating voltage and t is themigration time. Although a coated column was used, injectionFig. 2 The Bio-Rad Prep Cell.Table 1 A comparison of migration times and resolution of the enantiomersof piperoxan in different buffer systemsReagent(100 mm with Migration2% SCD) times/min ResolutionTES 2.69/4.41 3.22MES 3.47/5.52 3.25Glycine 4.20/6.43 3.11Citric acid 4.10/7.53 4.341478 Analyst, July 1998, Vol. 123of a neutral marker (nitromethane), revealed that there wassignificant electroosmotic flow (EOF) (meof = 1.92 31025cm2 V s21), but it was fairly constant over the SCDconcentration range investigated (±7.11 3 1028 cm2 V s21). Astable coating of SCD on the inside of the capillary resulting ina negative surface charge might account for this EOF. Thediffusion of the analytes led to ‘fronted’ peak shapes. Themigration times were therefore taken at the time where the peakis split into two portions of equal area. It should be noted thatthis does not compensate for the slight variation in responsefrom the leading to the tailing edge of the peak. Fig. 3 displaysthe graph used to determine the binding constants using theintercept/slope. The values of the binding constants were K1 =1150 ± 130 and K2 = 722 ± 50 for the first and second elutingenantiomers, respectively.The effect of the addition of methanol and acetonitrile to therun buffer is displayed in Fig. 4. SCD (2%) was used with 150mm citric acid adjusted to pH 3 using sodium hydroxide. It wasfound that migration times of the piperoxan enantiomersincreased with addition of the organic modifiers to the runbuffer. The difference in migration times observed with theorganic modifiers is believed to be a result of several factors.There could be competition of the organic modifiers andpiperoxan for the hydrophobic SCD cavity. In addition, it ispossible that the piperoxan enantiomers could have a greateraffinity for the background electrolyte which would decreasethe free energy driving the complexation. As can be seen fromFig. 4, at concentrations up to approximately 5% modifier, thereis no significant difference between methanol and acetonitrile.After additions above 5%, however, there is increased resolu-tion between the enantiomers using acetonitrile in comparisonwith methanol. It is therefore possible to enhance enantio-separation using acetonitrile. It is likely, however, that run timescould become impractical using the gel electrophoresis system.Conversely, no enantioseparation was found in the presence ofN,NAdimethylformamide (DMF). At a 10% concentration ofDMF, the migration time of the single peak (containing bothunseparated enantiomers) was greater than the migration time ofthe first eluting enantiomer, but lower than that of the secondFig. 3 Plot of (m-mf)21 versus [SCD]21 for the piperoxan enantiomers. 150mm citric acid was used with varying concentrations of SCD, the buffer pHwas adjusted with sodium hydroxide to pH 3.Fig. 4 Addition of organic modifiers [methanol (MeOH) and acetonitrile(ACN)] to the SCD system. The buffer was 150 mm citric acid, 2% SCD andthe respective concentration of organic modifier. The buffer pH wasadjusted with sodium hydroxide to pH 3 before addition of modifier.Fig. 5 Enantiomeric separation of piperoxan using CE. The buffer was 1%SCD adjusted to pH 3 with ≈ 54 mm citric acid.Fig. 6 Enantioseparation of piperoxan using the Prep Cell. The top andelution buffers were 1% SCD adjusted to pH 3 with ≈ 54 mm citric acid. Thebottom buffer was ≈ 54 mm citric acid adjusted with sodium hydroxide topH 3.Analyst, July 1998, Vol. 123 1479eluting enantiomer of piperoxan, for the corresponding bufferscontaining 10% methanol and acetonitrile, respectively.The buffer conditions for separation of the piperoxanenantiomers using CE were adapted to a Bio-Rad Prep Cell. Theagarose gel served as an anticonvective medium and was notconsidered to play a significant part in the enantioseparation ofthe piperoxan. Agarose gels were chosen in preference topolyacrylamide gels, owing to ease of preparation, readyavailability and because agarose is less toxic than the polymer-izing agents used to prepare the polyacrylamide gel. Theagarose gels were found to be stable over the voltage range usedand were mechanically stable when removed from the gelcylinder. Running the system at higher voltages, however, couldcause the agarose to melt as a result of joule heating (agarosegelling temperature 34–35 °C).The enantioseparation of piperoxan using CE is displayed inFig. 5. Owing to the lack of enantiomerically pure piperoxanstandards, it was not known which enantiomer eluted first. Fig.6 displays an electropherogram obtained from the Prep Cell for0.5 mg sample loading of piperoxan. Fronted peak shapes wereobtained, in accordance with CE, with a resolution of 1.34 (N =137 ± 8). It was found that by running with the entire systemplaced in a bowl of ice water (i.e., the bottom buffer solutionwas cooled), the current was reduced and the separation couldbe performed at a higher voltage (170 V). A separation ofsimilar resolution was achieved, but in a shorter run time(< 4 h). Fig. 7 displays the electropherograms of fractionscollected from the Prep Cell run, indicating that the fractionswere enantiomerically pure. Prior work with terbutaline failedto achieve the level of separation obtained with the gel systemin this study.In conclusion, CE was used to optimize parameters for scale-up of a chiral separation to preparative gel electrophoresis.Although more work is required to assess the general applicabil-ity and limits of this approach, preparative chiral gel electro-phoresis may offer a viable alternative to more traditionalmethods.The authors gratefully acknowledge the generous support of theNational Institutes of Health First Award (Grant 1R29GM48180-04) and Cerestar Inc., for donation of the sulfatedcyclodextrin.References1 St. Claire, R. L., Anal. Chem., 1996, 68, 569R.2 Nishi, H., and Terabe, S., J. Chromatogr. A, 1995, 694, 245.3 Terabe, S., Otsuka, K., and Nishi, H., J. Chromatogr. A, 1994, 666,295.4 Tait, R. J., Thompson, D. O., and Stobaugh, J. F., Anal. Chem., 1994,66, 4013.5 Stalcup, A. M., and Gahm, K. H., Anal. Chem., 1996, 68, 1360.6 Stalcup, A. M., Gahm, K. H., Gratz, S. R., and Sutton, R. M. C., Anal.Chem., 1998, 70, 144.7 Fanali, S., J. Chromatogr. A, 1996, 735, 77.8 Budavari, S., ed., Merck Index, Merck & Co., Rahway, N.J., 11thedn., 1989, pp. 1187–1188.9 Billmeyer, F. W., Textbook of Polymer Science, 3rd edn., Wiley, NewYork, 1984, p. 208.10 Kuhn, R., Frei, R., and Christen, M., Anal. Biochem., 1994, 218,131.11 Tanaka, Y., Yanagawa, M., and Terabe, S., J. High. Resolut.Chromatogr., 1996, 19, 421.12 Wren, S. A. C., and Rowe, R. C., J. Chromatogr., 1993, 635, 113.Paper 8/01340CReceived February 16, 1998Accepted April 14, 1998Fig. 7 Enantiomerically pure fractions of piperoxan run on CE. The bufferwas 1% SCD adjusted to pH 3 with ≈ 54 mm citric acid.1480 Analyst, July 1998, Vol. 123

Use of capillary electrophoresis as a method development tool for classical gel electrophoresis

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