Enantiomeric separation of a group of chiral dihydropyridines by electrokinetic chromatography

Enan iome ic sepa a ion o a g oup o chi al dihyd opy idines by elec okine ic ch oma og aphy Elec okine ic ch oma og aphy (EKC) was employed o achie e he enan iome ic sepa- a ion o a g oup o chi al 1,4-dihyd opy idines (DHPs) wi h pha macological ac i i y. Micelles o bile sal s alone o mixed wi h neu al cyclodex ins, micelles o sodium dodecyl sul a e (SDS) mixed wi h neu al cyclodex ins, and anionic cyclodex in de i - a i es, i.e., ca boxyme hyl-g-cyclodex in (CM-g-CD), ca boxyme hyl-b-cyclodex in (CM-b-CD), and succinyla ed b-cyclodex in (Succ-b-CD), we e employed as pseudo- s a iona y phases. The enan iome ic sepa a ion abili y o hese chi al selec o s wi h espec o DHPs was s udied in di e en expe imen al condi ions. CM-b-CD was shown o be he bes chi al selec o o pe o m he enan iome ic sepa a ion o DHPs by EKC. Nex , he in luence o he CM-b-CD concen a ion, he pH and na u e o he bu - e , he empe a u e, and he applied ol age on he enan iome ic esolu ion o DHPs was s udied. The use o a 50 mMammonium ace a e bu e , pH 6.7, 25 mMin CM-b- CD oge he wi h an applied ol age o 15 o 20 kV, and a empe a u e o 15 o C enabled he indi idual enan iome ic sepa a ion o wel e DHPs, each one in o i s wo enan io- me s, and hei sepa a ion in mul icomponen mix u es o up o six DHPs in o all hei enan iome s. Keywo ds: Elec okine ic ch oma og aphy / Enan iome ic sepa a ion / 1,4-Dihyd opy idines EL 3869 Ca men Ga cía-Ruiz M. Luisa Ma ina Depa amen o de Química Analí ica, Facul ad de Ciencias, Uni e sidad de Alcalµ, Alcalµ de Hena es (Mad id), Spain 1 In oduc ion Enan iosepa a ion o chi al compounds o pha maceu ical in e es is an impo an ask owing o he di e en pha - macological ac i i y ha can be exhibi ed by each enan- iome . The e o e, excellen chi al esolu ion echniques a e equi ed in o de o moni o he chi al compounds o hei syn he ic p ocesses. Because o i s high sepa a ion e iciency and lexibili y, capilla y elec opho esis (CE) has expe ienced eno mous g ow h in he ield o chi al sepa a ions [1]. I elec ically neu al compounds need o be enan iome i- cally sepa a ed by CE, an elec okine ic ch oma og aphy (EKC) echnique mus be employed. EKC [2] is based on he pa i ioning o he analy es be ween wo phases o di - e en eloci ies ela i e o each o he . One o hese phases is a bu e solu ion, which mig a es wi h he eloc- i y o he elec oosmo ic low (EOF), and he o he is a compound dis ibu ed homogeneously in he bu e solu- ion ha ac s as a pseudos a iona y phase. In o de o ob ain chi al sepa a ions in CE, his compound should be a chi al selec o which can ecognize bo h enan iome s s e eoselec i ely, i.e., wi h di e en binding cons an s [1]. Di e en pseudos a iona y phases we e employed o pe - o m enan iome ic sepa a ions, among hem: na i e cy- clodex ins [3±6], neu al and cha ged cyclodex in de i a- i es [3, 5, 7], bile sal s [4], monome ic and polyme ic chi al su ac an s [8], c own e he s [3], calixa enes [6], p o eins [9], mac ocyclic an ibio ics [10], polysaccha ides [6] o e go alkaloids [6]. To unde s and he mechanisms o chi al sepa a ions in CE, i is impo an o know he o ces which a o complex o ma ion and con ol he s e eoselec i i y in in e molecu- la selec o -selec and in e ac ions. Usually, se e al o ces and mechanisms ac join ly, which makes he iden i ica- ion o he main o ce di icul . The mos popula and bes s udied in his espec a e cyclodex in- ype chi al selec- o s, which o m inclusion complexes wi h se e al com- pounds [1]. The 1,4-dihyd opy idines (DHPs) s udied in his wo k a e Han zsch es e s, which a e o a pha macological in e es owing o hei calcium an agonis ac i i y [11]. Chemically, his amily o compounds con ains he pa e n s uc u e o Co espondence: M. Luisa Ma ina, Depa amen o de Química Analí ica, Facul ad de Ciencias, Uni e sidad de Alcalµ, C a. Ma- d id-Ba celona Km. 33.600, 28871 Alcalµ de Hena es (Mad id), Spain E-mail: [email p o ec ed]s Fax: +34-91-8854971 Abb e ia ions: CM-b-CD, ca boxyme hyl-b-cyclodex in; CM-g- CD, ca boxyme hyl-g-cyclodex in; DHPs, 1,4-dihyd opy idines; HP-b-CD, (2-hyd oxy)p opyl-b-cyclodex in; SC, cholic acid so- dium sal ; SDC, deoxycholic acid sodium sal ; STC, au ocholic acid sodium sal ; STDC, au odeoxycholic acid sodium sal ; Succ-b-CD, succinyla ed-b-cyclodex in. Elec opho esis 2000, 21, 1565±1573 1565  WILEY-VCH Ve lag GmbH, 69451 Weinheim, 2000 0173-0835/00/0808-1565 $17.50+.50/0 CE and CEC a benzene o me hyl subs i u e included in posi ion 4 o he e asubs i u ed DHP ing. These compounds con ain a chi al ca bon a om a his posi ion and ha e been syn- hesized as acemic mix u es o enan iome s. A ew epo s ha e appea ed in he li e a u e in which CE was used o achie e he enan iome ic sepa a ion o some chi- al DHPs. Na i e and subs i u ed cyclodex ins (CDs) we e employed as chi al selec o s in o de o compa e he abili y o high pe o mance liquid ch oma og aphy (HPLC) and CE o pe o m he enan iome ic sepa a ion o i e DHPs (amlodipine, ni edipine, nimodipine, is adipine, and nisoldipine) [12]. In his wo k, he use o ca boxy- me hyl-b-CD (CM-b-CD) in EKC was ound o be he mos sui able selec o . On he o he hand, hyd oxyp opyla ed g-CD appea ed o be he bes chi al selec o o sepa a e, by CE, he wo enan iome s o one DHP when compa ed o o he CD de i a i es [13]. Since i is no ye possible o p edic he success o an enan iome ic sepa a ion on he basis o he chemical s uc u e o a solu e and a chi al selec o , i is o p ima y in e es o compa e he abili y o di e en chi al selec o s o he sepa a ion o a pa icula chi al compound o o compa e he sepa a ion o di e en acemic analy es o a pa icula chi al selec o o unde s and chi al ecogni ion p inciples [14]. Fu he mo e, se e al expe imen al pa am- e e s, such as chi al selec o ype and concen a ion, pH, ionic s eng h and concen a ion o he backg ound elec- oly e, EOF, o ganic modi ie , e c. [15] should be aken in o accoun o ob ain a success ul enan iome ic sepa a- ion by CE. The aim o his s udy was he selec ion o he op imal expe imen al condi ions enabling he enan iome ic sepa- a ion o a g oup o DHPs o pha macological in e es . Se e al chi al selec o s, including cha ged CD de i a- i es, micelles o bile sal s alone o mixed wi h neu al CDs and micelles o sodium dodecyl sul a e (SDS) mixed wi h neu al CDs, we e e alua ed sys ema ically. The in luence o o he expe imen al condi ions (na u e and pH o he bu e , concen a ion o he chi al selec o , empe - a u e, and applied ol age) on he enan iome ic sepa a- ion o DHPs was s udied o ob ain he bes chi al selec- o . 2 Ma e ials and me hods 2.1 Chemicals and samples All eagen s we e o analy ical g ade. 2-(N-cyclohexylami- no)e hanesul onic acid (CHES), au ocholic acid sodium sal (STC) and cholic acid sodium sal (SC) we e pu - chased om Sigma (S . Louis, MO, USA); sodium dihy- d ogen phospha e, SDS, N,N-dime hyl o mamide (DMF), and sodium hyd oxide we e supplied om Me ck (Da m- s ad , Ge many); deoxycholic acid sodium sal (SDC), au odeoxycholic acid sodium sal (STDC), ammonium ace a e, b-CD, g-CD, and u ea we e om Fluka (Buchs, Swi ze land); (2-hyd oxy)p opyl-b-cyclodex in (HP-b- CD), CM-g-CD, CM-b-CD, and succinyla ed b-cyclodex- in (Succ-b-CD) we e ob ained om Cyclolab (Budapes , Hunga y). Wa e used o p epa e solu ions was pu i ied h ough a Milli-Q sys em om Millipo e (Bed o d, MA, USA). All solu ions we e il e ed h ough 45 mm po e size disposable nylon il e s om Scien i ic Resou ces (Ea on- own, NJ, USA). The 18 DHPs s udied we e syn hesized a he Depa men o O ganic Chemis y o he Uni e si y o Alcalµ, Spain. Table 1 shows iden i ica ion numbe s ( aken om [11]) and s uc u es o hese compounds as used h oughou his s udy. 2.2 Appa a us Two CE ins umen s we e used: (i) a P ince model om Laue Labs (Emmen, The Ne he lands), equipped wi h a Lambda 1000 UV de ec o and acquisi ion da a sys em Model S a 4.5 om Va ian Associa es (Suga Land, TX, USA) when bile sal s wi h o wi hou neu al CDs and SDS wi h neu al CDs we e used; and (ii) an HP 3D CE sys em (Hewle -Packa d, Waldb onn, Ge many) equip- ped wi h an on-column diode a ay de ec o (DAD) and HP 3D-CE Chems a ion so wa e when cha ged CD de- i a i es we e used. A 50 mm inne diame e (ID) and 375 mm ou e diame e (OD) used-silica capilla y wi h an e ec i e leng h o 50 cm (65 cm o al leng h o he Laue - Labs ins umen and 58.5 cm o he HP sys em) was employed (Polymic o Technologies, Phoenix, AZ, USA). The capilla y empe a u e was a ied om 15 o 45 o C and UV de ec ion was pe o med a 238 nm [12]. Elec o- ly ic solu ions we e degassed in an ul asonic cleane KM om Raypa (Ba celona, Spain). The pH o he sepa a ion bu e s was adjus ed wi h a 654 pH me e om Me ohm (He isau, Swi ze land). 2.3 P ocedu e When micelles we e used in he sepa a ion bu e , elec- oly ic solu ions we e p epa ed in wo s eps: (i) weighing and dissol ing he app op ia e amoun o bu e (CHES) in Milli-Q wa e o ob ain he equi ed concen a ion and adjus ing he pH o he desi ed alue wi h a concen a ed solu ion o sodium hyd oxide, and (ii) adding he adequa e olume o his bu e solu ion ( o ob ain he desi ed con- cen a ion o he bu e in he inal solu ion) o he app o- p ia e amoun o a bile sal , o a bile sal and a neu al CD, o SDS and a neu al CD, plus u ea and adding Milli- Q wa e o 10 mL. When cha ged CD de i a i es we e 1566 C. Ga cía-Ruiz and M. L. Ma ina Elec opho esis 2000, 21, 1565±1573 used in he sepa a ion bu e , solu ions we e p epa ed by a di e en p ocedu e because he bu e pH was modi ied by adding hese CDs wi h acid p ope ies. The app op i- a e amoun o he bu e and he cha ged CD was weighed and dissol ed in Milli-Q wa e . Then, he pH was adjus ed o he desi ed alue wi h a concen a ed solu ion o sodium hyd oxide o CHES and phospha e bu e s and wi h 1 Mo 0.1 MNH 3 solu ion o he ammonium ace- a e bu e . Sample solu ions we e p epa ed by dissol ing each DHP in DMF. In o de o ob ain good peak shapes and ep oducible e en ion da a, he capilla y was condi- ioned a he beginning o he day p io o each analysis, and a he end o he day. The capilla y was insed a he beginning o he day wi h Milli-Q wa e o 2 min, 0.1 M sodium hyd oxide o 2 min, and Milli-Q wa e o 2 min. Be ween injec ions, he washing ou ine consis ed o Milli- Q wa e o 2 min, 0.1 Msodium hyd oxide o 2 min, Milli- Q wa e o 2 min, and elec oly ic solu ion o 2 min p io o injec ion. A he end o he day, he capilla y was insed wi h Milli-Q wa e o 2 min, sodium hyd oxide o 2 min, Milli-Q wa e o 2 min, and hen s o ed in wa e . The injec ion was made by p essu e: 10 o 5 mba o 0.02 min in he Laue labs ins umen and 30 mba o 2 s in he HP CE sys em. The applied ol age anged om 15 o 25 kV. Enan iome ic esolu ion was calcula ed by he ol- lowing equa ion: R s = 1.18 ( 2 - 1 )/(w 1 +w 2 ) whe e 1 and 2 a e he mig a ion imes o he wo enan- iome s and w 1 ,w 2 co espond o he middle high wid hs o he co esponding peaks. Elec opho esis 2000, 21, 1565±1573 Enan iome ic sepa a ion o 1,4-dihyd opy idines 1567 Table 1. Iden i ica ion numbe s and s uc u es o DHPs s udied G oup 1 -R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 1 -COOCH 2 -(*1) -COOCH 3 6 -COOCH 2 CH 2 OCH 3 -COOlsp 7 -COOlsp -COOCH 2 CH 3 12 -COOCH 2 CH 3 -COOCH 3 17 -COOlsp -COOCH 3 G oup 2 4 -COOCH 2 CH 3 -COOCH 3 G oup 3 8 -COOCH 2 CH 3 -COOCH 3 -H -NO 2 -H -H -H 9 -COOCH 2 CH 3 -COOCH 3 -Cl -Cl -H -H -H 10 -COOCH 2 CH 3 -COOCH 3 -OCH 2 OCH 3 -H -H -H -H 11 -COOCH 2 CH 3 -COOCH 3 -H -OCH 3 -H -OCH 3 -H 13 -COOCH 2 CH 3 -COOCH 3 -H -H -H -H -H 15 -COOCH 2 CH 3 -COOCH 3 -H -OCH 3 -OCH 3 -H -H 16 -COOCH 2 CH 3 -COOCH 3 -OCH 3 -OCH 3 -H -H -H 18 -COOCH 2 CH 3 -COOCH 3 -H -OCH 3 -OH -OCH 3 -H 19 -COOCH 2 CH 3 -COOCH 3 -H -OCH 3 -OCH 3 -OCH 3 -H 25 -COOlsp -COOCH 2 CH 2 OCH 3 -H -NO 2 -H -H -H G oup 4 22 -COOCH 3 -COOCH 2 CH 3 -H 23 -COOCH 2 CH 2 -(*2) -COOCH 2 CH 3 -H Isp, isop opyl 3 Resul s and discussion Eigh een chi al DHPs we e injec ed in a CE sys em using di e en chi al selec o s in he sepa a ion bu e . Bile sal s alone o mixed wi h neu al CDs o mix u es o SDS and neu al CDs we e used in micella EKC (MEKC) and cha ged CD de i a i es we e employed in EKC. The na u e, concen a ion, and pH o he bu e , as well as he empe a u e, applied ol age, and concen a ion o he chi al selec o we e a ied. All condi ions used in his s udy o he enan iome ic sepa a ion o he compounds s udied a e summa ized in Table 2. 3.1 MEKC The eigh een DHPs we e injec ed in an MEKC sys em in which a 100 mMCHES bu e (pH 9), 100 mMin SDS, 2 M u ea and 50 mMin b-CD o g-CD was used. Unde hese condi ions, no chi al ecogni ion was obse ed o DHPs. Since mix u es o CDs may o igina e changes in selec i - i y, a mix u e o 50 mMin b-CD and 50 mMin g-CD was also used unde he same expe imen al condi ions de- sc ibed be o e bu no enan iome ic esolu ion (o any o he compounds s udied) was obse ed. These esul s could be explained acco ding o he hypo hesis o he compe i i e in e ac ion o DHPs and su ac an molecules wi h he CD ca i y p oposed by Gila e al. [12]. Fou bile sal s, SC, STC, SDC, and STDC we e employed nex . Since hese micella sys ems enable chi al ecogni- ion, he addi ion o a chi al selec o o he sepa a ion bu - e is no needed al hough i can be used in o de o enhance enan ioselec i i y. Fi s , bile sal s we e used alone in a 100 mMCHES bu e (pH 9) modi ied wi h 2 M u ea. A 50 mMconcen a ion o bile sal enabled he pa - ial enan iome ic esolu ion o DHPs 17 and 25 in he case o SC, and DHP 17 in he case o STC (see Table 2). Howe e , a his concen a ion, no chi al ecogni ion was 1568 C. Ga cía-Ruiz and M. L. Ma ina Elec opho esis 2000, 21, 1565±1573 Table 2. Expe imen al condi ions o he enan iome ic sepa a ion o DHPs s udied Bu e Addi i es Chi al selec o Cu en DHPs enan iome ically in ensi y (mA) sepa a ed 50 m M b-CD &18 a) None 100 m M CHES 100 m M SDS + 50 m M g-CD &19 a) None (pH 9) 2 M u ea 50 m M b-CD + 50 m M g-CD &21 a) None 100 m M CHES 2 M u ea 50 m M SC &22 b) 17, 25 (pH 9) 100 m M SC &36 b) 17 150 m M SC &54 b) 17 50 m M STC &20 b) 17 50 m M SDC &28 b) None 50 m M STDC &23 b) None 100 m M SC + 25 m M b-CD &32 b) 17 100 m M SC + 50 m M b-CD &31 b) None 20 m M CM-g-CD (s.d. c) ~3.2) &52 b) 9, 22 100 m M phospha e (pH 6) ± 20 m M CM-g-CD (s.d. ~3.2) &140 b) 8 50 m M phospha e (pH 6) ± 20 m M CM-b-CD (s.d. ~3) &64 b) 8, 12, 16, 22 50 m M phospha e (pH 6) ± 10 m M CM-b-CD (s.d. ~3) + 10 m M CM-g-CD (s.d. ~3.2) &80 b) 8 50 m M phospha e (pH 6) ± 20 m M CM-b-CD (s.d. ~3) + 20 m M HP-b-CD (s.d. ~3) &64 b) 9 50 m M CHES (pH 9) ± 10 m M CM-b-CD (s.d. ~3) + 10 m M CM-g-CD (s.d. ~3.2) &48 b) 25 50 m M CHES (pH 9) ± 18 m M CM-b-CD (s.d. ~3) + 20 m M CM-g-CD (s.d. ~3.2) &130 b) 8, 16, 22, 25 50 m M CHES (pH 9) ± 10 m M CM-b-CD (s.d. ~3) &30 b) 1, 12, 17, 19, 23, 25 50 m M CHES (pH 7.9) ± 10 m M CM-b-CD (s.d. ~3) &30 b) 1, 12, 17, 19, 23, 25 50 m M CHES (pH 6.7) ± 10 m M CM-b-CD (s.d. ~3) &30 b) 12, 17, 19, 23 50 m M ammonium ace a e 10 m M CM-b-CD (s.d. ~3) &60 b) 1, 8, 12, 17, 19, (pH 6.7) ± 22, 23, 25 10 m M CM-g-CD (s.d. ~3.2) &80 b) 10, 22 10 m M CM-b-CD (s.d. ~3) + 10 m M CM-g-CD (s.d. ~3.2) &90 b) 17, 19, 22, 25 10 m M Succ-b-CD (s.d. ~3.5) &55 b) 19 a) 15 kV b) 20 kV c) s.d., subs i u ion deg ee ob ained when SDC and STDC we e used in he sepa a- ion bu e . Addi ionally, hese wo bile sal s showed wo se peak e iciencies and longe analysis imes han SC and STC. Since SC seemed he bes chi al micelle o achie e he enan iome ic esolu ion o DHPs, o he con- cen a ions o his bile sal in he sepa a ion bu e we e ied. Howe e , concen a ions o 100 mMand 150 mM enabled only he pa ial esolu ion o DHP 17. In o de o check i he addi ion o na i e b-CD o SC could imp o e he enan iome ic sepa a ions ob ained wi h SC, mix u es o 100 mMSC and 25 o 50 mMb-CD we e employed o injec ing all he DHPs s udied in he MEKC sys em. Unde hese condi ions, only DHP 17 was pa ially sepa a ed in o i s wo enan iome s, dec easing he esolu ion wi h he addi ion o inc easing amoun s o b-CD. 3.2 EKC wi h cha ged CDs Th ee anionic CD de i a i es (CM-b-CD, CM-g-CD and Succ-b-CD) we e used unde di e en expe imen al con- di ions in o de o choose he bes chi al selec o o achie e he enan iome ic sepa a ion o DHPs. The ollow- ing expe imen al condi ions we e a ied: he na u e and concen a ion o he bu e and i s pH, and he concen a- ion o he chi al selec o ; o esul s, see Table 2. When 50 o 100 mMphospha e bu e s a pH 6 we e used, a concen a ion o 20 mMCM-b-CD enabled he sepa a ion o a highe numbe o DHPs han CM-g-CD alone a he same concen a ion, o CM-b-CD mixed wi h CM-g-CD o wi h a neu al CD de i a i e (HP-b-CD). In ac , in he la - e h ee cases only one DHP was enan iome ically sep- a a ed (DHP 8 o 9) while in he o me (20 mMCM-b-CD alone) ou DHPs (numbe s 8, 12, 16, and 22) we e enan- iome ically sepa a ed, each one in i s wo enan iome s. Then, he use o a mix u e o CM-b-CD and CM-g-CD dec eased he numbe o DHPs enan iome ically sep- a a ed (only DHP 8) wi h espec o he use o CM-b-CD alone, whe eas he mix u e o CM-b-CD and HP-b-CD, al hough also dec easing he numbe o DHPs sepa a ed, enabled he enan iome ic sepa a ion o a new DHP (num- be 9) which could no be sepa a ed unde any o he o he condi ions ied wi h phospha e bu e . When 100 mMo 50 mMCHES bu e s we e used a pH 9, CM-g-CD a a concen a ion o 20 mMand modi ied wi h 2Mu ea enabled he enan iome ic sepa a ion o only wo DHPs (numbe s 9 and 22) while a concen a ion o 10 mM Elec opho esis 2000, 21, 1565±1573 Enan iome ic sepa a ion o 1,4-dihyd opy idines 1569 Figu e 1. Va ia ion o he enan iome ic esolu ion o wel e DHPs as a unc ion o he CM-b-CD concen a ion. Expe imen al condi ions: 50 mMammonium ace a e (pH 6.7); applied ol age, 20 kV; UV de ec ion, 238 nm; em- pe a u e, 25 o C; injec ion by p essu e, 30 mba o 2 s; capilla y, 50 mm ID, 375 mm OD, 58.5 cm leng h (50 cm o he de ec o ). Figu e 2. Va ia ion o he enan iome ic esolu ion o wel e DHPs as a unc ion o he empe a u e. Expe i- men al condi ions as in Fig. 1 bu wi h a ying empe a- u e; 25 mMCM-b-CD. CM-b-CD enabled he sepa a ion o six DHPs (numbe s 1, 12, 17, 19, 23, and 25). Howe e , he numbe o DHPs enan iome ically sepa a ed wi h CM-b-CD dec eased when 10 mMCM-g-CD was added o 10 mMCM-b-CD (one DHP sepa a ed) o when 20 mMCM-g-CD was added o 18 mMCM-b-CD ( ou DHP enan iome ically sepa a ed). In addi ion, an in e es ing change in selec i - i y occu s when using mix u es o he anionic CD de i a- i es. In ac , DHPs 8 and 16 we e enan iome ically sep- a a ed wi h a mix u e o 18 mMCM-b-CD and 20 mMCM- g-CD and hey could no be sepa a ed wi h any o he chi- al selec o s alone o wi h he o he mix u e in es iga ed (10 mMCM-b-CD and 10 mMCM-g-CD). On he o he hand, when 10 mMCM-b-CD was used as chi al selec o wi h 50 mMCHES bu e s a pH 7.9 and 6.7, no changes in he esul s we e obse ed a pH 7.9 wi h espec o pH 9 and a dec ease in he numbe o DHPs enan iome ically sepa a ed was ob ained a pH 6.7 (DHPs 12, 17, 19, and 23). Al hough in he case o CHES bu e , a pH alue o 6.7 ga e poo e esul s han a highe pH, Table 2 shows ha when 50 mMammonium ace a e (pH 6.7) was used, a concen a ion o 10 mMCM-b-CD enabled he enan io- me ic sepa a ion o a highe numbe o DHPs (eigh DHPs numbe s: 1, 8, 12, 17, 19, 22, 23, and 25). Howe e , he use o he o he wo anionic CD de i a i es, CM-g-CD and Succ-b-CD a he same concen a ion (10 mM) only allowed he chi al sepa a ion o wo o one DHPs, espec- i ely. While he DHP sepa a ed wi h Succ-b-CD (numbe 19) was also sepa a ed wi h CM-b-CD, only one o he wo DHPs sepa a ed wi h CM-g-CD (DHP 22) was also sepa a ed wi h CM-b-CD, enabling he use o CM-g-CD o he chi al sepa a ion o DHP 10, which could no be sepa a ed when 10 mMCM-b-CD was used. Finally, he mix u e o 10 mMCM-b-CD and 10 mMCM-g-CD enabled he enan iome ic sepa a ion o ou DHPs ha had also been sepa a ed when 10 mMCM-b-CD alone was em- ployed in he sepa a ion bu e . The esul s p esen ed in Table 2 show ha he use o a 50 mMammonium ace a e bu e (pH 6.7) wi h CM-b-CD alone as chi al selec o seemed o be he bes condi ion 1570 C. Ga cía-Ruiz and M. L. Ma ina Elec opho esis 2000, 21, 1565±1573 Figu e 3. Va ia ion o he enan iome ic esolu ion o wel e DHPs as a unc ion o he applied ol age. Expe i- men al condi ions as in Fig. 2; empe a u e, 15 o C; a ying ol ages. Figu e 4. Enan iome ic sepa a ion o DHPs 12 and 15 by EKC. Expe imen al condi ions as in Fig. 3; applied ol age, 15 kV. o pe o m he enan iome ic sepa a ion o he g oup o DHPs s udied. Nex , he in luence o se e al expe imen al pa ame e s such as he concen a ion o he chi al selec- o , he empe a u e, and he applied ol age on he enan- iome ic sepa a ion o he DHPs was in es iga ed unde he abo e-men ioned condi ions. Figu e 1 shows he a ia ion o he enan iome ic esolu- ion ob ained o a g oup o DHPs as a unc ion o he CM-b-CD concen a ion in a 50 mMammonium ace a e bu e (pH 6.7). DHPs ha we e no enan iome ically sep- a a ed a any o he concen a ions o CM-b-CD employed ha e no been included in his igu e. When he concen- a ion o chi al selec o was inc eased om 5 o 25 mM, an inc ease in he numbe o DHPs enan iome ically sep- a a ed, as well as in he enan iome ic esolu ion, was gen- e ally ob ained. Howe e , he e a e some DHPs o which a maximum in esolu ion was obse ed a an in e media e concen a ion o CM-b-CD. Fo example, he enan io- me ic esolu ion o DHPs 23 and 25 was g ea e a 10 mMCM-b-CD han a a highe concen a ion. Al hough he inc ease in he CM-b-CD concen a ion in he sepa a ion bu e up o 25 mMenabled he chi al ec- ogni ion o wel e DHPs, o some o hem only a pa ial esolu ion could be ob ained (see Fig. 1). Fo his eason, and wi h he aim o inc easing he enan iome ic esolu ion o he compounds s udied, he e ec o he empe a u e was in es iga ed. The a ia ion o he enan iome ic eso- lu ion o he wel e DHPs chi ally sepa a ed a 25 mMCM- b-CD as a unc ion o he empe a u e is shown in Fig. 2. An inc ease in he enan iome ic esolu ion was ob ained o mos o he DHPs s udied when he empe a u e was dec eased. This ac could be due o he inc ease in he s abili y o selec o -selec and complexes ha akes place when he empe a u e is dec eased [1]. F om hese esul s, i can be concluded ha he use o a 50 mM ammonium ace a e bu e (pH 6.7), 25 mMin CM-b-CD a a empe a u e o 15 o C, enabled he enan iome ic esolu- ion o wel e DHPs al hough some o hem we e only pa ially esol ed. Since all he expe imen s desc ibed abo e we e ca ied ou a a cons an applied ol age o 20 kV, he in luence o his a iable on he enan iome ic esolu ion o he wel e Elec opho esis 2000, 21, 1565±1573 Enan iome ic sepa a ion o 1,4-dihyd opy idines 1571 Figu e 5. Enan iome ic sepa a ion o DHPs 7, 8, 10, and 16 by EKC. Expe imen al condi ions as in Fig. 4; applied ol age, 20 kV. DHPs sepa a ed was also in es iga ed in o de o see i a a ia ion o he applied ol age could enhance he chi al sepa a ion o hose DHPs ha had been only pa ially e- sol ed up o ha momen . Figu e 3 shows he a ia ion o he enan iome ic esolu ion o wel e DHPs as a unc ion o he applied ol age, which was a ied om 15 o 25 kV. In hese expe imen s, a 50 mMammonium ace a e bu e , pH 6.7, 25 mMCM-b-CD a 15 o C, was used. Fo mos DHPs a maximum in he enan iome ic esolu ion was ob- ained a an applied ol age o 20 kV. Fo DHPs 12 and 15, howe e , he con a y was obse ed, ha is, a mini- mum in he enan iome ic esolu ion was ob ained a 20 kV, 15 kV being he ol age ha enabled he bes chi- al sepa a ion o hese compounds. Figu e 4 shows he enan iome ic sepa a ion o DHPs 12 and 15 using a 50 mMammonium ace a e bu e , pH 6.7, 25 mMCM-b- CD a 15 o C and an applied ol age o 15 kV. Fo he emaining DHPs, he enan iome ic sepa a ion was pe - o med unde he same condi ions bu a an applied ol - age o 20 kV. Al hough unde hese condi ions some DHPs we e no baseline esol ed, as shown in Fig. 5, o he DHPs could be enan iome ically sepa a ed in mul i- componen mix u es as shown in Fig. 6. 4 Concluding ema ks An adequa e selec ion o chi al addi i e o he sepa a ion bu e was a c ucial ac o o achie e he enan iome ic sepa a ion o DHP de i a i es. The use o cha ged (anionic) CD de i a i es p esen ed he bes enan ioselec- i i y wi h espec o he compounds s udied. The esul s show ha DHPs can be enan iome ically be e sepa a ed wi h CM-b-CD han wi h CM-g-CD o Succ-b-CD, pe haps due o he mo e s able inclusion complexes o med be- ween DHPs and his CD. The use o mix u es o CD de- i a i es did no enable he enhancemen o he enan io- me ic esolu ion al hough in e es ing changes in selec i - i y we e obse ed wi h espec o he use o each CD de- i a i e alone in he sepa a ion bu e . In addi ion, he enan iome ic esolu ion ob ained wi h he bes chi al selec o o DHPs (CM-b-CD) was highly in luenced by i s concen a ion in he sepa a ion bu e , he bu e na u e and pH, he empe a u e, and he applied ol age. Fo mos DHPs, an inc ease in he enan iome ic esolu ion was ob ained upon inc easing he CM-b-CD concen a- ion in he sepa a ion bu e and dec easing he empe a- u e, acco ding o he dec ease in he s abili y o selec o - selec and complexes when inc easing he empe a u e. The use o a 50 mMammonium ace a e bu e , pH 6.7, 25 mMCM-b-CD a a empe a u e o 15 o C and an applied ol age o 15 o 20 kV enabled he enan iome ic esolu- ion o wel e o he DHPs s udied as well as hei enan io- me ic sepa a ion in mul icomponen mix u es o up o six DHPs in o all hei enan iome s. The au ho s hank he Comisión In e minis e ial de Cien- cia y Tecnologia (Spain) o p ojec PB98-0709 and D . Al a ez-Builla (Uni e sidad de Alcalµ, Spain) o he kind gi o DHPs used in his wo k. Recei ed Oc obe 1, 1999 5 Re e ences [1] Chank e adze, B., Capilla y Elec opho esis in Chi al Analy- sis, John Wiley & Sons, Chiches e 1997. [2] Te abe, S., O suka, K., Nishi, H., J. Ch oma og . 1994, 666, 295±319. [3] Nishi, H., Te abe, S., J. Ch oma og . A 1995, 694, 245±276. 1572 C. Ga cía-Ruiz and M. L. Ma ina Elec opho esis 2000, 21, 1565±1573 Figu e 6. Sepa a ion o a mix- u e o six chi al DHPs in o hei wel e enan iome s by EKC. Expe imen al condi ions as in Fig. 5. A, unknown peak. [4] Nishi, H., J. Ch oma og . A 1996, 735, 57±76. [5] Vespalec, R., Boc Æek, P., Elec opho esis 1997, 18, 843±852. [6] Gübi z, G., Schmid, M. G., J. Ch oma og . A 1997, 792, 179±225. [7] Lu ie, I. S., J. Ch oma og . A 1997, 792, 297±307. [8] Shamsi, S. A., Wa ne , I. M., Elec opho esis 1997, 18, 853±872. [9] Lloyd, D. K., Aub y, A. F., Lo enzi, E. D., J. Ch oma og . A 1997, 792, 349±369. [10] A ms ong, D. W., Nai , U. B., Elec opho esis 1997, 18, 2331±2342. [11] Beni o, I., Ga cía, M. 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