Separation of a group of N-phenylpyrazole derivatives by micellar electrokinetic chromatography: application to the determination of solute-micelle association constants and estimation of the hydrophobicity

Sepa a ion o a g oup o N-phenylpy azole de i a i es by micella elec okine ic ch oma og aphy: Applica ion o he de e mina ion o solu e-micelle associa ion cons an s and es ima ion o he hyd ophobici y Micella elec okine ic ch oma og aphy (MEKC) was applied o he sepa a ion o a g oup o N-phenylpy azole de i a i es. Sodium dodecyl sul a e (SDS) as micella sys- em and 2-(N-cyclohexylamino)e hanesul onic acid (CHES) as sepa a ion bu e (pH 10) we e employed in he absence and p esence o di e en pe cen ages o medium chain alcohols (n-p opanol o n-bu anol). The sepa a ion o mul icomponen mix u es o he solu es s udied enabled he apid de e mina ion o hei e en ion ac o s which, in u n, allowed he s udy o he sepa a ion selec i i y o compounds and he de e mi- na ion o hei solu e-micelle associa ion cons an s ( om he linea a ia ion o he e en ion ac o s as a unc ion o he o al su ac an concen a ion in he sepa a ion bu e ). Sepa a ion selec i i y was s udied acco ding o he elu ion ange and numbe o solu es sepa a ed in all he elec oly e solu ions employed (45 micella phases). The e ec o he bu e concen a ion (0.05, 0.08 and 0.10 M), he alcohol na u e (n-p opa- nol o n-bu anol) and he alcohol pe cen age (1, 3 o 5%) o he alues ob ained o he solu e-micelle associa ion cons an s was also s udied. The bes sepa a ion (12 sol- u es) was pe o med when a 0.08 MCHES bu e , pH 10, 0.02 MSDS modi ied by 5% n-bu anol was used. The possibili ies o using MEKC o e alua ing he hyd ophobici y o compounds was in es iga ed h ough he s udy o he co ela ion be ween he loga- i hm o he e en ion ac o s o N-phenylpy azole de i a i es and hei loga i hm o he oc anol-wa e dis ibu ion coe icien s es ima ed by high pe o mance liquid ch oma og- aphy (HPLC). Keywo ds: Micella elec okine ic ch oma og aphy / N-phenylpy azole de i a i es / Sepa a ion selec i i y / Solu e-micelle associa ion cons an s / Hyd ophobici y es ima ion EL 3935 Ca men Ga cía-Ruiz M. Angeles Ga cía M. Luisa Ma ina 1 Depa amen o de Química Analí ica, Facul ad de Química, Uni e sidad de Alcalµ, Alcalµ de Hena es (Mad id), Spain 1 In oduc ion In micella elec okine ic ch oma og aphy (MEKC), a su - ac an a a concen a ion abo e i s c i ical micella con- cen a ion (CMC) is added o he sepa a ion bu e o a capilla y elec opho esis sys em [1±3]. Wi h his ech- nique, solu es a e dis ibu ed be ween he micelle and he su ounding nonmicella phase acco ding o hei solu e- micelle associa ion cons an s, and only one pa i ion equilib ium (wa e -micelle) exis s. Fo his eason, an accu a e knowledge o solu e-micelle associa ion con- s an s is essen ial o a be e unde s anding o he sepa- a ion mechanism and, consequen ly, p edic ing he e en ion beha io o compounds [4±6]. I he micella concen a ion in he bu e is low (close o CMC) [2], he solu e e en ion ac o (k) in MEKC can be ela ed o he o al su ac an concen a ion in he bu e using he ollowing equa ion [2, 5]: k=(K 2 +n)(C- CMC) (1) whe e K 2 is he solu e-micelle associa ion cons an pe su ac an monome , nis he mola olume o he micelle and Cis he o al su ac an concen a ion in he bu e . Equa ion (1) allows he calcula ion o solu e-micelle asso- cia ion cons an s o he solu es om he slope o he s aigh line ob ained o he a ia ion o he e en ion ac- o as a unc ion o he o al su ac an concen a ion in Co espondence: D . M. Luisa Ma ina, Depa amen o de Quími- ca Analí ica, Facul ad de Química, Uni e sidad de Alcalµ, 28871 Alcalµ de Hena es (Mad id), Spain E-mail: [email p o ec ed]s Fax: +39-91-8854971 2424 Elec opho esis 2000, 21, 2424±2431  WILEY-VCH Ve lag GmbH, 69451 Weinheim, 2000 0173-0835/00/1212-2424 $17.50+.50/0 he sepa a ion bu e . Thus, a dec ease in he e o ob- ained in de e mining associa ion cons an s can be expec ed wi h espec o o he echniques such as micel- la liquid ch oma og aphy (MLC) [7±11]. In MEKC, all neu al solu es mig a e be ween he elec o- osmo ic low ( o ) and he micella ( m ) mig a ion imes. F om he pa ame e s o and m he e en ion ac o (k)o a neu al solu e can be calcula ed as ollows [1]: k=( - o )/ o [1 - ( / m )] (2) whe e is he mig a ion ime o he neu al solu e. I he mig a ion ime o he solu e is simila o o o m , he e o in he de e mina ion o he e en ion ac o inc eases exponen ially [4]; hen, he a ia ion o k o e y pola o e y hyd ophobic compounds as a unc ion o he su ac- an concen a ion (Eq. 1) could no be a s aigh line and i would no be possible o calcula e solu e-micelle associ- a ion cons an s [4]. Howe e , o o he solu es (which a e nei he e y pola no e y hyd ophobic), MEKC can ha e a g ea po en ial in de e mining solu e-micelle associa ion cons an s due o he high e iciency and esolu ion o his echnique. These cha ac e is ics make possible he apid de e mina ion o he e en ion ac o s o g oups o solu es by means o he con ol o he sepa a ion selec i i y which can be modi ied by a g ea numbe o ac o s such as su - ac an na u e, bu e pH, empe a u e, and addi ion o o ganic sol en s o o he addi i es. O ganic sol en s a e use ul addi i es since hey can ex en he elu ion ange ( m / o ) o imp o e he esolu ion o hyd ophobic com- pounds [12, 13]. On he o he hand, hyd ophobici y es ima ion is o g ea impo ance in di e en disciplines, such as d ug design and oxicology [14±16]. In his sense, MEKC has been u ilized o he indi ec es ima ion o he hyd ophobici y o se e al g oups o compounds [6, 18±22]. I solu es expe- ience a simila pa i ioning in micelle-wa e and oc anol- wa e sys ems, an app oxima ely linea ela ionship be- ween he loga i hm o he e en ion ac o (log k) and he loga i hm o he oc anol-wa e dis ibu ion coe icien (log P ow ) can be ob ained [6, 17]. In his s udy, a g oup o N-phenylpy azole de i a i es wi h a pha macological in e es has been sepa a ed in a MEKC sys em using a 2-(N-cyclohexylamino)e hanesul- onic acid (CHES) bu e (pH 10) wi h sodium dodecyl sul- a e (SDS) micelles wi h and wi hou he addi ion o alco- hols such as n-p opanol o n-bu anol. Py azoles and hei de i a i es ha e impo an applica ions in ag icul u al and medical ields [23]. In pa icula , hey a e o pha macologi- cal in e es owing o hei capaci y o ac as an i umo al agen s [24] o o be used o he ea men o leukemia, heuma oid a h i is, pso iasis, o epilepsy [25, 26]. De e - mina ion o he e en ion ac o s o hese compounds unde di e en expe imen al condi ions has enabled cal- cula ion o he solu e-micelle associa ion cons an s and he s udy o he possibili y o MEKC in e alua ing hei hyd ophobici y. 2 Ma e ials and me hods 2.1 Chemicals and samples All eagen s employed we e o analy ical g ade. CHES was pu chased om Sigma (S . Louis, MO, USA); SDS, dime hyl o mamide (DMF), benzo(a)py ene (BaP), and n- bu anol we e om Me ck (Da ms ad , Ge many); n-p opa- nol was ob ained om Scha lau (Ba celona, Spain) and sodium hyd oxide was om Pan eac (Ba celona, Spain). All solu ions we e p epa ed wi h HPLC-g ade wa e (Milli- Q sys em; Millipo e, Bed o d, MA, USA). The 26 N-phe- nylpy azole de i a i es 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, names, and s uc u es o hese compounds. 2.2 Appa a us A capilla y elec opho esis ins umen 279A-HT model om Applied Biosys ems (No walk, CT, USA), equipped wi h a UV de ec o , a empe a u e con olled capilla y compa men , and an au osample was used. Da a ea - men was pe o med wi h a Tu boch om acquisi ion sys- em (Pe kin Elme , No walk, CT, USA). Capilla y empe - a u e was se a 30 o C and UV de ec ion a 238 nm was employed. A used-silica capilla y (25 mm ID; 375 mmOD) om Polymic o Technologies (Phoenix, AZ, USA) was employed. The o al leng h was 75 cm and he e ec i e leng h was 50 cm. 2.3 P ocedu e Sepa a ion bu e s we e p epa ed by weighing and dis- sol ing he app op ia e amoun s o CHES and su ac an (SDS) in HPLC-g ade wa e . Nex , he pH was adjus ed o he selec ed alue (pH 10) wi h a concen a ed solu ion o sodium hyd oxide. These solu ions we e il e ed and degassed p io o hei in oduc ion in o he capilla y and he bu e ese oi s. Sample solu ions we e p epa ed by dissol ing 5 mL o each N-phenylpy azole de i a i e (p e- iously dissol ed in DMF a an app oxima e concen a ion o 10 mg/mL) in 0.75 mL o sepa a ion media ( inal con- cen a ion, app oxima ely 0.07 mg/mL). All N-phenylpy a- zole de i a i es analyzed we e injec ed as mix u es con- Elec opho esis 2000, 21, 2424±2431 MEKC o N-phenylpy azole de i a i es 2425 CE and CEC aining he maximum numbe o solu es ha could be sep- a a ed in each o he measu ing condi ions. Peaks o sol- u es in he mix u es we e iden i ied by compa ing hei mig a ion imes wi h hose o indi idual s anda ds injec ed unde he same condi ions. DMF and benzo(a)py ene we e used as elec oosmo ic low and micelle mig a ion ma ke s, espec i ely. Bu e s con aining 0.05, 0.08, and 0.10 MCHES wi hou alcohols and 0.08 MCHES modi ied by 1, 3, o 5% n-p opanol o n-bu anol we e used. All CHES bu e s, pH 10, we e adjus ed wi h 0.1 Msodium hyd oxide. SDS concen a ion in he bu e s anged om 0.01 o 0.05 M( i e SDS concen a ions o each bu e , 0.01, 0.02, 0.03, 0.04, and 0.05 M). A he beginning o he day, he capilla y was insed wi h HPLC-g ade wa e (5 min), ollowed by 0.1 Msodium hyd oxide (5 min), and inally wi h HPLC-g ade wa e (5 min). Typically, analyses we e pe o med au oma ically using a un sequence ha included he ollowing s eps: (i) 2 min inse wi h HPLC- g ade wa e ; (ii) 2 min inse wi h 0.1 Msodium hyd oxide; (iii) 2 min inse wi h HPLC-g ade wa e ; (i ) 4 min inse wi h he sepa a ion bu e , in a sepa a e inle ial; ( ) hyd odynamic sample injec ion (67.73 kPa o 1 s) om he sample ial; and ( i) sample sepa a ion un du - ing he necessa y ime ( ol age applied was 15 kV). 2.4 Da a ea men A alue o 0.246 Lmol ±1 was aken as mola olume o SDS. I was assumed ha his alue did no a y app eci- ably unde he expe imen al condi ions used in his s udy [27]. The e o in de e mining solu e-micelle associa ion cons an s was asce ained om he s a is ical pa ame e s o leas -squa es i ing and om e o p opaga ion [28]. Nonpa ame ic es s used in his wo k o compa e associ- a ion cons an alues ob ained unde di e en expe imen- al condi ions we e he Wilcoxon ma ched pai es and he Kolmogo o -Smi no wo-samples es , o a signi i- can le el o 95%. They we e ca ied ou by using he S a g aphics plus p og am [29]. 3 Resul s and discussion The N-phenylpy azole de i a i es s udied we e injec ed in a MEKC sys em using as sepa a ion media CHES bu e s a pH 10 con aining SDS micelles. CHES bu e a pH 10 o igina ed an elec oosmo ic low s ong enough o allow he elu ion o hyd ophobic compounds [4]. Concen a ions o CHES and SDS in he sepa a ion bu e we e a ied om 0.05 o 0.10 M( h ee di e en concen a ions, 0.05, 0.08, and 0.10 M) and om 0.01 o 0.05 M( i e di e en 2426 C. Ga cía-Ruiz, M. A. Ga cía and M. L. Ma ina Elec opho esis 2000, 21, 2424±2431 Table 1. Iden i ica ion numbe s, names and s uc u es o he N-phenylpy azole de i a i es s udied No. Name R 3 R 4 R 5 R ©2 S uc u e 1 DNPP H H H NO 2 2 3-Me hyl DNPP Me H H NO 2 3 4-Me hyl DNPP H Me H NO 2 4 4,5-Dime hyl DNPP H Me Me NO 2 5 3-E hyl DNPP E H H NO 2 6 4,5-Dime hyl pNPP H Me Me H 7 3,4,5-T ime hyl DNPP Me Me Me NO 2 8 4-Me hyl pNPP H Me H H 9 3-Me hyl-4-ni o-5-chlo o DNPP Me NO 2 Cl NO 2 10 3,5-Dime hyl pNPP Me H Me H 11 4-B omo pNPP H B H H 12 3-B omo-4-me hyl DNPP B Me H NO 2 13 3,5-Dime hyl-4-b omo DNPP Me B Me NO 2 14 5-Me hyl-4-b omo pNPP H B Me H 15 3- e -Bu yl pNPP -Bu H H H 16 3,4-Dib omo DNPP B B H NO 2 17 3-E hyl-4-b omo DNPP E B H NO 2 18 4-Me hyl-5-phenyl DNPP H Me Ph NO 2 19 3-Phenyl-4-b omo DNPP Ph B H NO 2 20 3,5-Di- e -bu yl-4-me hyl DNPP -Bu Me -Bu NO 2 21 3,4-Dime hyl-5-phenyl DNPP Me Me Ph NO 2 22 3,5-Dime hyl-4- e -bu yl DNPP Me -Bu Me NO 2 23 4-B omo-5-me hyl DNPP H B Me NO 2 24 3-Phenyl-4-b omo-5-me hyl DNPP Ph B Me NO 2 25 3-Me hyl-4-b omo-5-phenyl DNPP Me B Ph NO 2 26 3,5-Diphenyl DNPP Ph H Ph NO 2 DNPP: dini o phenylpy azole pNPP: pa ani o phenylpy azole Me: Me hyl E : E hyl -Bu: e -Bu yl Ph: Phenyl © concen a ions, 0.01, 0.02, 0.03, 0.04, and 0.05 M), espec i ely. n-P opanol and n-bu anol we e added o 0.08 MCHES bu e s a pe cen ages anging om 1 o 5%. Re en ion ac o s we e de e mined om mig a ion imes o solu es and mig a ion imes o elec oosmo ic low and micelle ma ke s. Mul icomponen mix u es o he compounds s udied we e employed in o de o dec ease he ime equi ed o de e mine e en ion ac o s. F om he esul s ob ained, sepa a ion selec i i y could be in es i- ga ed, solu e-micelle associa ion cons an s could be de e mined, and he possibili ies o MEKC echniques o hyd ophobici y es ima ion we e e alua ed. 3.1 Va ia ion o he sepa a ion selec i i y wi h bu e , SDS, and alcohol concen a ions in he elec oly ic solu ion Sepa a ion selec i i y was s udied on he basis o he elu- ion ange ( m / o ) ob ained o each bu e employed and he numbe o solu es which could be sepa a ed unde each o he expe imen al condi ions es ed in his wo k. Table 2 g oups he alues ob ained o m / o in each bu e employed a he i e SDS concen a ions indica ed abo e. An inc ease in m / o could be obse ed when inc easing CHES concen a ion due o he dec ease in he elec oos- mo ic low. When he CHES concen a ion was kep con- s an (0.08 M), an inc ease in he elu ion ange was ob- se ed by adding alcohols (n-p opanol o n-bu anol). Acco ding o hese esul s, an imp o emen in he sepa a- ion o N-phenylpy azole de i a i es by MEKC was ob- se ed when adding alcohols o he sepa a ion bu e . A s a is ical s udy o he esul s showed ha he alues o m / o ob ained o n-p opanol and n-bu anol a pe cen- ages o 1 o 5% we e no signi ican ly di e en ( o a 95% signi icance le el) while o a pe cen age o 3% he di e - ences be ween he esul s ob ained o he wo alcohols we e s a is ically signi ican (n-p opanol enabling a g ea e elu ion ange han n-bu anol). Pe cen ages o alcohol highe han 5% we e no used in o de o main ain he in eg i y o SDS micelles in he elec oly ic solu ion. The e ec o SDS concen a ion in he sepa a ion bu e on he elu ion ange ( o each SDS concen a ion, all m / o alues ob ained in di e en bu e s we e conside ed) was also s udied. As expec ed [4, 6], i could be obse ed ha when he SDS concen a ion was inc eased, an inc ease in he elu ion window (see Table 2) and in solu e e en ion was ob ained, esul ing in a loss in esolu ion o he la e mig a ing compounds. In ac , when a mix u e o 15 N-phenylpy azole de i a i es was injec ed in a MEKC sys em using SDS concen a ions om 0.01 o 0.05 M ( esul s no shown), i could be obse ed ha o he low- es SDS concen a ion (0.01 M) a poo sepa a ion selec- i i y was ob ained o he ea lie mig a ing compounds whose esolu ion inc eased wi h he SDS concen a ion. When he SDS concen a ion was inc eased, a loss in e iciency and hen in esolu ion o he mos hyd ophobic compounds was obse ed, oge he wi h a signi ican inc ease in he analysis ime. Thus, in e media e SDS concen a ions (0.02, 0.03 M) enabled be e sepa a ion selec i i y o he g oup o compounds conside ed han high micella concen a ions (0.04 M, 0.05 M). Figu e 1 shows he elec ophe og ams co esponding o he injec- ion o wo mix u es o he N-phenylpy azole de i a i es s udied in his wo k (a mix u e o 15 N-phenylpy azole de- i a i es in Fig. 1a and a mix u e o he emaining 11 N- phenylpy azole de i a i es in Fig. 1b) using a 0.08 M CHES bu e , pH 10, 0.02 Min SDS, wi h 5% n-bu anol. The compa ison o hese sepa a ions wi h hose ob ained a lowe o highe SDS concen a ions illus a es ha he in e media e concen a ion o SDS employed (0.02 M) can be a good comp omise be ween esolu ion o all com- pounds and analysis ime. 3.2 De e mina ion o solu e-micelle associa ion cons an s Calcula ion o solu e-micelle associa ion cons an s by MEKC was pe o med acco ding o Eq. (1). Di e en elec- oly ic solu ions (0.05, 0.08 and 0.10 MCHES in absence o modi ie s and 0.08 MCHES in he p esence o 1, 3, o 5% n-p opanol o n-bu anol) we e employed in o de o de e mine he a ia ion o he e en ion ac o o each sol- u e as a unc ion o he SDS concen a ion ( i e SDS con- cen a ions o each elec oly ic solu ion). Howe e , he calcula ion o he solu e-micelle associa ion cons an s pe Elec opho esis 2000, 21, 2424±2431 MEKC o N-phenylpy azole de i a i es 2427 Table 2. Elu ion ange ( m / o ) ob ained in all sepa a ion media employed a di e en SDS concen a- ions SDS concen a ion (M) 0.01 0.02 0.03 0.04 0.05 0.05 MCHES 3.31 3.14 3.31 3.09 3.14 0.08 MCHES 3.51 3.55 3.69 4.15 4.24 0.10 MCHES 4.36 4.43 4.69 5.02 5.59 0.08 MCHES 1% n-P opanol 3.86 4.41 4.44 4.35 4.76 0.08 MCHES 3% n-P opanol 5.19 5.39 6.51 6.28 7.35 0.08 MCHES 5% n-P opanol 5.97 5.17 5.83 6.56 6.55 0.08 MCHES 1% n-Bu anol 4.00 3.88 4.16 4.44 4.73 0.08 MCHES 3% n-Bu anol 4.40 4.81 4.61 4.99 5.22 0.08 MCHES 5% n-Bu anol 3.91 4.21 5.35 6.03 7.27 monome was possible only o hose compounds o which a linea a ia ion o he e en ion ac o was ob- ained as a unc ion o he SDS concen a ion (when he e en ion ac o could be de e mined in he medium con- side ed). Table 3 g oups he alues ob ained o he sol- u e-micelle associa ion cons an s o N-phenylpy azole de i a i es wi h SDS and he ela i e e o associa ed wi h his de e mina ion. I can be obse ed ha solu e- micelle associa ion cons an s could no be ob ained o all compounds in all elec oly ic solu ions employed. A 0.08 MCHES bu e (pH 10) modi ied wi h 5% n-bu anol enabled he calcula ion o he highes numbe o solu e- micelle associa ion cons an s. On he o he hand, Table 3 also shows ha solu e-micelle associa ion cons an s dec eased gene ally wi h inc eas- ing CHES concen a ion (0.05, 0.08, and 0.10 M) al hough he use o nonpa ame ic es s o compa e hese alues indica ed ha di e ences we e no s a is ically signi ican o a signi icance le el o 95%. Also, when nonpa ame ic es s we e used o compa e he alues o solu e-micelle associa ion cons an s ob ained wi h a 0.08 MCHES bu e in he absence and p esence o an alcohol, di e ences s a is ically signi ican we e no ound, excep when he alues ob ained in a 0.08 MCHES bu e in absence and p esence o 5% n-bu anol we e compa ed. This dec ease in he solu e-micelle associa ion cons an s in p esence o an alcohol can be jus i ied by conside ing ha , when he alcohol is abso bed in o he su ace o he micelle, i displaces some o he wa e molecules lying in he pali- sade laye . Since alcohols a e weake , hyd ogen bond accep o /dono s do no in e ac as s ongly wi h solu es pa i ioning in he su ace o he micelle [30]. 3.3 S udy o he possibili ies o MEKC in e alua ing hyd ophobici y Es ima ion o hyd ophobici y o compounds wi h pha ma- cological ac i i y such as N-phenylpy azole de i a i es is impo an due o he co ela ion o his p ope y wi h bio- logical ac i i y. The loga i hm o he oc anol-wa e dis i- bu ion coe icien (log P ow ) de e mined by he shake- lask echnique was adi ionally used o hyd ophobici y quan- i a ion [31]. Howe e , due o he disad an ages o his me hod, o he echniques (such as ch oma og aphic) we e employed o he indi ec es ima ion o hyd ophobic- i y. Usually, he in es iga ion o he possibili ies o a ch o- ma og aphic echnique in e alua ing hyd ophobici y was pe o med h ough he s udy o he co ela ion be ween he e en ion ac o s o he g oup o compounds unde s udy o hei loga i hm and he loga i hm o hei oc anol- wa e dis ibu ion coe icien s [32]. In he case o he g oup o N-phenylpy azole de i a i es s udied in his wo k, he log P ow alue de e mined by he shake- lask me hod was known only o wo o hese compounds (numbe s 12 and 22) while o he o he s he pa ame e known was he log P ow es ima ed by HPLC [33]. Nex we s udied he co ela ion be ween he loga i hm o he e en- ion ac o s ob ained o N-phenylpy azole de i a i es in he di e en micella phases in which he e en ion ac o s could be de e mined o he 26 compounds, and he log P ow alues aken om li e a u e. Bes co ela ions we e ob ained when a concen a ion o 0.01 Min SDS was used 2428 C. Ga cía-Ruiz, M. A. Ga cía and M. L. Ma ina Elec opho esis 2000, 21, 2424±2431 Figu e 1. Elec ophe og am co esponding o he injec- ion o wo mix u es o he N-phenylpy azole de i a i es. (a) Mix u e o 15 compounds; (b) mix u e o he 11 emaining N-phenylpy azole de i a i es. Expe imen al condi ions: 0.08 MCHES bu e (pH 10), 0.02 MSDS, 5% n-bu anol; de ec ion wa eleng h, 238 nm; applied ol age, 15 kV; injec ion by acuum, 67.73 kPa du ing 1 s; empe - a u e, 30 o C; capilla y, 75 cm (50 cm o he de ec o ) ´ 25 mm ID (375 mm OD). See Table 1 o peak iden i ica- ion. Elec opho esis 2000, 21, 2424±2431 MEKC o N-phenylpy azole de i a i es 2429 Table 3. Solu e-micelle associa ion cons an s pe monome (K 2 ) and ela i e e o s (RE, %) o he N-phenylpy azole de i a i es s udied wi h SDS as su ac an ( i e concen a ions) in di e en media Com- 0.05 M CHES 0.08 M CHES 0.10 M CHES 0.08 M CHES 0.08 M CHES 0.08 M CHES 0.08 M CHES 0.08 M CHES 0.08 M CHES pound K 2 (RE, %) K 2 (RE, %) K 2 (RE, %) 1% n-P opanol 3% n-P opanol 5% n-P opanol 1% n-Bu anol 3% n-Bu anol 5% n-Bu anol K 2 (RE, %) K 2 (RE, %) K 2 (RE, %) K 2 (RE, %) K 2 (RE, %) K 2 (RE, %) 1 37.85 (1.51) 37.85 (2.82) 39.65 a) (1.30) 31.85 (3.11) 23.55 (6.12) 22.95 (1.74) 28.85 (1.78) 19.05 (3.57) 14.15 (5.99) 2 67.75 (0.61) 66.25 (2.13) 66.75 a) (2.58) 54.35 (3.68) 41.75 a) (2.06) 37.85 (3.82) 48.75 (1.55) 32.65 (2.29) 22.35 (6.61) 3 93.05 (0.74) 95.45 (2.06) 91.45 a) (2.93) 73.45 (2.82) 52.85 a) (2.95) 53.95 (4.95) 71.95 (8.58) 44.45 (5.54) 28.95 (7.26) 4 112.55 (1.85) 108.65 (2.18) 108.55 a) (3.79) 83.05 (3.44) 59.95 (7.31) 53.95 (4.95) 73.98 a) (1.63) 44.45 (5.54) 28.95 (7.26) 5 158.55 (1.08) 161.05 (1.99) 152.45 a) (1.83) 132.75 (1.61) 93.65 a) (4.11) 81.35 (6.00) 114.85 (1.58) 67.45 a) (2.10) 42.25 a) (3.28) 6 195.85 (2.70) 188.05 (1.28) 172.25 a) (3.02) 129.05 (4.87) 93.65 a) (4.11) 84.92 a) (5.52) 114.85 (1.58) 67.45 a) (2.10) 48.95 (8.12) 7 203.45 (2.84) 195.85 (2.45) 160.89 a) (10.63) 148.25 (2.65) 103.75 a) (8.52) 102.05 (5.89) 132.95 (0.78) 84.05 (1.57) 58.95 (4.19) 8 225.35 a) (2.02) 323.15 (2.96) 218.45 a) (2.98) 182.05 (2.35) 130.05 a) (8.76) 129.45 (6.12) 164.25 (0.67) 107.75 (1.23) 77.85 (3.88) 9 274.05 a) (2.29) 235.55 (3.71) 228.05 a) (1.69) 185.25 (3.23) 132.25 a) (5.35) 110.21 (7.87) 164.65 (1.69) 98.55 (3.18) 54.25 a) (3.74) 10 256.45 (1.66) 266.85 (0.99) 243.05 a) (4.01) 313.05 (1.86) 137.65 a) (4.38) 127.85 (5.01) 164.65 (1.69) 98.55 (3.18) 60.75 a) (2.96) 11 560.55 a) (8.19) 358.55 a) (1.50) 348.15 a) (2.02) 362.85 (0.58) 222.45 a) (3.35) 203.65 (6.36) 265.55 (2.51) 175.65 (5.01) 101.25 a) (2.64) 12 487.15 (4.15) 402.65 (3.53) 386.05 a) (1.26) 399.05 (2.50) 271.75 a) (6.57) 268.65 (5.63) 329.05 (1.25) 222.85 (1.96) 152.85 (5.52) 13 456.35 a) (4.03) 475.15 (5.07) 463.05 a) (2.63) 324.45 (3.09) 287.15 a) (4.07) 261.45 (3.93) 343.15 (2.56) 221.05 (6.41) 117.35 a) (2.77) 14 ± 699.05 (5.38) 827.65 a) (1.02) 732.65 (0.99) 530.65 a) (7.97) 431.15 a) (1.77) 511.25 (3.21) 331.45 (2.59) 209.45 (6.07) 15 858.45 a) (3.83) 783.75 (6.93) 603.75 a) (1.37) 749.85 (0.85) 595.55 (8.47) 583.45 a) (6.63) 644.35 (2.26) 341.45 a) (4.57) 249.65 a) (2.76) 16 904.95 a) (3.78) 733.35 (3.81) 728.45 a) (2.14) 516.15 (4.68) 621.95 a) (8.67) 603.55 (7.88) 656.15 (2.75) 356.15 a) (2.81) 195.95 a) (2.51) 17 ± 1553.65 (8.68) 1515.65 a) (7.77) 1466.25 (8.03) 1186.55 a) (7.03) 1000.85 a) (7.03) 1349.25 (7.73) 769.65 (3.30) 410.15 a) (7.04) 18 ± ± 2133.92 a) (4.79) 1281.55 (11.69) 1096.35 a) (9.32) 952.15 a) (6.14) 1244.65 (7.61) 756.65 (4.04) 391.15 a) (5.74) 19 ± ± 1393.05 a) (5.98) ± 2119.75 a) (12.88) ± 2527.25 a) (4.76) 746.15 a) (2.76) 299.65 a) (4.65) 20 ± ± ± ± ± ± ± 2714.55 a) (7.43) 1742.35 a) (6.28) 21 ± ± 2905.25 a) (8.29) ± ± ± ± 2714.55 a) (7.43) 1742.35 a) (6.28) 22 ± ± ± ± ± ± ± ± 2409.55 a) (10.26) 23 ± ± ± ± ± ± ± ± 2409.55 a) (10.26) 24 ± ± ± ± ± ± ± ± 2409.55 a) (10.26) 25 ± ± ± ± ± ± ± ± 2594.55 a) (11.21) 26 ± ± ± ± ± ± ± ± 2358.35 a) (11.50) a) Values calcula ed wi h ou SDS concen a ions in bu e s 0.08 MCHES (pH 10) modi ied by 3 o 5% n- bu anol. Da a o e en ion ac o s ob ained when using hese wo las sepa a ion bu e s o 10 N-phenylpy azole de i a i es (numbe s 1, 2, 3, 9, 10, 12, 13, 14, 17, and 19) co e ing a wide ange o known log P ow [33] we e chosen o ob ain a s aigh line log k±log P ow which was employed o calcula e log P ow o he emaining N-phenyl- py azole de i a i es om hei e en ion ac o alues. The leas -squa es equa ion wi h he bes co ela ion coe - icien was ob ained o a 0.08 MCHES bu e (pH 10), 0.01 Min SDS modi ied by 5% n-bu anol: log k= ±4.708 + 1.969 log P ow (3) ( = 0.983, n= 10) The a ia ion o log P ow alues p edic ed by his equa ion and log P ow alues es ima ed by HPLC (o de e mined by he shake- lask me hod o compounds 12 and 22) [33] is shown in Fig. 2. A linea co ela ion be ween he wo g oups o alues was obse ed. Rela i e e o ob ained in he p edic ion o each log P ow by MEKC was calcula ed wi h espec o he co esponding alue es ima ed by HPLC. Al hough o solu e 23 he e o was close o 24% ( his compound was no included in Fig. 2), he e o s ob- ained o he o he 25 compounds anged om 0.3 o 14.1% (a e age e o 3.3%). 4 Concluding ema ks MEKC enabled he sepa a ion o mix u es o a g oup o N-phenylpy azole de i a i es wi h pha macological ac i - i y. The sepa a ion o mul icomponen mix u es using CHES bu e s (pH 10) wi h SDS as micella sys em in he absence and in he p esence o medium chain alcohols allowed he apid de e mina ion o e en ion ac o s o hese compounds. Sepa a ion selec i i y s udied in e ms o elu ion ange ( m / o ) and numbe o compounds sep- a a ed showed ha an inc ease in he CHES concen a- ion o he addi ion o low pe cen ages o n-p opanol o n- bu anol inc eased he elu ion ange enhancing he sepa- a ion. Howe e , al hough an inc ease in he elu ion ange was also obse ed when inc easing he SDS concen a- ion in he sepa a ion bu e , be e sepa a ions we e ob- ained o in e media e SDS concen a ions due o he inc ease in he e en ion o he solu es o high SDS con- cen a ions, which p o oked a loss in esolu ion o highly hyd ophobic compounds oge he wi h a signi ican inc ease in analysis ime. Mix u es o up o 12 N-phenyl- py azole de i a i es we e sepa a ed using a 0.08 MCHES bu e (pH 10), 0.02 Min SDS modi ied by 5% n-bu anol. Solu e-micelle associa ion cons an s o N-phenylpy a- zole de i a i es wi h SDS we e de e mined in di e en media. Di e ences s a is ically signi ican o he alues ob ained o hese associa ion cons an s we e no ob- se ed when a ying he CHES concen a ion o when adding alcohols o he sepa a ion bu e , excep in he case o he addi ion o 5% n-bu anol. A highe numbe o solu e-micelle associa ion cons an s could be calcula ed in media modi ied by n-bu anol, especially o a pe cen - age o his alcohol equal o 5%. Finally, he s udy o he co ela ion be ween he loga i hm o he e en ion ac o s ob ained by MEKC o N-phenyl- py azole de i a i es as a unc ion o hei oc anol-wa e dis ibu ion coe icien s (log P ow ) es ima ed by HPLC showed a linea a ia ion which enabled he calcula ion o log P ow alues by MEKC o he compounds s udied by using calib a ion wi h da a co esponding o 10 N-phenyl- py azole de i a i es. E o s ob ained o log P ow p edic ed by MEKC o 25 solu es wi h espec o hose p edic ed by HPLC anged om 0.3 o 14.1% wi h an a e age e o o 3.3%. The au ho s hank he Uni e si y o Alcalµ, Spain (p ojec E029/98) and he Comisión In e minis e ial de Ciencia y Tecnología, Spain (p ojec PB98-0709) o inancial sup- po and D . Al a ez-Builla o he kind gi o he N-phe- nylpy azole de i a i es employed in his wo k. 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