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.
Recei ed Oc obe 11, 1999; e ised e sion Feb ua y 24, 2000
2430 C. Ga cía-Ruiz, M. A. Ga cía and M. L. Ma ina Elec opho esis 2000, 21, 2424±2431
Figu e 2. Va ia ion o log P
ow
alues es ima ed by MEKC
o 25 N-phenylpy azole de i a i es in a 0.08 MCHES
bu e (pH 10), 0.01 MSDS, 5% n-bu anol as a unc ion o
hei log P
ow
alues es ima ed by HPLC o de e mined by
he shake- lask me hod (compounds 12 and 22).
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Elec opho esis 2000, 21, 2424±2431 MEKC o N-phenylpy azole de i a i es 2431