Fas enan iome ic sepa a ion o uniconazole and
diniconazole by elec okine ic ch oma og aphy
using an anionic cyclodex in: Applica ion o he
de e mina ion o analy e-selec o appa en binding
cons an s o enan iome s
The enan iome ic esolu ion o he ungicides uniconazole and diniconazole was pe -
o med using elec okine ic ch oma og aphy wi h cyclodex ins as pseudos a iona y
phase (CD-EKC). A sys ema ic e alua ion o se e al chi al selec o s was made. The
anionic de i a i e ca boxyme hyla ed-g-cyclodex in (CM-g-CD) was ound o be he
mos app op ia e o he enan iosepa a ion o ungicides among all cyclodex ins
es ed. The in luence o some expe imen al condi ions such as na u e and bu e pH,
chi al selec o concen a ion, and empe a u e on he enan iome ic sepa a ion o he
compounds s udied was also in es iga ed. The use o a 50 mMphospha e bu e
(pH 6.5) con aining 5 mMCM-g-CD and a empe a u e o 50
o
C enabled he baseline
enan io esolu ion o mix u es o uniconazole and diniconazole in less han 5 min. In
addi ion, appa en binding cons an s o each enan iome -CM-g-CD pai a se e al
empe a u es, as well as he modynamic pa ame e s o binding we e calcula ed.
Keywo ds: Uniconazole / Diniconazole / Elec okine ic ch oma og aphy / Ca boxyme hyla ed g-
cyclodex in / Complex mobili y / Inclusion complex binding cons an / Enan iosepa a ion / The -
modynamic pa ame e s EL 4119
Yolanda Ma ín-Biosca
2
Ca men Ga cía-Ruiz
1
Ma ia L. Ma ina
1
1
Depa amen o de Química
Analí ica, Facul ad de
Química, Uni e sidad de
Alcalµ, Alcalµ de Hena es
(Mad id), Spain
2
Depa amen o de Química
Analí ica, Facul ad de
Fa macia, Uni e si a de
Valncia,
Bu jasso (Valencia), Spain
1 In oduc ion
Uniconazole and diniconazole ha e ungicidal and plan
g ow h egula ing ac i i ies. In bo h cases, he R-enan-
iome shows s onge ungicidal ac i i y han he S-enan-
iome , whe eas he la e has highe plan g ow h egula -
ing ac i i y. On he o he hand, uniconazole has highe
plan g ow h egula ing ac i i y han diniconazole bu is
less ac i e as a ungicide. Consequen ly, p oduc s con-
aining a high p opo ion o he R- and S-enan iome o
diniconazole and uniconazole, espec i ely, ha e been
de eloped as a high-ac i i y ungicide and an e ec i e
plan g ow h egula o [1, 2]. Reliable and e icien me h-
ods o sepa a ing he enan iome s a e he e o e neces-
sa y.
Chi al sepa a ions a e one o he mos p omising opics in
he ield o capilla y elec opho esis (CE), which has been
ou lined by se e al ecen e iew a icles [3±8]. In CE,
chi al sepa a ions can be achie ed using a ious chi al
selec o s, by means o elec okine ic ch oma og aphy
(EKC). As chi al selec o s, neu al and cha ged cyclodex-
ins (CDs) [4±7, 9], bile sal s [5], monome ic and poly-
me ic chi al su ac an s [10], c own e he s [7], calixa enes
[9], p o eins [11], mac ocyclic an ibio ics [12], polysac-
cha ides [9], o e go alkaloids [9] ha e been employed.
The chi al sepa a ion p inciple elies on he di e en pa i-
ion o enan iome s be ween he bulk solu ion and he chi-
al pseudophase.
Inclusion in o he ca i y o CDs ep esen s he mos e-
quen ly used app oach o chi al sepa a ion by CE. No
only neu al CD de i a i es, bu also cha ged CDs ha e
been de eloped. The chi al ecogni ion mechanism is
based on inclusion o a bulky hyd ophobic pa o he mol-
ecules, p e e ably a oma ic moie ies, in he hyd ophobic
ca i y o he CD. An addi ional equi emen is ha secon-
da y in e ac ions ha e o ake e ec : hese include dipole-
dipole in e ac ions o hyd ogen bonds be ween he
hyd oxyl g oups a posi ion 2 o 3 a he mou h o he CD
and pola subs i uen s close o he chi al cen e o he
analy e. Since uncha ged CDs mig a e a he same eloc-
i y as he elec oosmo ic low (EOF), hey only allow he
sepa a ion o cha ged analy es. The chi al esolu ion o
Co espondence: D . M. L. Ma ina, Depa amen o de Química
Analí ica, Facul ad de Química, 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]
Fax: +34-91-8854971
Abb e ia ions: CM-b-CD, ca boxyme hyla ed-b-cyclodex in;
CM-g-CD, ca boxyme hyla ed-g-cyclodex in; HP-b-CD, (2-hy-
d oxy)p opyl-b-cyclodex in; Succ-b-CD, succinyla ed-b-cyclo-
dex in; Succ-g-CD, succinyla ed-g-cyclodex in
3240 Elec opho esis 2000, 21, 3240±3248
 WILEY-VCH Ve lag GmbH, 69451 Weinheim, 2000 0173-0835/00/1515-3240 $17.50+.50/0
uncha ged analy es can be pe o med by micella elec o-
kine ic ch oma og aphy (MEKC) o by using cha ged CD
de i a i es.
CE is a con enien echnique o he s udy o binding con-
s an s in eal sepa a ion sys ems wi hou any adap a ions
and app oxima ions. Thus, his echnique allows he
measu emen o binding cons an s unde he exac condi-
ions a which enan iosepa a ions a e pe o med. This
cons i u es i s main ad an age e sus o he sepa a ion
echniques. In addi ion, he small sample and bu e
amoun s needed in CE oge he wi h i s e sa ili y enable
he s udy o di e en possibili ies o analy e-selec o bind-
ings [13, 14].
Enan iome ic sepa a ion o he s uc u al analogs unico-
nazole and diniconazole by CD-modi ied MEKC (CD-
MEKC) by using as sepa a ion solu ion 100 mMsodium
dodecyl sul a e (SDS) and 2 Mu ea in 100 mMbo a e bu -
e , pH 9.0, con aining 50 mMg-CD and 5% 2-me hyl-2-
p opanol, has been epo ed [1, 2]. Unde hese condi-
ions, enan io esolu ion o ungicides was achie ed in 40
min. The aim o his wo k was he selec ion o he op imal
expe imen al condi ions o enable as enan iome ic sepa-
a ion o he ungicides uniconazole and diniconazole. A
sys ema ic e alua ion o se e al chi al selec o s, and a
s udy o he in luence o o he expe imen al condi ions,
such as chi al selec o concen a ion and empe a u e, on
he enan iome ic sepa a ion we e made. The use ulness
o anionic CD de i a i es in he sepa a ion o ungicides
was e alua ed.
2 Ma e ials and me hods
2.1 Chemicals and samples
All eagen s we e o analy ical g ade. Tau ocholic acid
sodium sal (STC) and cholic acid sodium sal (SC) we e
pu chased om Sigma (S . Louis, MO, USA); sodium
dihyd ogen phospha e, is(hyd oxyme hyl)aminome hane
hyd ochlo ide (T is), 2-(N-mo pholino) e hanesul onic
acid (MES), sodium dodecyl sul a e (SDS), and sodium
hyd oxide we e om Me ck (Da ms ad , Ge many); am-
monium o ma e, ammonium ace a e and b-CD we e om
Fluka (Buchs, Swi ze land); (2-hyd oxy)p opyl b-CD (HP-
b-CD; wi h a subs i u ion deg ee, s.d., o app oxima ely
3), ca boxyme hyla ed-b-cyclodex in (CM-b-CD, s.d.
~ 3), ca boxyme hyla ed-g-cyclodex in (CM-g-CD, s.d. ~
3), succinyla ed b-cyclodex in (Succ-b-CD, s.d. ~ 3.5)
and succinyla ed g-cyclodex in (Succ-g-CD, s.d. ~ 3) 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
ungicides uniconazole (be a-((4-chlo ophenyl)me hy-
lene)-alpha-(1,1-dime hyle hyl)-1H-1,2,4- iazole-1-e ha-
nol) and diniconazole (be a-((2,4-dichlo ophenyl)me hy-
lene)-alpha-(1,1-dime hyle hyl)-1H-1,2,4- iazole-1-e ha-
nol) we e pu chased om ChemSe ice (Wes Ches e ,
PA, USA) and D . Eh ens o e Re e ence Ma e ials
(Augsbu g, Ge many), espec i ely. Figu e 1 shows he
s uc u es o he ungicides uniconazole and diniconazole
[1, 2]. Me hanol o HPLC g ade (Labscan, Dublin, I eland)
was used o he p epa a ion o ungicide solu ions.
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 an acquisi ion da a sys em
Model S a 4.5 om Va ian Associa es (Suga Land, TX,
USA); and (ii) an HP
3D
CE sys em (Hewle -Packa d,
Waldb onn, Ge many) equipped wi h an on-column diode
a ay de ec o (DAD) and HP 3D-CE Chems a ion so -
wa e. A 50 mm inne diame e (ID) and 375 mm ou e di-
ame 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
3D
sys em) was employed (Polymi-
c o Technologies, Phoenix, AZ, USA). Injec ion was pe -
o med hyd odynamically a 50 o 30 mba o 2 s. Usual
CE condi ions o sepa a ions o he ungicides unicona-
zole and diniconazole we e: applied ol age, 20 kV;
de ec ion wa eleng h, 220 nm; and capilla y empe a u e
se a 20, 30, 40 o 50
o
C. Elec oly ic solu ions we e
degassed in an ul asonic cleane KM om Raypa (Ba ce-
lona, Spain). A 654 pH me e om Me ohm (He isau,
Swi ze land) was employed o adjus he pH o he sepa-
a ion bu e s.
2.3 P ocedu e
Bu e s we e p epa ed by dissol ing he app op ia e
amoun o sodium dihyd ogen phospha e, ammonium o -
ma e, ammonium ace a e, T is o MES in Milli-Q wa e ,
Elec opho esis 2000, 21, 3240±3248 Fas enan iome ic sepa a ion o uniconazole and diniconazole 3241
Figu e 1. S uc u es o he ungicides uniconazole and
diniconazole.
CE and CEC
and adjus ing he pH o he desi ed alue wi h a 1 Msolu-
ion o sodium hyd oxide ( o phospha e, T is, o MES bu -
e s), o mic acid ( o o ma e bu e ) o ace ic acid ( o
ace a e bu e ). In o de o ob ain he elec oly ic solu ions
o he enan iome ic sepa a ions, he chi al selec o (bile
sal o nega i ely cha ged CDs alone o mixed wi h he
neu al HP-b-CD) was dissol ed in he co esponding
bu e solu ion. Finally, a e adding addi i es such as
SDS, he pH was adjus ed o he desi ed alue. When
me hanol was used as addi i e, i was added a e elec o-
ly ic solu ion p epa a ion. 5 mg/mL s ock solu ions o uni-
conazole and diniconazole we e p epa ed by dissol ing
10 mg o compound in 2 mL o me hanol. The wo king so-
lu ions we e ob ained a 0.2 mg/mL by dilu ion o he
s ock solu ions in me hanol. 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
injec ion and a he end o he day. In all cases, he condi-
ioning un included he ollowing s eps: (i) 2-min inse
wi h Milli-Q wa e , (ii) 2-min inse wi h 0.1 Msodium
hyd oxide, and (iii) 2-min inse wi h Milli-Q wa e a
1000 mba . Be o e sample injec ion he capilla y was also
insed wi h he unning bu e o 2 min.
2.4 Da a ea men
Enan iome ic esolu ion was calcula ed by he ollowing
exp ession:
R
s
= 1.18 (
2
±
1
)/(w
1
+w
2
) (1)
whe e
1
and
2
a e he wo enan iome s mig a ion imes
and w
1
,w
2
a e he middle high wid hs o he co espond-
ing peaks. The inclusion complexes binding cons an s o
each enan iome -CM-g-CD pai we e calcula ed, om
elec opho e ic mobili ies, using linea plo ing ap-
p oaches by leas -squa es me hods. F om he expe i-
men al da a, he elec opho e ic mobili y o each enan-
iome (m
i
) was de e mined by he ollowing equa ion:
milL
V
1
i
1
0
 2
whe e Land Ia e he o al capilla y leng h and he leng h
o he de ec o , espec i ely, Vis he un ol age,
i
is he
enan iome mig a ion ime, and
o
is he mig a ion ime o
me hanol, used as neu al ma ke o co ec changes in
solu ion iscosi y caused by a ia ions in CD concen a-
ion. Expe imen al da a we e manipula ed and pa ame-
e s we e calcula ed using Excel 7.0 om Mic oso O ice
so wa e. The unce ain ies in he binding cons an s,
mobili ies, and he modynamic pa ame e s lis ed in
Tables 3±5 we e calcula ed by e o p opaga ion me hods
using he e o s in he slopes and in e cep s ob ained by
leas -squa es me hods [15].
3 Resul s and discussion
3.1 Enan iome ic sepa a ion o uniconazole
and diniconazole
In p elimina y expe imen s, he enan iome ic esolu ion o
ungicides was in es iga ed by using di e en chi al
selec o s in he sepa a ion bu e . Cha ged CD de i a-
i es we e employed in EKC; and bile sal s and mix u es
o SDS and neu al and cha ged CDs we e used in micel-
la EKC (MEKC). The use o anionic CD de i a i es p o-
ided sa is ac o y esul s. The de i a i es Succ-b-CD,
Succ-g-CD, CM-b-CD, and CM-g-CD, and he mix u es
HP-b-CD/CM-g-CD we e e alua ed wi h di e en bu e s
and pH alues (expe imen al condi ions a e summa ized
in Table 1). The use o Succ-b-CD and Succ-g-CD alone
as chi al selec o s in he sepa a ion bu e did no p oduce
he enan iosepa a ion o any o he ungicides. Howe e ,
when CM-b-CD and CM-g-CD we e employed as chi al
selec o s, he chi al ecogni ion o uniconazole and dinico-
nazole was achie ed. As can be seen in Table 1, he bes
esolu ions we e achie ed when he de i a i e CM-g-CD
in 50 mMphospha e bu e a pH 6.5 was used. The use
o a lowe pH ( o ma e bu e , pH 4) imp o ed he unico-
nazole esolu ion, bu a dec ease in he diniconazole es-
olu ion and an inc ease in he mig a ion imes we e also
p oduced.
Since mix u es o CDs may o igina e changes in selec i -
i y, mix u es o HP-b-CD and CM-g-CD we e es ed.
Unde hese condi ions, he esolu ions ob ained we e
wo se han hose achie ed when CM-g-CD was used as
unique chi al selec o . In his case, he loss in esolu ion
could be caused by bo h a compe i i e e ec be ween
bo h CDs and he inc ease in he EOF. The use o me h-
anol as addi i e in he CD mix u es (50 mM o ma e bu e ,
pH 6.5) ga e ise o a sligh dec ease in he uniconazole
esolu ion, while an imp o emen in he diniconazole es-
olu ion was achie ed wi h espec o ha ob ained in he
same bu e wi hou me hanol. A sligh inc ease in he
mig a ion imes was also p oduced in he p esence o
me hanol.
Uniconazole and diniconazole we e also injec ed in se -
e al MEKC sys ems in which di e en bu e s, 50 mMin
SDS and 10 mMin HP-b-CD, b-CD o Succ-b-CD we e
used. Unde all hese condi ions, no chi al ecogni ion
was obse ed o he ungicides s udied. When wo bile
sal s, SC and STC we e used as chi al selec o s ( wo
concen a ions es ed in 50 mMphospha e bu e a
3242 Y. Ma ín-Biosca, C. Ga cía-Ruiz and M. L. Ma ina Elec opho esis 2000, 21, 3240±3248
pH 6.5), no chi al ecogni ion was obse ed. Among he
di e en chi al selec o s conside ed, CM-g-CD was ound
o be he mos app op ia e o he uniconazole and dinico-
nazole enan iosepa a ion. Nex , a mo e exhaus i e s udy
on he in luence o se e al pa ame e s on he enan io-
me ic esolu ion, using CM-g-CD as chi al selec o , was
pe o med in o de o imp o e he esolu ion o he com-
pounds s udied.
3.2 In luence o he chi al selec o
concen a ion and empe a u e on he
enan iome ic sepa a ion o uniconazole
and diniconazole wi h CM-g-CD
A solu ion con aining 50 mMphospha e bu e a pH 6.5
wi h CM-g-CD as chi al selec o was chosen o s udy he
in luence o CD concen a ion and empe a u e on he
enan iome ic esolu ion o uniconazole and diniconazole.
The concen a ion o CM-g-CD was a ied in he ange o
2±20 mM. Chi al ecogni ion o uniconazole and dinicona-
zole was achie ed o all concen a ions and empe a-
u es es ed. In addi ion, se e al o hese condi ions ena-
bled he as sepa a ion o mix u es o he wo ungicides.
Table 2 shows he a e aged mig a ion imes ob ained o
uniconazole and diniconazole enan iome s, and he eso-
lu ion be ween he second-mig a ing enan iome o unico-
nazole and he i s -mig a ing enan iome o diniconazole
a di e en empe a u es and CM-g-CD concen a ions
unde expe imen al condi ions, enabling he enan iosepa-
a ion o he wo ungicides in mix u es. I can be ob-
se ed ha a e aged mig a ion imes dec eased when
he CM-g-CD concen a ion was dec eased a a cons an
empe a u e, acco ding o a dec ease in he iscosi y o
he solu ion. In addi ion, o a cons an CM-g-CD concen-
a ion, a e aged mig a ion imes dec eased wi h inc eas-
ing empe a u e due o he dec ease ob ained o he is-
cosi y a inc easing empe a u e alues. Table 2 also
shows ha low CM-g-CD concen a ions oge he wi h a
empe a u e o 50
o
C a e he mos use ul expe imen al
condi ions a which o pe o m he enan iome ic sepa a-
Elec opho esis 2000, 21, 3240±3248 Fas enan iome ic sepa a ion o uniconazole and diniconazole 3243
Table 1. Some expe imen al condi ions es ed o he enan iome ic sepa a ion o uniconazole and diniconazole
Chi al Chi al selec o BGE Addi i es Uniconazole Diniconazole
selec o concen a ion
a)
R
sc)
b)
R
sc)
CM-b-CD 10 mM50 mMphospha e/pH 6.5 ± 5.77 0.52 5.70 ±
20 mM50 mMphospha e/pH 6.5 ± 6.73 ± 6.75 ±
10 mM50 mMace a e/pH 4.5 ± 11.19 0.89 10.67 0.77
CM-g-CD 5 mM100 mMphospha e/pH 6.5 ± 3.90 0.63 4.04 0.62
10 mM100 mMphospha e/pH 6.5 ± 4.14 1.05 4.59 1.05
5m
M50 mMphospha e/pH 6.5 ± 4.72 1.09 4.93 0.82
10 mM50 mMphospha e/pH 6.5 ± 4.98 1.41 5.20 1.06
15 mM50 mMphospha e/pH 6.5 ± 5.76 2.26 6.17 1.61
20 mM50 mMphospha e/pH 6.5 ± 6.71 1.92 7.54 1.77
15 mM50 mM o ma e/pH 4 ± 12.96 3 15.45 1.2
10 mMHP-b-CD 50 mMphospha e/pH 6.5 ± 4.00 0.51 4.19 0.65
+10m
MCM-g-CD 50 mMMES/pH 6.5 ± 4.56 0.58 4.74 0.98
50 mM o ma e/pH 5 ± 6.92 1.46 7.31 1.27
50 mM o ma e/pH 6.5 ± 4.34 0.66 4.09 0.44
50 mM o ma e/pH 6.5 5% me hanol 4.79 0.51 4.89 0.77
50 mM o ma e/pH 6.5 10% me hanol 5.92 0.53 6.15 1.20
Succ-b-CD 10 mM50 mMphospha e/pH 6.5 ± 7.82 ± 7.68 ±
20 mM50 mMphospha e/pH 6.5 ± 15.19 ± 16.23 ±
10 mM50 mMMES/pH 6.5 ± 5.97 ± 5.87 ±
10 mM50 mMphospha e/pH 6.5 50 mMSDS ± ± 11.34 ±
10 mM50 mMphospha e/pH 6.5 2% me hanol 8.04 ± 8.03 ±
10 mM50 mMphospha e/pH 6.5 5% me hanol 9.65 ± 9.49 ±
Succ-g-CD 10 mM50 mMphospha e/pH 6.5 ± 4.92 ± 4.83 ±
a) A e aged mig a ion ime o uniconazole enan iome s, in min
b) A e aged mig a ion ime o diniconazole enan iome s, in min
c) Resolu ion be ween enan iome s
In all cases CE condi ions we e: injec ion, 50 mba o 2 s; un ol age, 20 kV; empe a u e, 40
o
C; de ec ion wa eleng h,
220 nm.
ion o mix u es o uniconazole and diniconazole.
Al hough a 2 mMconcen a ion o CM-g-CD a 50
o
C ena-
bled he indi idual baseline esolu ion o he wo enan-
iome s o each ungicide, a concen a ion o 5 mMo CM-
g-CD was chosen in o de o achie e a good esolu ion
be ween he second-mig a ing enan iome o uniconazole
and he i s -mig a ing enan iome o diniconazole in a
mix u e o hese wo ungicides. Figu e 2 shows he sepa-
a ion o uniconazole and diniconazole pe o med a
50
o
C, using an elec oly ic solu ion con aining 50 mM
phospha e bu e a pH 6.5 and 5 mMCM-g-CD. These
condi ions allow he as enan iosepa a ion o a mix u e o
he wo ungicides in abou 5 min.
3244 Y. Ma ín-Biosca, C. Ga cía-Ruiz and M. L. Ma ina Elec opho esis 2000, 21, 3240±3248
Table 2. CM-g-CD concen a ion and empe a u e condi-
ions enabling he enan iosepa a ion o unicona-
zole and diniconazole mix u es
[CM-g-CD] T(
o
C)
a)
b)
R
sc)
(mM)
2 20 6.87 7.14 2.00
5 20 8.40 8.95 1.74
2 30 5.81 6.15 1.58
5 30 6.91 7.28 1.74
2 40 4.95 5.19 1.85
5 40 5.74 6.05 1.85
2 50 4.36 4.52 1.58
5 50 5.04 5.10 1.88
10 50 5.35 5.62 1.75
a) A e aged mig a ion ime o uniconazole enan iome s,
in min
b) A e aged mig a ion ime o diniconazole enan iom-
e s, in min
c) Resolu ion be ween he second-mig a ion enan iome
o uniconazole and he i s -mig a ing enan iome o
diniconazole.
BGE, solu ion con aining 50 mMphospha e bu e a
pH 6.5. In all cases CE condi ions we e: injec ion,
50 mba o 2 s; un ol age, 20 kV; de ec ion wa eleng h,
220 nm.
Figu e 2. Enan iome ic sepa a ion o uniconazole (peaks
1 and 2) and diniconazole (peaks 3 and 4). BGE, 50 mM
phospha e bu e a pH 6.5 con aining 5 mMCM-g-CD.
Capilla y, 50 mm´50 cm uncoa ed used silica; injec ion,
50 mba o 2 s; un ol age, 20 kV; empe a u e, 50
o
C;
de ec ion wa eleng h, 220 nm.
Figu e 3. Dependence o ne mig a ion imes o unicona-
zole (uppe pa ) and diniconazole (lowe pa ) upon he
concen a ion o CM-g-CD. (*)20
o
C; (&)30
o
C; (^)40
o
C;
(~)50
o
C.

F om hese esul s, he enan iome ic a io could be de e -
mined o he wo ungicides in es iga ed. Thus, he enan-
iome p opo ions we e calcula ed om he co espond-
ing a eas in di e en elec ophe og ams, ob aining alues
o abou 80:20 (peak 1 : peak 2) o uniconazole and
20:80 (peak 3 : peak 4) o diniconazole. Finally, Fig. 3
shows plo s o he ne mig a ion ime o uniconazole
(uppe pa ) and diniconazole (lowe pa ) a se e al em-
pe a u es as a unc ion o he CM-g-CD concen a ion.
The ne mig a ion ime was de ined as ¢
i
±
o
, whe e ¢
i
is
he a e age o he mig a ion imes o bo h enan iome s,
and
o
is he mig a ion ime o he neu al ma ke . I can
be obse ed ha ne mig a ion imes inc eased wi h
inc easing CM-g-CD concen a ions by means o a linea
a ia ion. This e ec could be explained by he exis ence
o a g ea e p opo ion o solu e complexed unde hese
condi ions. In addi ion, slopes o he linea plo s inc eased
wi h dec easing empe a u e due o he longe ne mig a-
ion imes ob ained a lowe empe a u es o a cons an
concen a ion o CM-g-CD acco ding o a dec ease in he
EOF caused by changes in he iscosi y.
3.3 De e mina ion o appa en analy e-selec o
binding cons an s o enan iome s
Knowledge o binding cons an s when inclusion complex-
a ion occu s is o in e es in o de o app ecia e he ex en
o an analy e inclusion in o he ca i y o a CD. A heo e i-
cal model ela ing mobili y o he concen a on o he CD
selec o was de eloped by W en and Rowe [16, 17]. Chi-
Elec opho esis 2000, 21, 3240±3248 Fas enan iome ic sepa a ion o uniconazole and diniconazole 3245
Figu e 4. Plo s o he change in solu e mobili y and di e ences in enan iome s elec opho e ic mobi-
li ies as a unc ion o CM-g-CD concen a ion o uniconazole (uppe pa ) and diniconazole (lowe
pa ). The cu es we e d awn by hand o illus a e he gene al ends in he da a. (*)20
o
C; (&)30
o
C;
(^)40
o
C; (~)50
o
C.
al ecogni ion will depend on he di e ence be ween he
elec opho e ic mobili ies o each ee and complexed
enan iome . Binding cons an s can be de e mined using
he ollowing exp ession:
KL m mi
mimc
 3
whe e Kis he binding cons an , [L] is he equilib ium con-
cen a ion o uncomplexed ligand, m
and m
c
a e he elec-
opho e ic mobili ies o he ee and complexed solu e,
and m
i
is he solu e mobili y a he ligand concen a ion [L].
The mobili y o he complex, m
c
, mus be measu ed in
o de o use Eq. (3) di ec ly o es ima e binding cons an s.
Al hough i can be app oached o he solu e mobili y a
e y high concen a ions o CD, some imes i s measu e-
men is di icul o impossible due o he di icul y o inding
sui able CD ma ke s and eaching sa u a ing condi ions.
In addi ion, an inc ease in he chi al selec o concen a-
ion leads o some changes in elec opho e ic mobili y
(due o changes in bu e iscosi y, EOF, e c.) which a e
no ela ed o he selec o -selec and binding [13]. This
can be a sou ce o sys ema ic e o in his echnique. In
o de o a oid his p oblem, Eq. (3) can be ea anged in
many plo ing o ms ha do no equi e he di ec meas-
u emen o m
c
. The plo ing o ms o hese equa ions,
summa ized by Rundle and A ms ong [14], a e he ol-
lowing:
Double ecip ocal
y- ecip ocal
L
mim
1
mcm
L 1
mcm K5
x- ecip ocal
mim
LKmim Kmcm 6
To calcula e he CD-analy e binding cons an s, he mobili-
ies o enan iome s we e measu ed a di e en concen-
a ions o chi al selec o . Plo s showing he e ec o CM-
g-CD concen a ion on solu e mobili ies a se e al empe -
a u es a e shown in Fig. 4. A he ou alues o he em-
pe a u e checked o uniconazole and diniconazole, elec-
opho e ic mobili ies can be seen o inc ease when
inc easing he CD concen a ion in all anges es ed. On
he o he hand, di e ences in elec opho e ic mobili ies o
enan iome s, Dm
i
, ini ially inc eased wi h inc easing CD
concen a ions, and emained mo e o less cons an o
dec eased sligh ly a e a maximum was eached. Fu -
he mo e, hese di e ences we e la ge a highe empe -
a u es.
3246 Y. Ma ín-Biosca, C. Ga cía-Ruiz and M. L. Ma ina Elec opho esis 2000, 21, 3240±3248
Table 3. Appa en binding cons an s and enan ioselec i i ies o complexa ion o uniconazole wi h CM-g-CD in 50 mM
phospha e bu e (pH 6.5) a se e al empe a u es
Plo ing me hod log K
1a)
log K
2a)
a
b)
m
c
(1)
c)
m
c
(2)
c)
2
(1)
d)
2
(2)
d)
20
o
C
Double ecip ocal 2.458 0.008 2.629 0.009 1.48 ± 9.83 0.13 ±11.52 0.12 0.999 0.999
y-Recip ocal 2.47 0.01 2.64 0.01 1.49 ± 9.72 0.06 ±11.42 0.06 0.9999 0.9999
x-Recip ocal 2.46 0.01 2.63 0.01 1.48 ± 9.8 0.3 ±11.5 0.5 0.998 0.997
30
o
C
Double ecip ocal 2.410 0.009 2.568 0.009 1.44 ±11.8 0.2 ±13.8 0.2 0.999 0.999
y-Recip ocal 2.41 0.02 2.57 0.03 1.43 ±11.7 0.2 ±13.8 0.2 0.999 0.9995
x-Recip ocal 2.415 0.014 2.571 0.015 1.43 ±11.7 0.5 ±13.7 0.6 0.997 0.997
40
o
C
Double ecip ocal 2.364 0.005 2.506 0.006 1.39 ±13.64 0.12 ±16.00 0.15 0.9998 0.9996
y-Recip ocal 2.346 0.013 2.48 0.02 1.37 ±13.84 0.14 ±16.2 0.2 0.9997 0.9997
x-Recip ocal 2.36 0.01 2.50 0.01 1.39 ±13.7 0.4 ±16.1 0.5 0.999 0.998
50
o
C
Double ecip ocal 2.357 0.005 2.461 0.007 1.27 ±15.00 0.15 ±17.6 0.2 0.9999 0.9998
y-Recip ocal 2.344 0.013 2.48 0.02 1.36 ±14.8 0.2 ±17.4 0.2 0.9997 0.9997
x-Recip ocal 2.334 0.011 2.464 0.012 1.35 ±14.9 0.4 ±17.6 0.2 0.999 0.999
a) Binding cons an s (M
±1
) o he i s - and second-mig a ing enan iome s
b) Enan ioselec i i ies o complexa ion (a) we e calcula ed as a=K
2
/K
1
c) Elec opho e ic mobili ies o he enan iome -CD complex in cm
2
kV
±1
min
±1
o he i s - and second-mig a ing enan io-
me s
d) Squa ed co ela ion coe icien ob ained by he leas -squa es me hod
1
mim
1
mcm K
1
L1
mcm 4
Tables 3 and 4 lis he appa en binding cons an s, enan-
ioselec i i ies o complexa ion, and elec opho e ic mobi-
li ies o he complexed solu e, m
c
, calcula ed using Eqs.
(4)±(6) o he chi al compounds s udied and CM-g-CD.
The enan ioselec i i ies o complexa ion (a) [13] we e cal-
cula ed as a=K
2
/K
1
whe e K
1
and K
2
a e he binding con-
s an s o he i s - and second-mig a ing enan iome s
wi h CM-g-CD. In all cases, easonable esul s wi h
accep able e o limi s and good co ela ion coe icien s
we e achie ed; howe e , he co ela ion coe icien s ob-
ained by double and y- ecip ocal me hods we e, in gen-
e al, sligh ly highe han hose o he x- ecip ocal me h-
od.
As uniconazole and diniconazole a e s uc u ally simila ,
hey also ha e simila alues o he appa en binding
cons an s, hese cons an s being sligh ly lowe o unico-
nazole. In all cases, good enan ioselec i i ies o complex-
a ion we e achie ed (a³1.19). On he o he hand, bo h
binding cons an s and enan ioselec i i ies we e sligh ly
lowe wi h inc easing empe a u e. Finally, elec opho e ic
mobili ies o he complexed solu e, m
c
, inc eased wi h
inc easing empe a u e, in ag eemen wi h he inc ease in
he EOF caused by changes in he iscosi y.
Tempe a u e dependence o he binding cons an s can
be desc ibed as:
InKDHo
RT DSo
R7
whe e DH
o
is he en halpy change associa ed wi h inclu-
sion complex o ma ion, DS
o
is he co esponding en opy
change, and Ris he gas cons an . As shown by Guil-
laume and Pey in [18], he modynamic da a can gi e
in o ma ion abou he solu e-CD complexa ion mecha-
nisms. A e age loga i hms o appa en binding cons an s
de e mined om alues calcula ed by di e en plo ing
me hods a se e al empe a u es we e plo ed agains he
ecip ocal empe a u e, acco ding o Eq. (7), o gi e
s aigh lines. The e we e no a ailable he modynamic
da a in he li e a u e conce ning inclusion complex o ma-
ion o hese ungicides wi h CM-g-CD. The alues o DH
o
and DS
o
calcula ed om he abo e plo s a e lis ed in
Table 5 o uniconazole and diniconazole enan iome s
along wi h co ela ion coe icien s o he plo s and Gibbs
Elec opho esis 2000, 21, 3240±3248 Fas enan iome ic sepa a ion o uniconazole and diniconazole 3247
Table 4. Appa en binding cons an s and enan ioselec i i ies o complexa ion o diniconazole wi h CM-g-CD in 50 mM
phospha e bu e (pH 6.5) a se e al empe a u es
a)
Plo ing me hod log K
1
log K
2
am
c
(1) m
c
(2)
2
(1)
2
(2)
20
o
C
Double ecip ocal 2.700 0.006 2.83 0.01 1.36 ±9.78 0.06 ±11.42 0.05 0.9996 0.999
y-Recip ocal 2.700 0.013 2.82 0.02 1.31 ±9.78 0.05 ±11.47 0.05 0.9999 1.0
x-Recip ocal 2.701 0.008 2.831 0.014 1.35 ±9.8 0.2 ±11.4 0.5 0.999 0.998
30
o
C
Double ecip ocal 2.631 0.008 2.721 0.008 1.23 ±11.71 0.10 ±13.78 0.11 0.999 0.999
y-Recip ocal 2.62 0.03 2.71 0.03 1.23 ±11.8 0.2 ±13.8 0.2 0.999 0.9996
x-Recip ocal 2.631 0.014 2.721 0.014 1.23 ±11.7 0.5 ±13.8 0.6 0.997 0.997
40
o
C
Double ecip ocal 2.555 0.005 2.635 0.006 1.20 ±13.74 0.08 ±16.15 0.11 0.9998 0.9996
y-Recip ocal 2.540 0.014 2.61 0.02 1.19 ±13.87 0.10 ±16.32 0.14 0.9998 0.9998
x-Recip ocal 2.552 0.008 2.63 0.01 1.20 ±13.8 0.3 ±16.2 0.5 0.999 0.998
50
o
C
Double ecip ocal 2.509 0.008 2.588 0.009 1.20 ±15.2 0.2 ±17.8 0.2 0.9996 0.999
y-Recip ocal 2.53 0.02 2.61 0.03 1.20 ±15.0 0.2 ±17.6 0.2 0.9995 0.9995
x-Recip ocal 2.512 0.015 2.590 0.016 1.20 ±15.2 0.6 ±17.8 0.8 0.998 0.997
a) See Table 3 o abb e ia ions
Table 5. The modynamic pa ame e s o binding be-
ween uniconazole and diniconazole enan io-
me s and CM-g-CD in 50 mMphospha e bu e
a pH 6.5
Analy e DH
o
DS
o
a)
DG
o298
(kJ mol
±1
) J mol
±1
K
±1
) (kJ mol
±1
)
Uniconazole
b)
± 7.7

1.2 21

4 0.98 ±14

2
Uniconazole
c)
±10.5

1.1 15

3 0.99 ±14.8

1.4
Diniconazole
b)
±11.4

1.2 13

4 0.99 ±15

2
Diniconazole
c)
±14

25

7 0.98 ±16

3
a) Co ela ion coe icien ob ained by he leas -squa es
me hod
b) Fi s -mig a ing enan iome
c) Second-mig a ing enan iome
ee ene gies DG
o
o inclusion complex o ma ion a
25
o
C. Thus highe en opy (DS
o
) and lesse en halpy
(DH
o
) can be obse ed o he CM-g-CD complex wi h uni-
conazole enan iome s han o diniconazole enan iome s.
On he o he hand, he inclusion in o he ca i y o he CM-
g-CD is a o ed o diniconazole compa ed wi h he unico-
nazole (as DG
o298
alues show) in ag eemen wi h a
majo mig a ion ime o diniconazole enan iome s.
4 Concluding ema ks
The pai s o enan iome s o bo h uniconazole and dinico-
nazole ha e been success ully sepa a ed by EKC
employing CM-g-CD as a chi al selec o . The use o an
elec oly ic solu ion con aining 50 mMphospha e bu e a
pH 6.5 and 5 mMCM-g-CD a 50
o
C allows he as enan-
iosepa a ion o uniconazole and diniconazole mix u es in
abou 5 min. These esul s no ably imp o e hose
epo ed in he li e a u e desc ibing he enan iosepa a ion
o hese ungicides by CD-MEKC in 40 min. On he o he
hand, he high e iciency o CE combined wi h plo ing
me hods makes he es ima ion o appa en binding con-
s an s o enan iome s o uniconazole and diniconazole
wi h CM-g-CD a simple, s aigh o wa d p ocess. Binding
cons an s o bo h compounds a e simila , being sligh ly
lowe o uniconazole. Good enan ioselec i i ies o com-
plexa ion we e achie ed (a³1.19) in all cases. The es i-
ma ion o binding cons an s a se e al empe a u es has
enabled he de e mina ion o he modynamic pa ame e s
ela ed o he enan iome CM-g-CD complexa ion.
The au ho s hank he Comunidad Au ónoma de Mad id
(Spain) o p ojec 07M/0049/1998.
Recei ed Ap il 18, 2000
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3248 Y. Ma ín-Biosca, C. Ga cía-Ruiz and M. L. Ma ina Elec opho esis 2000, 21, 3240±3248