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. A., Diez-Masa, J. C., Ma ina, M. L.,
J. Mic ocol. Sep. 1997, 9, 87±95.
[12] Gila , M., Uh o µ, M., Tesa o µ, E., J. Ch oma og . B 1996,
681, 133±141.
[13] Schmi , T., Engelha d , H., J. High. Resol. Ch oma og .
1993, 16, 525±529.
[14] Leli e, F., Ga eil, P., Ja dy, A., Anal. Chem. 1997, 69,
385±392.
[15] Fanali, S., J. Ch oma og . A 1997, 792, 227±267.
Elec opho esis 2000, 21, 1565±1573 Enan iome ic sepa a ion o 1,4-dihyd opy idines 1573