Dynamic micellar electrokinetic chromatography. Determination of the enantiomerization barriers of oxazepam, temazepam, and lorazepam

Article abstract of DOI:10.1021/ac991439g

The temperature-dependent enantiomerization barriers of oxazepam, temazepam, and lorazepam have been determined between 0 and 30 °C by dynamic micellar electrokinetic chromatography (DMEKC) in an aqueous 20 mM borate/phosphate buffer system at pH 8 with 6

Full text of DOI:10.1021/ac991439g

Anal. Chem. 2000, 72, 2758-2764
Dyn a m ic Mic e lla r Ele c t ro k in e t ic Ch ro m a t o g ra p h y.
De t e rm in a t io n o f t h e En a n t io m e riza t io n Ba rrie rs o f
Ox a ze p a m , Te m a ze p a m , a n d Lo ra ze p a m
Ga brie le Sc hoe tz, Olive r Tra pp, a nd Volke r Sc hurig*
Universita¨t Tu¨bingen, Institut fu¨r Organische Chemie, Auf der Morgenstelle 18, 72076 Tu¨bingen, Germany
tography is defined as a method to study reversible interconversion
processes proceeding during the time scale of partitioning, and it
has previously been applied for the determination of enantiomer-
ization barriers.5-17 In enantioselective chromatography, peak
profiles such as plateau formation or peak broadening are
occasionally observed. This phenomenon is sometimes considered
as a complication of the separation process, and the undesired
peak shapes are suppressed by the addition of organic modifiers
or a change of the chiral stationary phase (CSP).18 Yet in dynamic
chromatography, the characteristic peak profiles not only repre-
sent a diagnostic tool for an interconversion process itself, but
are the prerequisite for the quantitative determination of enanti-
omerization barriers.
Dynamic chromatography allows the measurement of barriers
above 80 kJ mol-1 in a short time. In contrast to classical
enantiomerization experiments (chiroptical methods), which re-
quire large quantities of single or enriched enantiomers, the
determination of enantiomerization barriers by dynamic chroma-
tography can be performed with minute amounts of the racemic
sample. An indispensable requirement for the application of this
method is the quantitative on-column separation of the enanti-
omers, whereby the interconversion process takes place in a chiral
environment.5 Enantiomerization leads to peak broadening, plateau
formation, and eventually peak coalescence, depending on the
stereochemical lability of the chiral compound investigated. The
shape of the chromatogram of the resolved racemate is therefore
subject to the rate of interconversion of the two enantiomers and
the chromatographic time scale of enantioseparation. Depending
The temperature-dependent enantiomerization barriers of
oxazepam, temazepam, and lorazepam have been deter-
mined between 0 and 3 0 °C by dynamic micellar electro-
kinetic chromatography (DMEKC) in an aqueous 2 0 mM
borate/ phosphate buffer system at pH 8 with 6 0 mM
sodium cholate as chiral surfactant. Interconversion
profiles featuring plateau formation and peak broadening
were observed and sim ulated by the new program
ChromWin based on the theoretical plate as well as on
the stochastic model using the experimental data plateau
height, h plateau, peak width at half-height, wh , total reten-
tion times, tR, and electroosmotic breakthrough time, t0 .
P eak form analysis yielded rate constants k and kinetic
activation parameters, ∆Gq, ∆Hq, and ∆Sq, of the enan-
tiomerization of oxazepam, temazepam, and lorazepam.
At 2 5 °C, the enantiomerization barrier, ∆Gq, was deter-
mined to be ∼9 0 kJ mol-1 and the half-lives, τ, were
determined to be approximately 2 1 min. The new ap-
proach allows the fast and precise determination of
enantiomerization barriers in a biogenic environment and
it mimics physiological conditions, as no organic modi-
fiers or abiotic chiral stationary phases (CSP ) are em-
ployed.
Policy statements of regulatory authorities such as the Ameri-
can Food and Drug Administration (FDA) not only restrict the
sale of racemic drugs but demand unambiguous data on the
configurational stability of pure enantiomers. Consequently, drug
manufacturers are bound to assess the “stereochemical integrity”
of enantiomers and to examine the “potential for interconver-
sion...of the individual isomers”.1,2
Enantiomerization, in contrast to racemization, constitutes a
reversible first-order reaction.3 It arises from the interconversion
of a stereogenic element in a particular molecule. Evaluation of
the stereochemical integrity of chiral compounds can be achieved,
inter alia, by employing enantioselective chromatography on chiral
stationary phases (CSPs),4 a technique which is generally used
for the determination of enantiomeric ratios. Dynamic chroma-
(5) Bu¨ rkle, W.; Karfunkel, H.; Schurig, V. J. Chromatogr. 1 9 8 4 , 288, 1-14.
(6) Jung, M.; Schurig, V. J. Am. Chem. Soc. 1 9 9 2 , 114, 529-534.
(7) Trapp, O.; Schurig, V. J. Am. Chem. Soc. 2 0 0 0 , 1424-1430.
(8) Wolf, C.; Ko¨ nig, W. A.; Roussel, C. Liebigs Ann. Chem. 1 9 9 4 , 781-786.
(9) Wolf, C.; Pirkle, W. H.; Welch, C. J.; Hochmuth, D. H.; Ko¨ nig, W. A.; Chee,
G.-L.; Charlton, J. L. J. Org. Chem. 1 9 9 7 , 62, 5208-5210.
(10) Mannschreck, A.; Zinner, H.; Pustet, N. Chimia 1 9 8 9 , 43, 165-166.
(11) Veciana, J. I.; Crespo, M. Angew. Chem., Int. Ed. Engl. 1 9 9 1 , 30, 54-77.
(12) Cabrera, K.; Jung, M.; Fluck, M.; Schurig, V. J. Chromatogr., A. 1 9 9 6 , 731,
315-321.
(13) Friary, R. J.; Spangler, M.; Osterman, R.; Schulman, L.; Schwerdt, J. H.
Chirality 1 9 9 6 , 8, 364-371.
(14) Gasparrini, F.; Misiti, D.; Pierini, M.; Villani, C. Tetrahedron: Asymmetry
1 9 9 7 , 8, 2069-2073.
(15) Oxelbark, J.; Allenmark, S. J. Org. Chem. 1 9 9 9 , 64, 1483-1486.
(16) Jung, M. Program Simul, No. 620, Quantum Chemistry Program Exchange.
QCPE Bull. 1 9 9 2 , 3, 12.
* Corresponding author:Phone:+49-7071-2976257. Fax:+49-7071-295538.
e-mail:volker.schurig@uni-tuebingen.de
(1) FDA. Chirality 1 9 9 2 , 4, 338-340.
(2) De Camp, W. H. Chirality 1 9 8 9 , 1, 2-6.
(3) Reist, M.; Testa, B.; Carrupt, P. A.; Jung, M.; Schurig, V. Chirality 1 9 9 5 , 7,
(17) Schoetz, G.; Trapp, O.; Schurig, V. Enantiomer, accepted for publication.
(18) Boonkerd, S.; Detaevernier, M. R.; Michotte, Y.; Vindevogel, J. J. Chro-
matogr., A 1 9 9 5 , 704, 238-241.
396-400.
(4) Schurig, V.; Bu¨ rkle, W. J. Am. Chem. Soc. 1 9 8 2 , 104, 7573-7580.
2758 Analytical Chemistry, Vol. 72, No. 13, July 1, 2000
10.1021/ac991439g CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/16/2000

on the enantiomerization temperature, different chromatographic
techniques (e.g., DGC, DHPLC, DMEKC) may be applied for the
quantification of the enantiomerization process at a given time
and temperature.
on-column UV detector (Bischoff Lamda 1000, Leonberg, Ger-
many) and a thermostated homemade water cooling system with
integrated temperature control (Haake D8-GH, Haake, Karlsruhe,
Germany). The effective length of the fused silica capillary
(Microquartz, Munich, Germany) was 95 cm (total length 112),
the temperature-regulated length was 76 cm, and the inner
diameter was 50 µm. Unless otherwise stated, 60 mM sodium
cholate (SC), as a chiral surfactant, introduced by Terabe et
al.,18,23-26 was used dissolved in 20 mM borate/ phosphate buffer
solution at a pH of 8. The temperature of the thermostated zone
ranged from 0 to 30 °C. UV detection was performed at 230 nm.
Peak integration was carried out with a Chromatopak C-R6A
integrator (Shimadzu, Kyoto, Japan).
Untreated silica capillaries were conditioned for 30 min with
0.1 M NaOH solution. Afterward, the capillary was purged with
running buffer for 30 min. Between injections, the capillary was
rinsed with running buffer for 5 min. Before any change of analyte
or buffer concentration, the capillary was treated with 0.1 M NaOH
solution for 10 min, followed by running buffer for 30 min.
Injections were performed hydrodynamically at the anodic side
by applying a pressure of 20 mbar for two seconds. A voltage of
+25 kV was used for separation.
Computer Simulation. For the calculation of the enantiomer-
ization barrier, the plateau height, hplateau; peak width at half-height,
wh; total retention times, tR, of the enantiomers; and the electroos-
motic break through time, t0, (using dimethylformamide as a
marker) of the experimental chromatograms were used. Since
the peak profiles of interconverting enantiomers are broadened
(tailing of the first eluted enantiomer and fronting of the second
eluted enantiomer), plate numbers have to be adjusted for
simulation to fit the experimental chromatogram. While the use
of effective plate numbers, Neff, in the simulation leads to peak
widths of the simulated chromatogram that are too large, the use
of the theoretical plate numbers, Nth, leads to peak widths that
are too small. Therefore, as a compromise, mean plate numbers
Nh, calculated from the modified eq 1,15 were applied. This equation,
established for gas-chromatographic two-phase systems,16 can be
adopted here because of the long retention time of the micellar
pseudo stationary phase (using Sudan III as a marker) and the
relative insensitivity of the simulation program toward errors in
plate numbers, N.
By comparison of experimental and computer-simulated chro-
matograms, kinetic activation parameters (∆Hq and ∆Sq) of
enantiomerization are obtained. The first simulation program was
published in 19845 and based on the theoretical plate model; later
it was extended to simulations of up to 120 000 effective plates
(SIMUL).6,16 The stochastic model,19-21 which treats peaks as a
Gaussian curve and utilizes a probability distribution to describe
the interconverting species of the enantiomers, has also been
applied for the determination of enantiomerization barriers.10,11
The new program ChromWin22 allows simulation with the theo-
retical plate model, the stochastic model, and a modified stochastic
model and is running on a personal computer under Windows. It
allows the simulation of chromatograms with large plate numbers
usually obtained by capillary gas chromatography and capillary
electromigration methods.
In the present study, dynamic micellar electrokinetic chroma-
tography (DMEKC) was applied to determine the enantiomeriza-
tion barrier of the chiral benzodiazepine drugs oxazepam,
temazepam, and lorazepam in a single-phase system whereby the
chiral surfactant is dissolved in the running buffer. The enantio-
merization barriers were determined by comparing elution profiles
obtained experimentally by DMEKC in the presence of sodium
cholate with elution profiles simulated with the ChromWin
program. The present examples were selected (i) to show the
applicability of DMEKC as a general method for the determination
of enantiomerization barriers, (ii) to demonstrate the efficiency
of the simulation program at large plate numbers, and (iii) to prove
the applicability of both the theoretical plate model and the
stochastic model to one-phase systems involving chromatographic
partitioning in the presence of micelles (MEKC).
EXPERIMENTAL SECTION
Materials. The tranquilizers, oxazepam, temazepam, lorazepam,
and the sodium phosphate (NaH2PO4 99%) and sodium borate
(Na2B4O7 ‚10 H2O 99%) buffer salts were purchased from Sigma-
Aldrich (Deisenhofen, Germany). The sodium salt of 3R,7R,12R-
trihydroxy-5â-cholic-acid (purum) was obtained from Fluka (Buchs,
Switzerland). One molar HPLC grade sodium hydroxide solution
(E. Merck, Darmstadt, Germany) was diluted with 18.2 MΩ high
purity water obtained from a Millipore-Q System (Millipore,
Marlborough, Massachusetts). High-purity water, 18.2 MΩ, was
also used to prepare the borate and phosphate buffer solutions.
All benzodiazepine samples were dissolved in HPLC-grade metha-
nol (E. Merck, Darmstadt, Germany), and the concentration was
adjusted to 0.1 mg/ mL. Before use, all sample and buffer solutions
were passed through a 0.45-µm disposable filter cartridge (Chro-
mafil, Machery and Nagel, Du¨ ren, Germany).
t (tR - tM)
Nh ) 5.545 R
(1)
(wh)2
The initially injected amounts of the enantiomers were set equal
for the racemic benzodiazepine samples. Peak form analysis was
performed with ChromWin according to the theoretical plate
model and the modified stochastic model using an improved
Newton algorithm in order to find the best agreement of the
simulated and experimental elution profiles in only a few simula-
tion steps. The program furnishes the apparent rate constants,
Capillary Electrophoresis. The separation of the enantiomers
of the three benzodiazepines was carried out with a Prince Unicam
Crystal 300/ 31 capillary electrophoresis system equipped with an
(23) Terabe, S.; Otsuka, K.; Ando, T. Anal. Chem. 1 9 8 5 , 57, 834-841.
(19) Keller, R. A.; Giddings, J. C. J. Chromatogr. 1 9 6 0 , 3, 205-220.
(20) Kramer, R. J. Chromatogr. 1 9 7 5 , 107, 241-252.
(24) Terabe, S.; Shibata, M.; Miyashita, Y. J. Chromatogr., A 1 9 8 9 , 480, 403-
411.
(21) Cremer, E.; Kramer, R. J. Chromatogr. 1 9 7 5 , 107, 253-263.
(22) Trapp, O.; Schurig, V. Comput. Chem. 2 0 0 0 , 24, in press. Information
available from the authors upon request.
(25) Otsuka, K.; Terabe, S. J. Chromatogr., A 1 9 9 0 , 515, 221-226.
(26) Muijselaar, P. G.; Otsuka, K.; Terabe, S. J. Chromatogr., A 1 9 9 7 , 780, 41-
61.
Analytical Chemistry, Vol. 72, No. 13, July 1, 2000 2759

Fig u re 1 . Principle of microscopic reversibility. Equilibria in a
theoretical plate: A is the first eluted enantiomer, B is the second
eluted enantiomer, k represents the rate constant, and K represents
the distribution constant.
app
ka1pp and
micelle.
k
k
, of enantiomerization in the presence of the chiral
was calculated via eq 2 from ka1pp according to the
Fig u re 2 . Ohm’s Law plot at temperatures from 10 to 25 °C of a
60 mM borate/phosphate buffer solution at pH 8.
-1
app
-1
principle of microscopic reversibility3,5 shown in Figure 1. The
Internal Joule heating also leads to peak broadening, loss in
resolution, and random enantiomerization of the enantiomers.
Therefore, to gain temperature constancy, low ionic strength
running buffers, small inner diameters of the capillaries, and low
voltages may be used. However, despite the fact that all of these
steps lead to a reduction in Joule heat, they do so at the expense
of longer running times, increased peak broadening, decreased
sensitivity, and lower resolution. Another strategy to reduce the
side effects of Joule heating is an effective external cooling system.
A standard air-ventilated system can hardly cope with the exces-
sive heat generated within the capillary; this frequently causes
temperature fluctuations, especially at higher buffer concentra-
tions. Even commercial liquid cooled systems27 cannot entirely
compensate for heating effects, because the heat is not dissipated
rapidly enough or the heat capacity of the coolant is too low. Water
cooled systems, which have a larger heat capacity, are usually
avoided in CE, because of the risk of a capillary breaking at high
voltages.
apparent rate constants, ka1pp and
k
, arise from the rate
app
-1
constant kdiss in the dissolved state and kmic in the micellar state
according to eqs 3 and 4. The retention factors k′A of the first
eluted enantiomer A and k′B of the second eluted enantiomer B
have been calculated from the total retention time tR and the
electroosmotic break through time t0 according to k′ ) (tR - t0)/
t0.
ka1pp
k′B
)
(2)
app
k′A
k
-1
k′A
1
ka1pp
)
)
kdis
+
km1 ic
(3)
(4)
1 + k′A
1 + k′A
k′B
1
app
mic
k
kdis
+
k
-1
-1
1 + k′B
1 + k′B
In the present study a very effective and inexpensive home-
made aqueous cooling system was used in order to perform
reliable temperature-dependent measurements. The hazard of
accidental capillary breaking was minimized by coating the outer
wall of the fused silica capillary with nonconductive PTFE before
inserting it into a water-filled PE tube which was connected to
the thermostat. Because of the high pump efficiency (12 L/ min),
the generated heat was removed instantaneously. As a result, the
resolution factor, Rs, of the separations increased and the enan-
tiomerization conditions remained stable and reproducible as
compared with those of the conventional methodology.
The apparent rate constants were evaluated from as many as
187 chromatograms. As the process of enantiomerization is
defined as a reversible first-order microscopic process with 2 kenant
) krac, a statistical transmission factor, κ, of 0.5 was used in
calculating the Gibbs activation energy, ∆Gq(T),3,12 from the rate
constant ka1pp according to the Eyring equation, eq 5.
Enantiomerization studies were performed at different tem-
peratures. According to the Gibbs-Helmholtz equation, ∆Gq(T)
) ∆Hq - T∆Sq, the activation enthalpy, ∆Hq, was obtained via
the slope and the activation entropy, ∆Sq, via the intercept of the
Eyring plot (T-1 versus ln(kapp/ T)).
Evaluation of the efficacy of the developed cooling system was
performed by the Ohm’s Law relationship. Linearity in the Ohm
plot is only achieved at a constant resistance. As Joule heating
increases resistance, the current will deviate from linearity at
increased voltage. Figure 2 shows highly linear Ohm plots (Figure
2, agreement factor > 0.9999) at different temperatures for a 60
mM borate/ phosphate buffer solution at pH 8, thus indicating the
absence of Joule heating in the thermostated system. The current
was measured 5 min after the voltage was applied. Following each
measurement, the capillary was flushed with buffer solution for 5
min at 1 bar. The temperature of the buffer solution was adjusted
to 20 °C for all experiments.
ka1pp
h
∆G(T) ) -RT ln
(5)
(
)
κ kBT
with κ as the transmission factor (κ ) 0.5), kB as the Boltzmann
constant (kB ) 1.380662 × 10-23 J‚K-1), T as the enantiomerization
temperature [K], h as Planck’s constant (h ) 6.626176 × 10-34
J‚s), and R as the gas constant (R ) 8.31441 J‚K-1‚mol-1).
RESULTS
Temperature Regulation. To conduct reliable kinetic studies,
Optimization of Surfactant and Buffer Concentrations. To
optimize conditions of enantiomer separation, various buffer and
precise control of the temperature is mandatory. Temperature
control in capillary electrophoresis is difficult because of the strong
internal Joule heating within the capillary, which influences the
mobility of the analytes and the reproducibility of the separation.
(27) Palmieri, R. H. Technical information T-1823-A; Beckman Instruments:
Fullerton, California, USA 1996.
2760 Analytical Chemistry, Vol. 72, No. 13, July 1, 2000

Fig u re 3 . Concentration dependency of the separation factor R of
the enantiomer separation of oxazepam. Chiral surfactant: sodium
cholate. Buffer: borate/phosphate at pH 8. Column: 95 cm × 50 µm
(i.d.). U ) 25 kV. λ ) 230 nm. T ) 10 °C.
Fig u re 6 . Experimental and simulated chromatograms of the
simultaneous enantiomer separation of temazepam (Tm, left) and
oxazepam (Ox, right) at different temperatures by DMEKC. Chiral
surfactant: 60 mM sodium cholate. Buffer: 60 mM borate/phosphate
at pH 8. Column: 95 cm × 50 µm (i.d.). U ) 25 kV. λ ) 230 nm.
Fig u re 4 . Concentration dependency of retention times, tR, of the
enantiomer separation of oxazepam. Chiral surfactant: sodium
cholate. Buffer: borate/phosphate at pH 8. Column: 95 cm × 50 µm
(i. d.). U ) 25 kV. λ ) 230 nm. T ) 10 °C.
Fig u re 5 . Structural formulas of the investigated benzodiazepines.
From left to right: oxazepam, temazepam, and lorazepam.
Fig u re 7 . Experimental and simulated chromatograms of the
simultaneous enantiomer separation of lorazepam at different tem-
peratures by DMEKC. Chiral surfactant: 60 mM sodium cholate.
Buffer: 20mM borate/phosphate at pH 8. Column: 95 cm × 50 µm
(i. d.). U ) 25 kV. λ ) 230 nm.
surfactant concentrations were examined. Figure 3 shows the
variation of buffer concentrations (borate/ phosphate, pH 8), at a
constant surfactant concentration (sodium cholate, SC) of 60 mM
and for increasing SC concentrations at a buffer concentration of
20 mM. Whereas increase in buffer concentration leads to an
increase in the separation factor R, a higher surfactant concentra-
tion results in a deterioration of the separation process. The
optimum separation conditions were achieved at low surfactant
and high buffer concentrations.
The concentration dependency of the retention times is shown
in Figure 4. Elevated concentrations of buffer and surfactant lead
to increasing retention times, whereby the rising buffer concentra-
tion shows a larger effect.
Finally, a buffer concentration of 20 mM and a surfactant
concentration of 60 mM were chosen as a compromise between
long retention times and a satisfactory enantiomer separation.
Enantiomerization. According to Figures 6 and 7, enanti-
omerization of oxazepam, temazepam, and lorazepam is recog-
nized by characteristic plateau formation between 5 and 25 °C.
The plateau arises from molecules which interconvert during the
chromatographic time scale. For all investigated benzodiazepines
(cf. Figure 5), peak coalescence commences at 30 °C, and a single
broadened peak is observed for the enantiomers, whereas below
0 °C the usual baseline separation occurs. Chromatograms
measured at these borderline temperatures were not utilized for
computer simulation.
Computer Simulation. The chromatograms were simulated
both with the theoretical plate model16 and the modified stochastic
model of ChromWin using the experimental data plateau height,
h
plateau; peak width at half-height, wh; total retention times, tR, and
electroosmotic break through time, t0. Kinetic activation data of
enantiomerization were obtained by peak form analysis and
iterative comparison of experimental and simulated chromato-
grams. The application of the principle of microscopic reversibility4
Analytical Chemistry, Vol. 72, No. 13, July 1, 2000 2761

Ta b le 1 . S e le c t e d Ex p e rim e n t a l Da t a a n d Ca lc u la t e d Ra t e Co n s t a n t s o f t h e En a n t io m e riza t io n s o f Ox a ze p a m ,
Te m a ze p a m , a n d Lo ra ze p a m Ob t a in e d b y Ch ro m Win o n t h e Ba s is o f t h e Mo d ifie d S t o c h a s t ic Mo d e l1 6
app
no.
compound
T [°C]
tR1
tR2
Nh1
Nh2
hplateau[%]
ka1pp
k
-1
1
2
3
4
5
6
7
8
9
oxazepam
oxazepam
oxazepam
temazepam
temazepam
temazepam
lorazepam
lorazepam
lorazepam
10
15
20
10
15
20
10
15
20
57.8
52.9
45.7
53.2
49.6
43.2
44.5
38.4
34.7
59.4
54.3
46.7
54.5
50.7
44.1
45.1
39.0
35.2
179 000
192 000
190 000
157 000
225 000
280 000
209 000
214 000
236 000
225 000
248 000
221 000
149 000
240 000
320 000
241 000
268 000
270 000
15
25
44
11
18
34
9
1.12 × 10-4
2.05 × 10-4
3.29 × 10-4
7.30 × 10-5
1.48 × 10-4
3.05 × 10-4
9.64 × 10-5
1.86 × 10-4
3.07 × 10-4
1.07 × 10-4
1.79 × 10-4
3.15 × 10-4
6.94 × 10-5
1.41 × 10-4
2.92 × 10-4
7.38 × 10-5
1.80 × 10-4
2.97 × 10-4
25
42
Ta b le 2 . Kin e t ic Ac t iva t io n P a ra m e t e rs o f t h e En a n t io m e riza t io n o f Ox a ze p a m , Te m a ze p a m , a n d Lo ra ze p a m a t 2 9 3
K a n d p H 8
compound
∆Gq [kJ mol-1
]
∆Hq [kJ mol-1
]
∆Sq [J (K mol)-1
]
ka1pp [10-4 s-1
]
t1/ 2 [min]
oxazepam
temazepam
lorazepam
91.3 ( 0.1
91.6 ( 0.2
91.4 ( 0.1
72.0 ( 0.5
90.5 ( 0.7
76.5 ( 0.6
-65.8 ( 9
-3.9 ( 12
-50.9 ( 8
3.3
2.9
3.1
34.9
39.8
37.3
Ta b le 3 . Re s u lt s o f Kin e t ic Ac t iva t io n Ba rrie rs o f In d e p e n d e n t S t u d ie s
compound
oxazepam
method/ conditions
T [K]
∆Gq[kJ mol-1
]
ka1pp [10-4 s-1
]
t1/ 2[min]
ref
DHPLC/ methanol/
phosphate buffer/ pH 2.8
ethanolysis/
298
89.0
11
30
oxazepam
298
92.1
11.8 ( 0.2
CD spectropolarimetry
racemization/ HPLC
ethanolysis/ HPLC/
0.1 M H2SO4
oxazepam
oxazepam
273
298
0.268
∼430
32
35
94.6
95.0
90.8
55.5 ( 0.2
temazepam
temazepam
ethanolysis/
298
298
38.6 ( 0.3
12.7 ( 0.3
30
34
CD spectropolarimetry
ethanolysis/ HPLC/
CD spectropolarimetry/
0.5 M H2SO4
lorazepam
ethanolysis/ HPLC
298
16.8 ( 0.8
28
requires that the rate constants of interconversion of the two
enantiomers are different in the presence of the chiral surfactant.
Thus, whereas the second eluted enantiomer is enriched at the
chromatographic time scale as it is formed more rapidly than the
app
-1
first eluted enantiomer (k1app
>
k
), no overall deracemization
occurs because the second enantiomer is depleted to a greater
extend due to its longer residence time in the column. Examples
for the experimental data used for the simulations are depicted
in Table 1. The apparent rate constants obtained from the
simulation are displayed graphically as an Eyring plot (Figure 8,
agreement factor > 0.993). From the slope of this graph, the
kinetic activation parameters given in Table 2 are obtained. The
errors for the activation parameters ∆Hq and ∆Sq were determined
from the standard deviations due to equations given by Sand-
stro¨ m.28
It is noteworthy that the values for ∆Gq of oxazepam,
temazepam, and lorazepam are comparable to each other, whereas
the ∆Hq and ∆Sq values are different. The better agreement
between the enthalpy and entropy values for oxazepam and
lorazepam may be explained by their close structural relationship.
However, the methyl group with its electron donating properties
Fig u re 8 . Eyring plot of the simulated apparent rate constant, k1a
of enantiomerization of oxazepam, temazepam, and lorazepam.
,
pp
in position 1 in temazepam seems to influence the interconversion
in regard to ∆Hq and ∆Sq.
In principle, the calculated rate constant also allows the
determination of the enantiomeric excess (ee) of nonracemic
samples before separation even if the enantiomers are intercon-
verting during the time scale of partitioning. Figure 9 displays
the simulated chromatogram of the pure enantiomer A, intercon-
app
-1
verting at a rate constant of
example 3 in Table 1).
k
) 3.15 × 10-4 s-1 (data from
(28) Sandstro¨ m, J. Dynamic NMR Spectroscopy; Academic Press: London, 1982;
111-112.
2762 Analytical Chemistry, Vol. 72, No. 13, July 1, 2000

Fig u re 9 . Simulated chromatograms of the first eluted enantiomer of oxazepam with an enantiomeric excess (ee) of 100% (left), 80% (middle),
and 60% (right) (data from example 3 in Table 1) with interconversion taking place during the separation process.
are compatible with a one-phase system involving partitioning
equilibria in a micelle (DMEKC).
An explanation for this phenomenon is the existence of a
pseudo-stationary phase, as already proposed by Terabe et al.,23
which serves as an interphase within the one-phase system and
is therefore comparable to a CSP in an ordinary enantiomeric
separation.
Accuracy and P recision. The determined enantiomerization
barriers ∆Gq of oxazepam compare well with data previously
reported from independent methods,12,29,34 e.g., DHPLC,12 pola-
rimetry,27,33 and the investigation of the racemization kinetics of
hydrolysis of optically pure salts,29 provided the difference between
the terms enantiomerization and racemization is considered.3,12
A comparison of the enantiomerization barriers of benzodiazepines
obtained by other methods described in the literature is given in
Table 3. As the data compare well to those determined, the
accuracy and applicability of the present approach employed in a
chromatographic one-phase system based on electromigration
(DMEKC) is demonstrated.
The precision of the DMEKC experiment was determined as
follows. The enantiomerization of all three benzodiazepines was
repeated at least 5 times at exactly the same experimental
conditions. The repeatability of the method is quite satisfactory
(2.93 σ (standard deviation); error of ∆Gq(19 °C) ) 0.41 kJ‚mol-1
at 10 °C). The precision of the enantiomerization experiment was
then rechecked after 6 months with an identical system, but a
Fig u re 1 0 . Comparison of simulations obtained by the modified
stochastic model (dotted line) and the theoretical plate model (plain
line) (data from example 3 in Table 1). The differences of the two
chromatograms arise from the different plate numbers used for
simulation. For the theoretical plate model the average of the mean
plate numbers was used, whereas true mean plate numbers were
used for the stochastic model.
(29) Yang, S. K. Enantiomer 1 9 9 8 , 3, 485-490.
(30) Barret, J.; Franklyn Smyth, W.; Davidson, I. E. J. Pharm. Pharmacol. 1 9 7 2 ,
25, 387-393.
(31) Yang, S. K.; Lu, X.-L. Chirality 1 9 9 2 , 4, 443-446.
(32) Yang, S. K.; Tank, R.; Pu, Q.-L. J. Labelled Compd. Radiopharm. 1 9 9 6 , 38,
753-759.
Applicability to One-P hase Systems. As demonstrated in
Figure 10, both the theoretical plate model and the stochastic
model yield essentially the same simulated chromatograms. Thus,
although both models have been derived for chromatographic two-
phase systems, the obtained results demonstrate that both models
(33) Yang, S. K. Chirality 1 9 9 9 , 11, 179-186.
(34) Yang, T. J.; Yang, S. K. J. Food Drug Anal. 1 9 9 5 , 3, 173-183.
Analytical Chemistry, Vol. 72, No. 13, July 1, 2000 2763

new capillary. Within the given errors the same enantiomerization
barriers as previously observed were determined.
between 80 and 120 kJ mol-1. The temperature constancy
throughout each run, achieved through efficient thermostating,
permits the use of high ionic strength running buffers and thus
helps to improve peak resolution.
CONCLUSION
DMEKC represents a useful tool for the measurement of
enantiomerization barriers under physiological conditions. This
is an advantage compared with DHPLC, which frequently uses a
large amount of organic solvents or aqueous systems containing
organic modifiers. In contrast to classical enantiomerization
studies, the method described does not require enantiomerically
enriched material or even the pure enantiomers.
ACKNOWLEDGMENT
This work was supported by the Deutsche Forschungsgemein-
schaft, Fonds der Chemischen Industrie and the Graduiertenkol-
leg “Chemistry in Interphases”. O.T. thanks the Stiftung Stipendien-
Fonds der Chemischen Industrie for a doctorate scholarship.
The temperature applied is only limited by the boiling and
freezing points of the buffer solution and the solubility of the chiral
surfactant. The flexible temperature range from -10 to +80 °C
allows the determination of enantiomerization barriers, ∆Gq,
Received for review December 15, 1999. Accepted March
20, 2000.
AC991439G
2764 Analytical Chemistry, Vol. 72, No. 13, July 1, 2000