Download CHROMATOGRAPHIC SEPARATION OF SECNIDAZOLE

CHROMATOGRAPHIC
SEPARATION
OF
SECNIDAZOLE
RACEMATE USING A SIMULATED MOVING BED
A. C.NASCIMENTO1, W.M.FERRARI1, R.F.PERNA2, M. A. CREMASCO1
1
University of Campinas, Chemical Engineering School, Department of Process Engineering
2
University of Alfenas, Institute of Science and Technology
Contact email: [email protected]
ABSTRACT – The simulated moving bed continuous separation process that increases
throughput, purity, and yield compared to batch chromatography is considered as an
important preparative technique to purify pharmaceutical drugs. Secnidazole is a chiral
drug marketed in the racemic form but pharmacological effects due to molecule chirality
indicate that one of the enantiomers is more potent. Preparative separation of the
secnidazole enantiomers was performed by a continuous simulated moving-bed (SMB)
chromatographic unit. It was used an experimental technique called shortcut technique,
that allows the rigorous solution of the Equilibrium Theory model to be approximated
without knowing the adsorption equilibria. Amylose tris(3,5-dimethylphenylcarbamate)
functioned as the stationary phase and acetonitrile-isopropanol mixtures as mobile
phases. The enantiomeric purity obtained were 72.5 % for R-(-)-secnidazole and 85.5 %
for S-(+)-secnidazole in the raffinate and extract streams, respectively.
1. INTRODUCTION
The interest in chirality and its consequences is not a new phenomenon. However, during the
last decade it has raised increasing expectations due to scientific and economic reasons, being the
pharmaceutical industry the main contributor and driving force (Maier et al., 2001; Nascimento et al.,
2012). The study in this area contributed to the design and synthesis of new pharmaceutical products,
since the use of enantiomerically pure chiral drugs can be advantageous by allowing a reduction in the
total dose administered, the simplification in the dose-response relationships, the removal of the
source of intersubject variability and reduction in toxicity due to inactive isomer (Caldwell, 1996).
The percentage of chiral commercially available drugs is increasing and now, 9 of the top 10 drugs
have ingredients chiral active, and consequently, the development of various approaches to the
separation of optically active chiral compounds has become a growing focus of research (Fu et al.,
2013).
Secnidazole (1-(hydroxypropyl)-2-methyl-5-nitroimidazole) is one of the newest nitroimidazole
derivatives (Figure 1) and it is used in hepatic amoebiasis, giardiasis and bacterial vaginosis. It has
been reported that some imidazole enantiomers are characterized as pharmacologically active and the
other toxic or inactive (Nascimento et al. 2012).
The use of chromatographic technique to obtain significant quantities of enantiomerically pure
drug intermediates is well established and a simulated moving bed (SMB) chromatography in recent
years has become a routine technique for the separation of enantiomers (Wang and Ching, 2002). The
selection of SMB operating conditions is not a straightforward task, because it depends on each
separation process in particular, and role information on the balance and aspects of mass transfer. The
main problem is the correct choice of solid flows (represented by the time of exchange of power and
removal of positions) and liquid (Rodrigues and Pais, 2004). Obtaining these parameters is of
paramount importance, even before starting the unit. Herein are included the determination of the
power flow of the sample and desorbent and the flow of refined and extract withdrawal, besides swap
time the positions of the currents in the system. Designed for high throughput separation, the SMB
units often operate with high concentration of power, which leads to non-linear behavior in the
competitive adsorption of the enantiomers. Therefore, modeling and simulation tools are crucial
before carrying out experiments on the system. Different techniques for obtaining these parameters
are reported in the literature, it may be noted the triangle method, the method of separation volume,
the concept of standing waves, and finally, the shortcut technique. The method of the triangle, while a
greater spread in the literature, is not able to unite, at the operating conditions, the purity of the
desired product flow rates and lengths of the separating zones and mass transfer parameters, because
they and the effects of axial dispersion are neglected. Therefore, the methodology can be applied to
obtain only an initial guess for the optimization of SMB. Moreover, the methodology of the
separation volumes, also based on equilibrium theory, improves the triangle method, since they are
considered mass transfer effects and importance of the flow rates in zones I and IV SMB unit when
determining the full area of separation. The concept of standing waves, in turn, has been developed in
order to overcome the disadvantages mentioned by methods based on equilibrium theory, it unites the
purity and recovery of products of axial dispersion, the resistance to mass transfer, the lengths and
superficial velocity of the separation zones and the speed of the moving bed, providing good
performance and reduced solvent consumption. Moreover, its implementation is more complex than
the ones mentioned in the previous paragraph. Finally, the shortcut technique, it becomes more
simple, practical in terms of implementation, once the operational parameters of a SMB unit may be
determined by a long pulse tests at different flow rates, without prior knowledge of the transfer
parameters mass and adsorption isotherm (Nascimento, 2012).
1.1. Shortcut technique
The shortcut technique is characterized as an empirical strategy for obtaining operational
parameters of a SMB unit (Mallmann et al., 1998;. Migliorini et al., 2002; Xie et al., 2003). Their
application depends solely on the analysis of the concentration distribution of the components of a
binary mixture, generated from long pulse tests (high amount of solute) in different flows. Obtaining
of the adsorption isotherm parameters and mass transfer for each component is unnecessary, the
method by imposing more practical and applications in less than ideal separation systems.
Mallmann et al., 1998 and Migliorini et al., 2002 proposed similar shortcut experimental
methods for SMB design, which did not require the isotherm parameters. In both studies, frontal
experiments and hodograph analysis were used, and mass-transfer effects were neglected. In this
study, an experimental design procedure is proposed to include the mass-transfer effects in the design.
The following equations are needed to derive the design equations for an SMB process: (1)
correlation between the shock-wave velocity and the flow rate; (2) correlation between the masstransfer zone length and the flow rate; and (3) correlation between the diffuse wave velocity and the
flow rate. These empirical correlations, which can be estimated from the long pulse including frontal
and elution tests of a binary mixture, are given with unknown coefficients as follows
u sh,1  x1 F
(1a)
u sh, 2  x 2 F
(1b)
LMTZ ,1  x3 F  y 3
(2a)
LMTZ , 2  x 4 F  y 4
(2b)
u diff ,1  x5 F  y 5
(3a)
u diff , 2  x 6 F  y 6
(3b)
where u is the wave velocity (m/s) and x and y are coefficients estimated from long pulse tests;
subscripts sh, diff, and MTZ denote shockwave, diffuse wave, and mass-transfer zone, respectively.
On the basis of the above correlations, the SMB design procedure is as follows:
(1) The concentration waves migrate by one column length between two consecutive switchings
in SMB. To maintain the shockwave of component 2 in zone III, LIII is set equal to LMTZ,2 + Lc . As an
SMB approaches a true moving bed, Lc approaches zero and LIII equals LMTZ,2. The flow rate of zone
III (FIII) can be estimated from Eq. 4 with the LMTZ,2. (2) Calculate the shockwave velocity (ush) of
component 2 from Eq. 1b by setting F equal to FIII. (3) In order to let the adsorption wave of
component 2 stand in zone III, the average port velocity (v) is set equal to (ush) of component 2. (4)
Similarly,v is set equal to ush of component 1 and the zone IV flow rate (FIV) is estimated from Eq.
1a. (5) For a given feed flow rate (FF), the flow rate of zone II (FII) is given by the difference between
FIII and FF. (6) The zone I flow rate (FI) is estimated from Eq. 6 by setting the diffuse wave velocity
(udiff ) of component 2 equal to v , such that the diffuse wave of component 2 stands in zone I.
The following design equations are derived based on the above procedure:
FI 
v  y6 
x6
F II  F III  FF
(4a)
(4b)
F III 
F
III

 Lc  y4
x4
(4c)
v
x1
(4d)
v  u sh  x 2 F III
(4e)
F IV 
According to the preceding design step 5, the zone II flow rate (FII) is determined by the
difference between the zone III flow rate (FIII) and the given feed flow rate (FF). The estimated FII,
however, cannot guarantee that the desorption wave of component 1 will stand in zone II. In order to
prevent component 1 from contaminating the extract, the desorption or diffuse wave velocity of
component 1 (udiff,1), which can be calculated from Eq. 3a with the estimated FII, should be greater
than or equal to the average port velocity (v). This is given by:
F II 
v  y5
x5
(5)
If Eq. 5 cannot be satisfied with the estimated FII, the feed flow rate should be reduced or the
length of zone III should be increased. Eq. 5 can thus be used in combination with Eqs. 4b and 4c to
estimate the maximum feed flow rate for a given SMB equipment. Note that pressure drop limitation
is not considered here. The estimation of the zone IV flow rate (FIV) from the design step 4 is based
on the standing condition of the component 1 shockwave (ush,1 equals v ). To prevent component 1
from entering zone I and cross-contaminating the extract, the spreading of the adsorption wave should
be confined within zone IV. For this reason, the mass-transfer zone length of component 1 (LMTZ,1),
which is calculated from Eq. 2a with the estimated FIV, should be less than or equal to LIV - Lc
F IV 
L
IV

 Lc  y 3
x3
(6)
If Eq. 6 cannot be satisfied with the estimated FIV, the length of zone IV should be increased.
2. EXPERIMENTAL
2.1. Racemic Mixture, Stationary Phase and Mobile Phase
The racemic secnidazole separated in this work was kindly donated by EMS® Pharmaceutical
Industries (Hortolândia, São Paulo, Brazil). Semi-preparative columns (10 cm x 1.0 cm I.D.,20 μm)
packed with amylose tris(3,5-dimethylphenylcarbamate), commercially known as Chiralpak® AD
The chromatographic experiments columns, for use in the Varicol system were purchased from Chiral
Technologies Europe. The eluent used as the mobile phase was a mixture of isopropyl alcohol
/acetonitrile (60:40%, by volume), Isopropyl alcohol was purchase from Tedia (USA) and acetonitrile
purchase from J.T.Baker (Mexico), all HPLC grade.
Equipment: A Shimadzu.high performance liquid chromatography (HPLC) system. VARICOLMicro unit was purchased from Novasep Company, France, which can operate alternatively along
with two different modes, SMB process and Varicol process. The shifting of the inlet and outlet lines
is realized by several groups of pneumatic valves. The whole system is controlled by specific
software, which is designed to monitor purification according to quality rules and maximum security.
Both UV (KNAUER, Germany) and Polar monitor (IBZ, Germany) detectors are located in the
recycling line between the last column and the first column, which can online detect the
concentrations of secnidazole enantiomers. Six columns were used in SMB and the zone
configuration (allocation of the 6 columns in the four zones) was 1-1-2-2.
Long Pulses of the Secnidazole Mixture: The long pulse experiment included a frontal
containing a secnidazole mixture 2,5 mg/mL, followed by elution with mobile phase. The effluent
stream was monitored with a UV detector at 398 nm and collected every minute. The collected
sample size was 2.0 mL. The samples were assayed with the HPLC. Three long pulse experiments at
different flow rates 2.0 mL/min, 2.5 mL/min, 5.0 mL/min were conducted.
3. RESULTS AND DISCUSSION
3.1. Long Pulses of the Secnidazole Mixture
The long pulse chromatograms are shown in Figure 1. From the chromatograms of the long
pulse experiments, one can obtain several empirical correlations (Eqs. 1-3), which are needed in the
shortcut design of SMB. The shockwave velocities are calculated by dividing the column length by
the mass center times of the adsorption waves (or frontal waves). The mass-transfer zone length for
each component equals the product of the shockwave velocity and the time span associated with the
5-95% plateau concentration portion of the adsorption wave. The diffuse wave velocities are
estimated from the column length and the 5% plateau concentration points on the diffuse waves. The
mass-transfer zone length (LMTZ), shockwave velocity, and the diffuse wave velocity are plotted
against the flow rate in Figure 2. The coefficients x and y in Eqs. 1-3 are regressed from the data
shown in Figure 2 and listed in Table 1.
SMB experiment based on the shortcut design: The empirical correlations obtained from the
long pulse experiments were used to calculate the zone flow rates and switching time from Eqs. 15a15e. The calculated values are listed in Table 2. The high affinity enaniomer (+)-Sec was collected in
the extract and the low affinity enantiomer (-)-Sec was collected in the raffinate. The purities of the
raffinate and extract were 72.5% and 85.5%, respectively. The SMB experimental results proves that
the experimental shortcut design method is feasible, efficient, and practical for nonideal systems. It
does not require the knowledge of either the isotherm parameters or the mass-transfer parameters.
Only three or more long pulse experiments at different flow rates are needed. Note that this method is
limited to a constant feed composition. If the feed composition changes over a wide range, a series of
long pulse experiments with different feed compositions will be needed.
Figure 1 – Long pulse experimental results at the flow rate of (a) 2.0 mL/min, (b) 2.5 mL/min, and (c)
3.0 mL/min.
Figure 2 – Summary of long pulse experimental results as a function of flow rate.
Table 1 – Coefficients of the Correlations from the Long Pulse Experiments
x1(m-2)
x2(m-2)
x3(s/m2)
x4(s/m2)
x5(m-2)
x6(m-2)
7598.1
4486.7
4.00x106
4.00x106
7142.9
6302.5
y3(m)
y4(m)
y5(m)
y6(m)
0.0475
-0,0566
-4.00x10-5
-0.0001
Table 2 – Summary of Operation Conditions for SMB
Inlet and outlet
Feed
0,6
flow rate (mL/min)
Desorbent
1,2
Extract
0,9
Raffinate
1,0
I
2,6
II
1,7
III
2,3
IV
1,4
Zone Flow rate (mL/min)
Switching time (min)
9,5
Column dimension(I.D x Lc cm)
1.0 x 10
Zone configuration
1-1-2-2
4. CONCLUSION
This study showed that it is possible to obtain enriched enantiomers secnidazol by shortcut
technique using Chiralpak AD column and the mobile phase consisted of 60% isopropyl alcohol and
40% acetonitrile. The separation of the racemic secnidazol in continuous chromatography process was
successfully demonstrated experimentally and the purities obtained could be enhanced through an
additional crystallization process.
5. ACKNOWLEDGMENT
The authors acknowledge the financial support obtained from CNPq, including that one from Proc. n.
304875/2013-9 for this research project, and secnidazole’s donation by EMS company.
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