Colloids and Surfaces A: Physicochem. Eng. Aspects 317 (2008) 431–437
On the use of colloid-enhanced ultrafiltration in view of enantiomericenrichments and limiting conditions
C. Tondre ∗, S. Parant, P. Lemiere, C. GerardinGroupe de Chimie Physique Organique et Colloıdale, Unite Mixte de Recherche n◦ 7565, Nancy Universite, CNRS,
B.P. 239, 54506 Nancy-Vandoeuvre Cedex, France
Received 12 April 2007; received in revised form 4 October 2007; accepted 8 November 2007Available online 22 November 2007
bstract
We review here the different techniques which have been used during the past twenty years to achieve enantiomer separation, distinguishingetween analytical and preparative methods, and stressing upon the place of techniques based on surfactants and colloidal particles. Due to thereat progress of membrane technologies, special emphasis will be put on the different attempts resting on the use of ultrafiltration techniques.he main successes in enantiomer separations (or at least enantiomer enrichment) obtained with these techniques involved the formation of a
ernary complex including a metal ion (Cu2+ in most cases). Our concern in this work was to investigate the feasibility of performing enantiomernrichments through micellar ultrafiltration, when only weak enantiomer/selector complexes were formed, not requiring a bridging metal ion. Aodel system (ephedrine enantiomers and (S)-dodecoxycarbonylvaline micelles as chiral selector), for which successful separations were achieved
n micellar electrokinetic chromatography, was tested for this purpose. The lack of significant enrichment tend to demonstrate, that, in such a case,he difference in the binding of the enantiomers to the chiral selector (measured from UV spectroscopy and circular dichroism) is too weak to thinkbout an application of ultrafiltration techniques, even in a multistage mode. 2007 Elsevier B.V. All rights reserved.
It is well known that the biological and pharmaceutical activ-ties of chiral compounds may be very different depending onhe enantiomer: sometimes one of the enantiomers is activehereas the other one has no effect, but it may also happen thatne of the enantiomers has undesired, or even worth, extremelyarmful effects [1]. For this reason the obtaining of opticallyure products is a requirement of the utmost importance in theharmaceutical industry [2–5]. During the past twenty years,esides the involvement of organic chemists in developing newsymmetric syntheses, many efforts have been done to improvehiral separation techniques. These techniques make use of chi-al selector molecules chosen (or synthesized) to specifically
ecognize one of the enantiomers in an optical pair [6]. This isften a big challenge, especially when considering the fact thathe differences in the Gibbs free energies involved in the for-
ation of diastereoisomers are usually very small (typically inhe order of 0.1–0.6 kJ/mol) [7]. It should also be emphasizedhat depending on whether we are looking for a simple analyt-cal technique, or for a preparative technique on a large scale,enerally implies rather different approaches.
.1. Analytical techniques
From the strict point of view of enantiomer analysis, micel-ar electrokinetic chromatography (MEKC) has proved to be aowerful tool and abundant results have been reported in theiterature [7–14], which often demonstrate a total resolution ofacemates. In these techniques colloidal particles including chi-al recognition sites are used as pseudo-stationary phases andhe separation is based on the slight preferential partitioningf one of the enantiomers in favor of this chiral pseudo-phase.
lthough the larger parts of these works have involved micellesf chiral surfactants, there has been some attempts to use otherypes of colloidal particles. Vesicles [9,15–17] as well as chiral
icroemulsions [18] have been tested for this purpose with more
r less success, depending on the particle chemical structure. Theccessibility of the chiral site to the enantiomer to be recognizeds of course of primary importance. This is well demonstrated inhe work of Pascoe et al. [9] who have used mixed micelles con-tituted of a standard surfactant (sodium dodecylsulfate (SDS)r cetyltrimethylammonium bromide (CTAB)) and a chiral sur-actant ((S)- or (R)-dodecoxycarbonylvaline (DDCV)). Whereasn enantiomeric selectivity could be demonstrated for a seriesf pharmaceutical analytes with mixed micelles of SDS andDCV, both negatively charged, no selectivity was found with
atanionic vesicles made of DDCV (−) and CTAB (+). This isikely to be due to the differences in the electrostatic interactionsetween the two surfactants inside the particle: the attractiventeractions between DDCV (−) and CTAB (+) makes difficulthe approach of the analyte towards the chiral site of DDCV,hereas the repulsive interactions between DDCV (−) and SDS
−) has no such effect.Some advantages of chiral vesicular systems prepared from
ingle-tailed amino acid derivatized amphiphiles have beenointed out by Mohanty and Dey [15], namely the low criti-al vesicular concentration (cvc) and the high hydrophobicityacilitating the partitioning of the analytes between aqueousnd vesicle phase. On the other hand, Pascoe and Foley [18]bserved that using an oil-in-water microemulsion incorporat-ng chiral DDCV, in place of simple DDCV chiral micelles,lightly improved the enantioselectivity and the rapidity of theeparation.
The efficiency of other chromatographic methods such asapillary electrophoresis (CE) and high-pressure liquid chro-atography (HPLC) in view of enantioseparations has also
een investigated [19–21]. Addition of micelles to glycopeptidentibiotics [20] or to cyclodextrines [19] has demonstrated theeal potential of CE techniques. Molecular imprinting technolo-ies have been parallely developed in combination with HPLC22,23]: biomimetic polymeric recognition sites were used inhis case as chiral stationary phase. For instance, the chiral res-lution of (±)-ephedrine was successfully achieved with thisechnique [23].
.2. Preparative techniques
The production of pure enantiomers, from the resolution ofacemates, on a large scale, is a matter of chemical engineer-ng. We will certainly not be totally exhaustive in examininghe different techniques which have been considered to solvehis problem, but we will try to mention here those which haveemonstrated a significant potential. They can essentially be sep-rated into chromatographic and membrane techniques, with theatter including a large variety of devices.
.2.1. Chromatography processesEnantiomer separation by chromatography processes in liq-
id and supercritical phases was reviewed by Perrut et al. [24]
n 1993, date at which they pointed out the fact that if largecale separations remained very limited this could probably bettributed to (i) the low capacity of available stationary phasesnd (ii) to their high cost. Since that time the development of
etre
ochem. Eng. Aspects 317 (2008) 431–437
imulated moving bed (SMB) chromatography has permittedaximization of productivity as well as reduction of eluent
onsumption [25,26]. More recently, the use of supercriticaluid associated with SMB technology (SF-SMB) has provedspecially effective for scaling up enantiomer separations to con-inuous operations [27,28]. Another approach was considered byan der Ent et al. who have developed a system, which can beegarded as a scaling up of CE techniques [29].
.2.2. Membrane processes
.2.2.1. Liquid membranes. Although liquid membrane tech-iques are quite often only considered for laboratory scalexperiments [30], some devices may have potential for perform-ng separations at a larger scale. The chiral colloidal particle issed here to selectively transport the enantiomers from a sourcehase containing the racemate, towards a receiving phase wheren enrichment in one of the enantiomers is expected. Essentialonditions are that the liquid/liquid interfaces should be perme-ble to the enantiomers to be separated but non-permeable tohe chiral selector. A very attractive device derived from the liq-id membrane principle can be found in the “resolving machine”eveloped by Newcomb et al. [31]. In this machine a W-tube wassed to achieve a simultaneous transport of two enantiomers, onef them going in the left arm of the tube and the other one in theight arm, with optical purities ranging from 70 to 90%.
Chiral emulsion liquid membranes have been investigated byickering et al. for the enantioseparation of phenylalanine, withaximum enantioselectivity not passing 2.4 [32,33].Polymer-supported liquid membranes containing a chiral
rown ether [34] or chiral alcohols [35] have been used fortudying the selective transport of amino acids. An enantiomeresolution ratio of 22.7 was obtained for dl-phenylglycine [34].
Hollow fibers modules offer another type of approach whichan be considered similar to polymer-supported liquid mem-ranes. In that case the pores of the hollow fibers are filled upither with a liquid immiscible with the liquid phase circulatingnside and outside the fiber [36,37], or with a polymeric gel [38]note that such utilization of hollow fibers is different from theirse in cross-flow ultrafiltration devices, where only the size ofhe pores controls the solutes permeation [see below]).
A convincing demonstration of the efficiency of hollow fiberxtraction was reported by Ding et al. [38], who have achievedhe separation of racemic leucine, with an isomer yield tremen-ously enhanced compared to other techniques. The selectoras an alkyl hydroxyproline associated with cupric ion and theores of the fibers were filled with cross-linked polyvinylalcoholel (which does not prevent diffusion). Using a rather similarevice, Keurentjes et al. [37] have determined the enantiose-ectivities of a variety of racemic drugs (including ephedrine)btained with different chiral selectors, mainly tartaric aciderivatives. In that case, two liquids, containing the opposinghiral selectors, were flowing counter currently, separated byliquid membrane filling the pores of the fibers. Bench-scale
xperiments were performed by the authors after calculation ofhe number of transfer units required for a high degree of sepa-ation. Optical purities higher than 99% were obtained for bothnantiomers.
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.2.2.2. Ultrafiltration membranes. We finally come to enan-ioseparations based on colloid-enhanced ultrafiltration, whichas motivated undertaking the present work. Micellar-enhancedltrafiltration has proved to be an efficient method to removeetal ions from aqueous solutions. Selective hydrophobic
xtracting molecules can be dissolved in micellar particles,hich can themselves be retained by the smaller pore sizef ultrafiltration membranes [39–42]. The same principle haseen applied for performing enantiomer enrichments, usingydrophobic chiral selectors in place of metal ion extractingolecules. This was apparently a very promising approach,hich has resulted in several interesting reports [1,43–48].
n addition, membrane technologies greatly improved duringecent years with the availability of all kinds of ultrafiltrationodules, with a large variety of pore size or chemical structures.
t is also well known that even with a basic enantioselectivity ofnly a few percents, one can reach high optical purities by themplementation of cascaded (or multi stage) devices [37,45,47].
Surprisingly there were only few investigations in this direc-ion.
One should mention that biological macromolecules havelso been considered in this optic. Several authors have reportedn the optical resolution of amino acids by ultrafiltration mem-ranes containing serum albumin (BSA) [49–51]. Higuchi et al.nvestigated the use of immobilized DNA membranes for chiraleparations of amino acids, showing that, in that case, the poreize of the membrane regulated the preferential permeation ofne enantiomer [52,53].
Coming back to micellar-enhanced ultrafiltration, a firsteport on enantiomer separation by Creagh et al. [43] appearedn 1994. The authors used a chiral selector, l-5-cholesterollutamate included in a non-ionic surfactant. The mixedicelles formed preferentially bind d-phenylalanine over l-
henylalanine in the presence of copper(II). Depending on thexperimental conditions, a selectivity as high as 4.2 could bebtained. The cholesterol tail from the selector was assumed toecrease the exchange dynamics and to rigidify in some wayhe micellar edifice. Several other papers published by the sameesearch group have appeared later on: Overdevest et al. [44–47]ave extensively investigated the previous system in order to testts industrial feasibility, including the examination of cascadedperations. They concluded that, within a certain experimentalindow defined as the enantiomer feed concentration multipliedy its affinity constant, ultrafiltration in cascaded systems com-ines the benefits of chromatographic and distillation processes47]. They also shown that the enantiomer complexation to enan-ioselective micelles can be described by Langmuir isotherms44].
We concluded from these results, as well as from other works,hat micelle-enhanced ultrafiltration appears a suitable techniqueo achieve the separation (or at least a significant enrichment) ofacemic mixtures, but we noticed that in many cases where a sig-ificant selectivity was obtained a copper ion was involved in the
ormation of a ternary complex selector site: Cu2+: enantiomer21,33,38,43–48,54].
Considering the preceding points and the increasing com-ercial availability of all kinds of ultrafiltration membranes
[caN
icochem. Eng. Aspects 317 (2008) 431–437 433
especially hollow fiber modules), from the laboratory scale tohe industrial one, we wanted to examine up to what extent a sep-ration of enantiomers obtained from MEKC, can also be carriedut with ultrafiltration devices. We wanted to avoid the presencef copper ions because we assume that their elimination in thenal product will necessarily be a complicating factor. Doing
hat we were faced with lower discriminating factors between thewo enantiomers to be separated. To the best of our knowledgee are not aware of previous reports in which it was attempted
o use ultrafiltration to perform enantiomer enrichments in suchifficult conditions.
We have chosen as model system the enantiomers ofphedrine, which were previously the object of many studies7,9,10,18,23,37] and whose UV-absorbance spectrum makeshe analysis very easy. We used as chiral surfactant (S)-N-odecoxycarbonylvaline (a standard selector commonly utilizedn MEKC) [7,9,10,13,18] which was shown to have a lowerV-absorbance and to give higher enantioselectivities than (S)--dodecanoylvaline [13]. The enantioselectivities reported in
he literature for ephedrine were ranging from 1.1 to 1.2 at pHsbove 7.0. We show here that in such conditions we reach theimits of the application of the ultrafiltration technique in thisomain.
. Experimental
.1. Chemicals
Pure ephedrine enantiomers were obtained from Ega-Chemieor (−)(1R, 2S) ephedrine and from Sigma–Aldrich for (+)phedrine, obtained in the form of (+)(1S, 2R) ephedrine hemi-ydrate. The first sample was used as received and the second oneas recrystallized in hexane to remove an absorbing impurity.oth samples were carefully dried before utilization. Their ele-ental analysis was checked with a Thermofinnigan Flash EA
112 apparatus. Their optical purity was checked by circularichroism. (S)-dodecoxycarbonylvaline (DDCV) was synthe-ized from (S)-valine and the corresponding chloroformate,ollowing the indications given by Mazzeo et al. [13]. Theurity of the product was checked by elemental analysis, IRnd 1H NMR. Elemental analysis: calcd: C, 65.62; H, 10.71;, 4.25; found: C, 65.23; H, 10.72; N, 4.12. IR: ν (OH):000–3450 cm−1; ν (C O): 1716 cm−1. 1H NMR: 0.87 (t, 3H,J = 7 Hz); 0.94 (d, 3H, 3J = 6.5 Hz); 1.01 (d, 3H, 3J = 6.5 Hz);.24 (m, 14 H); 1.60 (m, 2H); 2.21 (m, 1H); 4.06 (t, 2H,J = 7 Hz); 4.31 (m, 1H); 5.09 (m, 1H). [α]22
D 2.12 (c 0.08 inHCl3).
The range of concentration of ephedrines (1–8 mM) was cho-en so as to ensure a convenient measurement of UV-absorption.he concentration of DDCV (5 mM) was adjusted so as to allow
he ratio between bound enantiomer and DDCV to vary in theange 0–1. This concentration is equivalent to 10 times the cmc
9]. Buffers were avoided in order to ensure a total absence ofompetition between the enantiomers and buffer molecules forbinding site. The pHs were adjusted by dropwise additions ofaOH or HCl.
434 C. Tondre et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 317 (2008) 431–437
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ig. 1. Example of UV-absorbance analysis of retentate and permeate afterltrafiltration. Initial concentrations: ephedrine (+) 3.9 mM; DDCV 5 mM; pH.21.
.2. Techniques
The ultrafiltration of the micellar solutions of DDCV, contain-ng one or the other ephedrine enantiomer at pH 8.2–8.3, waserformed at room temperature in a stirred cell of volume 10 mlamicon 8010). Cellulosic disk membranes (Millipore PLGC)ith a molecular weight cut-off of 10,000 Da were used, with
n applied pressure of 3.9 bar and a 600 rpm agitation. 50% ofhe initial solution was collected as permeate and both the lat-er and the retentate were analyzed from UV-absorption (λmax56–257 nm), using standard curves established in similar con-itions. The concentrations of free ephedrine were obtained fromhe measurements on the permeate. The analysis of ephedrineinding to DDCV micelles has taken into account the existencef free ephedrine in the retentate at the same concentration as inhe permeate.
The UV–vis spectra were recorded on a Cary 3E Varianpectrophotometer.
Circular dichroism was measured with a Biologic MOS 450pparatus (Grenoble, France). The conditions were as follows:lit widths: 1 nm; data acquisition: 0.5 nm/point and 10 s/point;verage over 4 spectra to improve the signal to noise ratio (eachverage spectrum takes an acquisition time of 1 h in the range40–285 nm).
. Results and discussion
We have given in Fig. 1 an example of UV spectroscopy anal-sis of both the permeate and retentate in the initial conditionsndicated in the caption of the figure. The small contribution ofDCV was evaluated in independent experiments: it is expected
hat the DDCV is present in the permeate at a concentration ofhe order or its cmc, evaluated at 0.5 mM [9]. On the other hand,he concentration of micelles is increased by a factor of 2 when0% of the solution is collected as permeate. From the analy-
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ig. 2. Binding curve of ephedrine (+) and S-DDCV. Concentrations: DDCVmM; ephedrine (+) 1–8 mM; pH = 8.25 (±0.05). Full line: best fit according
o Langmuir isotherm (see text) with qs = 4.72 mM, K = 3.31 mM−1.
is of a whole set of curves comparable to those in Fig. 1, weould determine, for each of the enantiomers, the binding curvesepresented in Figs. 2 and 3.
These curves were analyzed analogously to Langmuirdsorption isotherms, as was done before, for another system,y Overdevest et al. [44]. The experimental data were fitted byhe equation:
i = qsiKici
1 + Kici
here i represents either the (−) or the (+) ephedrine enan-iomer, K (mM−1) is the Langmuir affinity constant, c and qmM) are the equilibrium concentrations of free and bound enan-iomers, respectively. qs is the Langmuir saturation constant (i.e.
ig. 3. Binding curve of ephedrine (−) and S-DDCV. Concentrations: DDCVmM; ephedrine (−) 1–8 mM; pH = 8.25 (±0.05). Full line: best fit according
o Langmuir isotherm (see text), with qs = 5.40 mM, K = 2.36 mM−1.
Fig. 4. CD spectra of pure ephedrine enantiomers at concentration 3 mM, anddifference spectrum.
C. Tondre et al. / Colloids and Surfaces A
for ephedrine (+):
qs(+) = 4.72 mM
K(+) = 3.31 mM−1
for ephedrine (−):
qs(−) = 5.40 mM
K(−) = 2.36 mM−1
(+) is slightly larger than K(−) as it could be expected from thehermodynamic measurements of Peterson and Foley [7], whoad used R-DDCV (instead of S-DDCV here) and consequentlyound a reverse affinity, with K(−) slightly higher than K(+). Thebsolute values of K reported by these authors are not directlyomparable to ours, because their measurements were made athigher pH, in the presence of buffers. However, the optimized
heoretical fits shown in Figs. 2 and 3 are not totally satisfactory.he uncertainties of our measurements are related to (i) the accu-
acy of the optical density measurements, (ii) the accuracy onhe volume filtrated, which influences the concentrations in theetentate, and (iii) the pH adjustments, in the absence of buffers,ven though concentration corrections were applied to take intoccount the dropwise additions of acid or base. The optimizedalues obtained for qs(+) and qs(−), especially for the latter,re higher than the expected concentration of chiral sites in theicelles (in the order of 4.5 mM, when taking into account the
mc value of DDCV [9]). If we fix the qs parameter at 4.5 the bestts obtained are of poorer quality and K(−) is found larger than(+). This proves how much it is difficult to establish a clear dis-
inction between the affinities of the two ephedrine enantiomersor S-DDCV micelles by the ultrafiltration method.
We were perfectly aware that measurements performed withhe pure enantiomers, considered separately, will not necessar-ly reflect the situation encountered when a racemic mixture isonsidered, and thus when two enantiomers of opposite sign cane in competition for a same recognizing site. For this reasone have also performed some experiments with 50/50 mixturesf the two enantiomers. The previous method, resting on UV-bsorbance measurements, cannot be utilized in that case and weave analyzed the solutions using circular dichroism (CD). Theesults appear to confirm the slightly higher affinity of ephedrine+) for S-DDCV. They are represented in Figs. 4 and 5. The firstigure shows the CD-spectrum of 3 mM solutions of ephedrine−) and ephedrine (+), respectively. In the presence of DDCV,he analysis is much more complex, due to the contribution of-DDCV itself to the CD-spectrum. We have compared in Fig. 5
he spectra obtained when the initial concentration of the race-ate mixture was 8 mM (with 5 mM of S-DDCV, when it was
resent). Again we see how close to the limit of the apparatusesolution we must go: curve (a) represents the CD-spectrum of
he initial mixture of ephedrine enantiomers, before the additionf DDCV. We expected an horizontal line, but we had to useuch a small scale for comparison with the other solutions, thatslightly curved line appears (note that the total deviation is not
Fig. 5. CD spectra of (a) initial 50/50 mixture of ephedrine enantiomers; (b) afteraddition of S-DDCV to the preceding mixture; (c) rententate after ultrafiltration;(d) permeate. Initial conditions: ephedrine racemate 8 mM; DDCV 5 mM; pH8.25 (a 3-points smoothing was applied to the curves).
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36 C. Tondre et al. / Colloids and Surfaces A: P
arger than one millidegree !); curve (b) was obtained after theddition of S-DDCV and before ultrafiltration. The characteris-ic peaks of ephedrine (+) show up in this spectrum, suggestinghe presence of traces of a DDCV/ephedrine (+) association inlight excess; curve (c) is the spectrum of the rententate after0% ultrafiltration of the initial volume and, as expected, theoncentration of the preceding complex is increased by a fac-or of about 2, confirming a slightly preferential retention ofphedrine (+); finally curve (d) corresponds to the analysis ofhe permeate, in which, we can guess the presence of the char-cteristic peaks of the other enantiomer, ephedrine (−). Theseesults confirm the difficulty to produce significant enantiomericnrichment from ultrafiltration, when the enantioselectivity is inhe order of 1.1–1.2 (i.e. when the association constants of thewo enantiomers for the chiral selector differ by only 10–20%).or the sake of comparison, we can notice, that the associationonstants reported in the case of ternary complexes (selector:u2+: enantiomer) were differing by a factor larger than 7 [44]nd in the case of tryptophan/BSA [50] by a factor about tenimes more (76).
Although the lack of significant enrichment observed inhis work could have been anticipated from the low values ofnantioselectivities reported from MEKC, nothing can replacessertions based on experiments, not forgetting that rather lowelectivities can sometimes be exploited in multistage processes.
. Conclusion
In conclusion, the results reported here have shown the lim-ts of the application of colloid-based ultrafiltration in view ofnantiomeric separations and it is hoped that, in spite of theiregative side, they will be useful to people interested in applyingltrafiltration for similar purposes. The chiral recognition whichppears sufficient in MEKC to lead to satisfying resolution ofhe system investigated here, is not discriminating enough toermit a significant enrichment using ultrafiltration technology.he chromatographic analytical method takes advantage of a
arge number of plates, favoring a progressive shift of the equi-ibria involved. One-stage ultrafiltration cannot be competitiveith this respect. However cascaded systems proved to be effi-
ient provided that the association constants between the chiralelector and the enantiomers are sufficiently different. From thisoint of view recognition through diastereo complexes involvingmetal ion appears to produce much better discrimination.
cknowledgements
The authors thank Sandrine Adach for performing the ele-ental analysis and Eric Dumortier for his valuable help in
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