Rapid enantiomeric separation of polychlorinated biphenyls by electrokinetic chromatography using mixtures of neutral and charged cyclodextrin derivatives

Journal of Chromatography A, 910 (2001) 157–164 www.elsevier.com/locate/chroma Rapid enantiomeric separation of polychlorinated biphenyls by electrokinetic chromatography using mixtures of neutral and charged cyclodextrin derivatives * ´´ C. Garcıa-Ruiz, Y. Martın-Biosca, A.L. Crego, M.L. Marina ´´ ´ ´ Departamento de Quımica Analıtica , Facultad de Quımica , Universidad de Alcala , Ctra . Madrid–Barcelona Km . 33.600, ´ 28871 Alcala de Henares ( Madrid ), Spain Received 14 July 2000; received in revised form 19 October 2000; accepted 20 November 2000 Abstract Electrokinetic chromatography with cyclodextrin derivatives (CD-EKC) was used to achieve the rapid enantiomeric separation of chiral polychlorinated biphenyls (PCBs). Thirteen of the 19 chiral PCBs stable at room temperature were individually separated into their two enantiomers by using 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.5) containing carboxymethylated g-cyclodextrin (CM-g-CD) as pseudostationary phase mixed with b-cyclodextrin (b-CD) or permethylated b-cyclodextrin (PM-b-CD). Urea was also added to increase the solubility of PCBs and cyclodextrins in the aqueous separation buffer. Several experimental parameters such as the nature, concentration, and pH of the buffer, nature and concentration of the cyclodextrin derivatives used, and the addition of different additives were studied in order to improve the enantiomeric separation. In addition, the effect of some instrumental parameters such as separation temperature and applied voltage was also investigated. PCBs were enantiomerically separated in less than 12 min by using a 50 mM MES buffer (pH 6.5) containing 20 mMCM-g-CD, 10 mMb-CD or 20 mMPM-b-CD, and 2 Murea at a temperature of 458C and an applied voltage of 20 kV. 2001 Elsevier Science B.V. All rights reserved. Keywords : Enantiomer separation; Buffer composition; Polychlorinated biphenyls; Cyclodextrins 1. Introduction over, although biologically active chiral compounds are introduced into the environment as racemates, Polychlorinated biphenyls (PCBs) represent an their uptake and metabolism by an organism may be important class of priority pollutants due to their selective for enantiomers [3,4]. Therefore, it is very persistence, toxicity and bioaccumulation [1,2]. The important to develop analytical methods enabling the enantiomeric separation of chiral PCBs is of special enantiomeric separation of chiral PCBs. relevance because each enantiomer may have differGas chromatography (GC), usually employed to ent toxicity, activity or metabolic pathways. Moreseparate PCB congeners, has also been employed to perform the separation of chiral PCBs [5–11]. Nevertheless, the characteristics of capillary electro- *Corresponding author. Tel.: 134-91-8854-935; fax: 134-91phoresis (CE) techniques (high separation efficiency 8854-971. E-mail address : [email protected] (M.L. Marina). and flexibility) make this technique competitive with 0021-9673/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0021-9673(00)01176-6 ´ 158 C . Garcıa-Ruiz et al . /J . Chromatogr . A 910 (2001) 157 – 164 GC in the field of the enantiomeric separations [12]. plied voltage was also studied in order to improve Prior to this work, a mode of CE, cyclodextrinthe resolution obtained between the enantiomers of modified micellar electrokinetic chromatography chiral PCBs. (CD-MEKC) has been employed for the enantiomeric separation of chiral PCBs [13–16]. In fact, 12 PCBs (IUPAC numbers 45, 84, 88, 91, 95, 132, 136, 2. Experimental 139, 149, 171, 183 and 196) were separated each one into its two enantiomers using sodium dodecyl sulfate (SDS) in 2-(N-cyclohexylamino)ethane2.1. Reagents sulfonic acid (CHES) buffer (pH 10) containing 2 M urea and g-cyclodextrin (g-CD) [13] or mixtures of All reagents employed were of analytical grade. band g-CD as chiral modifiers [14]. On the other CHES was purchased from Sigma (St. Louis, MO, hand, chiral separations of PCBs 131, 135, 144, and USA); 2-morpholinoethanesulfonic acid (MES), so175 along with other seven chiral PCBs described dium dihydrogenphosphate dihydrate, butanol before were obtained using a borate buffer (pH 9) (BuOH), dimethylformamide (DMF), and sodium containing also SDS and g-CD, but with a higher hydroxide were supplied by Merck (Darmstadt, concentration of urea (5 M) [15]. The above-menGermany); ammonium acetate, b-CD, g-CD, and tioned group of 15 PCBs were also enantiomerically urea were from Fluka (Buchs, Switzerland); carseparated by CD-MEKC using a mixed micellar boxymethylated g-cyclodextrin [CM-g-CD, degree of system bile salt–SDS with g-CD [16]. Finally, the substitution (D.S.)|3], carboxymethylated b-cyclopossibilities of using bile salts alone as chiral dextrin (CM-b-CD, D.S.|3), and permethylated bsurfactant in MEKC to achieve the enantiomeric cyclodextrin (PM-b-CD, D.S.|12–13) were obtained separation of chiral PCBs were investigated [17]. from Cyclolab (Budapest, Hungary); methanol If micelles are not added to the electrolyte solu- (MeOH) was from Lab Scan (Dublin, Ireland).Water tion, the separation of electrically neutral compounds used to prepare solutions was purified through a (such as PCBs) can also be achieved by CE using Milli-Q system from Millipore (Bedford, MA, USA). charged cyclodextrins alone or mixed with neutral All solutions were filtered prior use through 0.45-mm CDs. In this type of electrokinetic chromatography pore size disposable nylon filters from Scientific (EKC), named cyclodextrin electrokinetic chromaResources (Eatontown, NJ, USA). tography (CD-EKC), the charged cyclodextrin acts The 19 PCBs studied were supplied by Dr. as pseudostationary phase [12,18]. As in other modes Ehrenstorfer Reference Materials (Augsburg, Gerof CE, the enantioselectivity of this chiral pseudomany). Table 1 shows the basic structures and stationary phase can be modified using various chiral IUPAC numbers of the compounds used throughout additives [19,20], with several types of cyclodextrin this study according to Ballschmiter and Zell nomenderivatives (neutral and charged) the most used in clature [23]. the last few years [21,22]. The purpose of this work was to study the possibilities of CD-EKC with a charged cyclodextrin 2.2. Apparatus as chiral pseudostationary phase alone or mixed with 3D a neutral cyclodextrin as chiral additive to achieve A HP CE system (Hewlett-Packard, Waldbronn, the enantiomeric separation of a group of chiral Germany) equipped with an on-column diode array PCBs. The influence of the nature, concentration and detection (DAD) system and HP 3D-CE Chemstation pH of the buffer, as well as the nature and consoftware was used. Separations were performed on centration of the cyclodextrin derivatives used and uncoated fused-silica capillaries of inner diameter the addition of several organic additives (methanol, (I.D.) 50 mm and outer diameter (O.D.) 375 mm, butanol, and urea) on the enantiomeric separation of purchased from Composite Metal Services (WorcesPCBs was investigated. The effect of other exter, UK). Capillaries had a total length of 58.5 cm perimental parameters such as temperature and apand a length of 50 cm to the detector. Capillary ´ C . Garcıa-Ruiz et al . /J . Chromatogr . A 910 (2001) 157 – 164 159 Table 1 Sample solutions were prepared by dissolving Basic structure, systematic names and IUPAC numbers (according each PCB in DMF to achieve a final concentration of to Ballschmitter and Zell nomenclature [23]) for the chiral PCBs 200 ppm approximately. studied Before first use, a new capillary was rinsed with 1 MNaOH for 30 min, followed by a 30-min rinse with water. Between introduction of samples, the capillary was conditioned with water for 2 min, 0.1 MNaOH for 2 min, water for 2 min and separation PCB name IUPAC number buffer for 2 min. Injections were made by pressure (from 50 mbar for 1.2 s to 20 mbar for 1 s). For 2,29,3,6-Tetrachlorobiphenyl 45 2,29,3,39,6-Pentachlorobiphenyl 84 separation, voltages between 15 and 25 kVwere used 2,29,3,4,6-Pentachlorobiphenyl 88 and the detection took place at 230 nm. At the end of 2,29,3,49,6-Pentachlorobiphenyl 91 the day the capillary was rinsed with water for 2 2,29,3,59,6-Pentachlorobiphenyl 95 min, 0.1 MNaOH for 2 min and water for 2 min, 2,29,3,39,4,6-Hexachlorobiphenyl 131 then stored in water. The conditioning method was 2,29,3,39,49,6-Hexachlorobiphenyl 132 2,29,3,39,59,6-Hexachlorobiphenyl 135 used to obtain good peak shapes and reproducible 2,29,3,39,6,69-Hexachlorobiphenyl 136 retention data. 2,29,3,4,49,6-Hexachlorobiphenyl 139 2,29,3,4,59,6-Hexachlorobiphenyl 144 2,29,3,49,59,6-Hexachlorobiphenyl 149 3. Results and discussion 2,29,3,39,4,49,6-Heptachlorobiphenyl 171 2,29,3,39,4,5,69-Heptachlorobiphenyl 174 2,29,3,39,4,59,6-Heptachlorobiphenyl 175 All chiral PCBs stable at ambient temperature (19) 2,29,3,39,4,6,69-Heptachlorobiphenyl 176 were injected into a CE system using charged CD 2,29,3,4,49,59,6-Heptachlorobiphenyl 183 derivatives alone or mixed with neutral CD deriva2,29,3,39,4,49,59,6-Octachlorobiphenyl 196 tives in the separation buffer to perform their en2,29,3,39,4,49,6,69-Octachlorobiphenyl 197 antioseparation by CD-EKC. The type of buffer without and with additives (methanol, butanol and temperature was varied from 25 to 608C and UV urea), and some instrumental parameters such as detection was performed at 230 nm. separation temperature and applied voltage were also Electrolytic solutions were degassed in an ulvaried. trasonic bath KM from Raypa (Barcelona, Spain). A 654 pH meter from Metrohm (Herisau, Switzerland) 3.1. CD-EKC using charged cyclodextrins alone was employed to adjust the pH of the separation buffers. CM-g-CD and CM-b-CD were used at concentrations ranging from 5 to 50 mM(five values, 5, 10, 2.3. Procedure 20, 30, 50 mM) in the case of CM-g-CD and from 10 to 20 mMfor CM-b-CD (two values). Two pH Separation buffers were prepared by dissolving the values were tested using different buffers: pH 9 appropriate amount of buffer, CD derivatives, and using a 50 mMCHES buffer and pH 6.5 using 50 urea into water to achieve the desired concentration, mMammonium acetate or 50 mMMES buffers. adding in some cases adequate aliquots of liquid Concentrations ranging from 10 to 50 mMin CM-gadditives (methanol or butanol) prior to the addition CD were also tested at pH 6.5 (50 mMMES buffer) of water. Finally, the pH was adjusted to 6.5 with 1 using 1% and 5% methanol, 1% n-butanol or 2 M Mor 0.1 Msodium hydroxide solution for MES urea as additives. In all these experiments an applied buffer, with 1 Mor 0.1 MNH solution for voltage of 20 kV and a temperature of 258C were 3 ammonium acetate buffer or with 1 Mhydrochloric employed. Under any of these experimental conacid for phosphate buffer prior to obtain the final ditions the chiral recognition of PCBs was achieved. volume. However, since the use of CM-g-CD enabled the ´ 160 C . Garcıa-Ruiz et al . /J . Chromatogr . A 910 (2001) 157 – 164 obtention of a peak for a higher number of PCBs different concentrations of g-CD (20, 30, or 40 mM) (one single peak) than CM-b-CD, CM-g-CD was were employed, none of the PCBs studied were chosen in order to study the potential of using enantiomerically separated. These results show that mixtures of this charged CD and neutral CDs to g-CD is not a good chiral additive in the CD-EKC perform the chiral separation of PCBs. A value of system described. However, when b-CD or PM-bpH equal to 6.5 was also chosen because at pH 9 the CD were used as chiral additives, several chiral electroosmotic flow was much too high and the PCBs were enantiomerically separated using 20 mM elution window was very small. In addition, MES or 30 mMCM-g-CD as pseudostationary phase (see buffer was preferred instead of ammonium acetate Table 2). When b-CD was used up to 12 of the 19 buffer because the latter generated higher current chiral PCBs (45, 88, 91, 95, 131, 132, 136, 139, 144, intensity values than MES buffer. 149, 171 and 176) were enantiomerically resolved, while the use of PM-b-CD only enabled the enantio3.2. CD-EKC using mixtures of charged and separation of eight chiral PCBs (45, 88, 91, 95, 131, neutral cyclodextrins 136, 144 and 197). Although the use of PM-b-CD enabled the enantiomeric separation of a lower The use of different neutral cyclodextrins (g-CD, number of PCBs, this CD derivative allowed the b-CD or PM-b-CD) as chiral additives in the enantioseparation of PCB 197 that was not possible separation buffer containing CM-g-CD was studied using b-CD as chiral additive. Furthermore, it is the in order to achieve the enantiomeric separation of first time that PCB 197 is separated into its two chiral PCBs (see Table 2). Urea (2 M) was also enantiomers by CE (see Fig. 1a), and although it was added because it was shown that the shape of peaks only partially resolved (R50.8), this PCB has only s improved under these conditions probably due to a been partially separated by GC and high-performbetter solubilization of PCBs and CDs in the aqueous ance liquid chromatography (HPLC) but in very long buffer. When mixtures of 30 mMCM-g-CD with analysis times [11,24]. In fact, the partial enantiorTable 2 Chiral PCBs enantioseparated, from the 19 chiral PCBs studied, using 50 mMMES buffer (pH 6.5) and 2 Murea with mixtures of a CM-g-CD, as pseudostationary phase, and several neutral CDs, as chiral additives Charged CD Neutral CD Temperature Current intensity Chiral PCBs enantiomerically separated (pseudostationary phase) (chiral additive) (8C) (mA) 30 mMCM-g-CD 20 mMg-CD 25 115 None 30 mMg-CD 25 115 None 40 mMg-CD 25 115 None 30 mMCM-g-CD 40 mMb-CD 25/45 115/150 45, 95 20 mMCM-g-CD 40 mMb-CD 45 97 45, 95 30 mMb-CD 45 97 45, 95, 136 20 mMb-CD 25/45/60 70/97/120 45, 88, 91, 95, 136, 144, 149 10 mMb-CD 45 97 45, 88, 91, 95, 131, 132, 136, 139, 144, 149, 171, 176 5mMb-CD 45 97 45, 88, 91, 95, 131, 132, 136, 139, 144, 149, 171, 176 20 mMCM-g-CD 10 mMPM-b-CD 45 97 45, 88, 91, 95, 131, 136, 144 20 mMPM-b-CD 45 97 45, 88, 91, 95, 131, 136, 144, 197 40 mMPM-b-CD 45 97 45, 88, 95, 136 60 mMPM-b-CD 45 97 45, 88, 95, 136 30 mMCM-g-CD 20 mMPM-b-CD 45 150 45, 88, 91, 95, 131, 144, 197 a Current intensity is also included. Instrumental conditions: uncoated fused-silica capillary, 58.5 cm (50 cm to the detector)350 mm I.D.; injection, 20 mbar, 1 s; applied voltage, 20 kV; and UV detection at 230 nm. ´ C . Garcıa-Ruiz et al . /J . Chromatogr . A 910 (2001) 157 – 164 161 MEKC [15,16], longer analysis times (between 15 and 25 min) were required in contrast to the result described in this work (analysis time less than 8 min) (see Fig. 1b). These chiral separations were also reported by GC [8,9] but in general very long analysis times were required (more than 1 h). On the other hand, these results do not agree with those obtained by CD-MEKC [13,14], in which g-CD was more effective in the enantiomeric separation of chiral PCBs than b-CD due to the larger size of the cavity of g-CD compared to b-CD. This different behavior between CD-MEKC and CD-EKC could be due to the formation of a ternary complex PCB– surfactant monomer–CD (demonstrated for other several solutes and surfactants [25]), which does not take place in CD-EKC. In addition, some authors believe that this ternary complex inside the g-CD cavity may account for its effectiveness [26]. The enantiomeric resolutions obtained for chiral PCBs when the concentration of b-CD was varied from 5 to 40 mMare shown in Fig. 2a. It can be observed that the best enantioresolution corresponds to 10 mMfor PCBs 45, 91, 95, 132, 144, 149 and 171, whereas for PCBs 88, 131, 136, 139 and 176 the best resolution was achieved at 5 mMof b-CD. On the other hand, the best results obtained for all the chiral PCBs enantiomerically separated with PMb-CD were achieved at a concentration of 20 mM (see Fig. 2b). Using a constant concentration of Fig. 1. Enantiomeric separation of PCBs 197, 144 and 131. b-CD or PM-b-CD, similar results were obtained Separation buffer: 50 mMMES (pH 6.5), 2 Murea, and 20 mMwhen 20 mMor 30 mMCM-g-CD was employed. CM-g-CD with (a) 20 mMPM-b-CD and (b) 10 mMb-CD. Then, a 20 mMconcentration of CM-g-CD was Injection by pressure, 20 mbar for 1 s. Capillary, 58.5 cm (50 cm chosen in order to decrease the current intensity (up to the detector)350 mm I.D. Temperature, 458C. Applied voltage, 20 kV (|97 mA); UV detection at 230 nm. to 150 mA with 30 mMCM-g-CD). Fig. 2 also shows that the enantiomeric resolutions obtained for esolution (R50.7) of PCB 197 has recently been PCBs 45, 88, 91, 95, 131, 136 and 144 were better s performed using a chiral capillary column (with with b-CD than with PM-b-CD and generally better hydroxypropyl-permethyltrifluroacetyl g-cyclodexwith 10 mMthan with 5 mMof b-CD. trin) by GC in an analysis time close to 60 min [11]. The effect of the temperature and the applied On the other hand, the use of a PM-b-CD derivatized voltage on the enantioseparation of PCBs was also silica as chiral stationary phase in reversed-phase studied in this work. Temperatures of 25, 45 and HPLC has allowed one to partially resolve PCB 197 608C and applied voltages of 15, 20 and 25 kV were (R50.8) in about 50 min [24]. These results seem to employed for the best conditions selected [50 mM s confirm that PM-b-CD is a good chiral additive for MES buffer (pH 6.5), 2 Min urea, 20 mMin the enantioseparation of highly hydrophobic comCM-g-CD and 10 mMin b-CD]. An increase in the pounds such as octachlorobiphenyl (PCB 197). In temperature or applied voltage did not produce a addition, although the chiral separations of PCBs 131 significant effect on the enantioresolution except for and 144 have previously been reported by CDPCBs 95 and 136. For PCB 95 an increment in the ´ 162 C . Garcıa-Ruiz et al . /J . Chromatogr . A 910 (2001) 157 – 164 Fig. 3. (a) Electropherogram corresponding to the chiral separation of a mixture of PCBs 176, 88, 91, 95 and 45 by CD-EKC using a 50 mMMES buffer (pH 6.5) containing 2 Murea, 20 mM CM-g-CD, and 10 mMb-CD. (b) Electropherogram corresponding to the chiral separation of a mixture of PCBs 131, 95, 45 Fig. 2. Enantiomeric resolution of chiral PCBs as a function of (a) and 136 by CD-EKC using a 50 mMMES buffer (pH 6.5) b-CD concentration; (b) PM-b-CD concentration. Other concontaining 2 Murea, 20 mMCM-g-CD, and 20 mMPM-b-CD. ditions as in Fig. 1. Other conditions as in Fig. 1. temperature or voltage drives to a decrease in the CD-EKC into all their 10 enantiomers in less than 12 enantioresolution. In the case of PCB 136 the min, using 20 mMCM-g-CD as pseudostationary contrary effect was observed, that is, enantiomeric phase and 10 mMb-CD as chiral additive. Fig. 3b resolution increased when increasing the temperature shows the enantiomeric separation of four PCBs (45, or the applied voltage. On the other hand, an increase 95, 131 and 136) in only 9 min, using 20 mM in the temperature or the applied voltage produced a CM-g-CD as pseudostationary phase and 20 mM decrease in the analysis time but an increase in the PM-b-CD as chiral additive. In both separations the current intensity (data not shown). Therefore, 458C analysis time is smaller than that obtained by CDand 20 kV were chosen as good compromise among MEKC (between 20 and 40 min) [13–16], but a enantioresolution, analysis time and current intensity. smaller number of PCBs can be separated simulUnder these conditions, the simultaneous enantiotaneously because the separation window is wider meric separation of mixtures of chiral PCBs were when a CD-MEKC system is used. Other separations performed. of chiral PCBs (132, 139, 149 and 171) are shown in Two examples of multicomponent separations are Fig. 4, being in all cases the analysis time sigshown in Fig. 3. In Fig. 3a a mixture of up to five nificantly smaller than that obtained with CD-MEKC PCBs (45, 88, 91, 95 and 176) was separated by [13–16]. ´ C . Garcıa-Ruiz et al . /J . Chromatogr . A 910 (2001) 157 – 164 163 the enantiomeric separation of several PCBs. Up to 12 of the 19 chiral PCBs stable at room temperature (45, 88, 91, 95, 131, 132, 136, 139, 144, 149, 171 and 176) were enantioresolved with b-CD, while eight chiral PCBs (45, 88, 91, 95, 131, 136, 144 and 197) were enantiomerically separated with PM-bCD. This work presents for the first time the enantioresolution of the PCB 197 by CE, and although it was only partially resolved, similar resolutions were obtained by GC or HPLC but using very longer analysis times. In addition, although the chiral separations of the other PCBs have been reported by CD-MEKC, longer analysis times were required. The best enantioresolutions were obtained with concentrations between 5 and 10 mMof b-CD and 20 mMof PM-b-CD. On the other hand, in general, the enantioresolution of the chiral PCBs did not have a significant change when increasing the temperature (upto608C) or the applied voltage (up to 25 kV), being 458C and 20 kV a good compromise among enantioresolution, analysis time, and current intensity.Finally, multicomponent mixtures of chiral PCBs were enantiomerically separated by CD-EKC using 20 mMCM-g-CD as pseudostationary phase in a MES buffer (pH 6.5) with 2 Murea and b-CD or PM-b-CD as chiral additive. Fig. 4. Electropherograms corresponding to the chiral separation Acknowledgements of PCBs 132, 149, 171 and 139 by CD-EKC using (a) 50 mM MES buffer (pH 6.5), 2 Murea, 20 mMCM-g-CD, and 10 mM b-CD and (b) 50 mMMES buffer (pH 6.5), 2 Murea, 20 mM´ The authors thank the Comunidad Autonoma de CM-g-CD, and 5 mMb-CD. Other conditions as in Fig. 1. Madrid (Spain) for project 07M/0049/1998. 4. Conclusions References The enantiomeric separation of chiral PCBs was ´´ ´ [1] M.J. Gonzalez, M.A. Fernandez, L.M. Hernandez, Arch. not possible by CD-EKC using an anionic cycloEnviron. Contam. Toxicol. 20 (1991) 343. dextrin alone (CM-b-CD or CM-g-CD) as chiral ˜´´ [2] G. Font, J. Manes, J.C. Molto, Y. Pico, J. Chromatogr. A 733 pseudostationary phase in a MES buffer (pH 6.5) (1996) 449. with 2 Murea. The addition of a neutral cyclodextrin ¨¨ [3] J. Faller, H. Huhnerfuss, W.A. Konig, R. Krebber, P. Ludwig, such as g-CD (concentrations ranging from 20 to 40 Environ. Sci. Technol. 25 (1991) 676. ¨ [4] H.R. 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