Journal of Chromatography A, 1161 (2007) 322–326
Separation of aromatic hydrophobic sulfonates by micellar electrokinetic chromatography Sille Ehala ∗ , Merike Vaher, Mihkel Kaljurand Department of Chemistry, Faculty of Science, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia Received 8 January 2007; received in revised form 15 May 2007; accepted 24 May 2007 Available online 7 June 2007
Abstract Two different buffer systems for the separation of 12 aromatic hydrophobic sulfonates by micellar electrokinetic chromatography (MEKC) were developed. The following buffer systems were used: aqueous phosphate buffers containing either cetyltrimethylammonium bromide (CTAB) or sodium dodecyl sulfate (SDS). Eleven aromatic sulfonates were simultaneously separated in less than 35 min employing 20 mM phosphate buffer, pH 7.0 containing 50 mM SDS and 10% of acetonitrile. © 2007 Elsevier B.V. All rights reserved. Keywords: Aromatic hydrophobic sulfonates; Micellar electrokinetic chromatography
1. Introduction The determination of hydrophobic sulfonates studied in this work is of interest for various reasons. These compounds have been used as substrates for the synthesis of bactericides, herbicides and fungicides, as well as intermediates in the production of synthetic dyes, conductive gels and chemicals for organic synthesis. Trace levels of sulfonic acid esters may be formed as by-products during the manufacture of pharmaceuticals. GC–MS has been used for the determination of methyl and ethyl esters of methanesulfonic, benzenesulfonic and ptoluenesulfonic acids in active pharmaceutical ingredients . Methyl, ethyl and isopropyl esters of p-toluenesulfonic acid and methyl, ethyl, isopropyl and n-butyl esters of benezenesulfonic acid in drug substances have also been analysed by HPLC–MS . According to our knowledge, hydrophobic aromatic sulfonates have not earlier been studied by capillary electrophoresis (CE). However, hydrophilic aromatic sulfonates have been separated by capillary zone electrophoresis (CZE)
Corresponding author. Present address: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo namesti 2, CZ 166 10 Prague 6, Czech Republic. Tel.: +420 220183239; fax: +420 220283592. E-mail address: [email protected]
(S. Ehala). 0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.05.098
in aqueous [3–5] and non-aqueous media  as well as by micellar electrokinetic chromatography (MEKC) . Different surfactants, including sodium dodecyl sulfate (SDS), Brij 35  and cetyltrimethylammonium bromide (CTAB) , have been earlier employed in the separation of water-soluble benzeneand naphthalenesulfonates in MEKC. Naphthalenesulfonate isomers have also been separated by the cyclodextrin-mediated CE . The separation of hydrophilic benzene- and naphthalenesulfonates by capillary electrophoresis has been reviewed by Cugat et al. . The separation of hydrophobic sulfonates by CZE is, however, impossible because these compounds are neutral. Therefore, MEKC must be used to separate them. The problem of uncharged analytes was first solved in 1984 by Terabe et al. , who introduced an MEKC method. In MEKC the low-molecular-mass surfactants are added to the buffer as pseudo-stationary phases, which enable neutral solutes as well as charged solutes with the same charge-to-mass ratio to be separated. The separation by MEKC is based on the differential partitioning of analytes between the micelle and the surrounding aqueous phase. The present work reports the development of methods for the determination of a variety of hydrophobic aromatic sulfonates (Fig. 1) by MEKC. This was done by investigating the effect of CTAB and SDS concentrations, the amount of an organic solvent, the electrolyte concentration, the voltage and the pH of the buffer on the separation of 12 sulfonates.
S. Ehala et al. / J. Chromatogr. A 1161 (2007) 322–326
Fig. 1. Molecular structures of the sulfonates studied.
2.2. Instrumentation and conditions
2.1. Chemicals and reagents
CE was performed using an HP3D CE capillary electrophoresis system (Hewlett-Packard) equipped with an on-column diode-array detection (DAD) system. The operation of the instrument, the data collection and analyses were controlled by 3D-CE ChemStation B.01.03 software (Agilent Technologies). The detection was performed at 214 nm. Uncoated fused-silica capillaries (Polymicro Technologies, Phoenix, AZ, USA) of 75 m I.D. × 375 m O.D. were used with an effective length of 73 cm and total length of 82 cm. The capillary was thermostated to 25 ◦ C. The samples were injected by applying a pressure of 50 mbar for 2 s. The applied voltage for separation was 20 kV (positive or negative polarity). The pH of aqueous buffers was adjusted by using 0.1 M NaOH. New capillaries were rinsed with 1 M sodium hydroxide for 20 min, with 0.1 M sodium hydroxide for 5 min, with water for 5 min and with the separation electrolyte for 20 min. Before injections, the capillary was conditioned by washing it with 0.1 M sodium hydroxide for 5 min, with water for 3 min, and with the separation buffer for 5 min. When using the buffer containing SDS the capillary was rinsed only with the separation buffer for 5 min. Hydrophilic 1-methylpyridinium-3-sulfonate (1) was used as an electroosmotic flow (EOF) marker. The migration time of the micelle was determined using dodecylbenzene as marker.
Acetonitrile was obtained from Romil (Cambridge, UK), sodium dihydrogen phosphate dihydrate from Reahim (Russia) and sodium hydroxide from Lachema-Chemapol (Brno, Czech Republic). Sodium dodecyl sulfate and cetyltrimethylammonium bromide were from Sigma (Steinheim, Germany). 3butynyl p-toluenesulfonate, ␣-chloro-␣-hydroxy-o-toluenesulfonic acid ␥-sultone, methyl p-toluenesulfonate, propargyl benzenesulfonate were obtained from Aldrich (Steinheim, Germany). 2-Chloroethyl p-toluenesulfonate, cyanomethyl benzenesulfonate, phenyl methanesulfonate and phenyl trifluoromethanesulfonate were from Aldrich (St. Louis, MO, USA). Methyl 4-nitrobenzenesulfonate and phenyl vinylsulfonate were from Fluka (Buchs, Switzerland). Cyclohexyl p-toluenesulfonate, ethyl benzenesulfonate and 1-methylpyridinium 3-sulfonate were from Sigma–Aldrich Library of Rare Chemicals (Milwaukee WI, USA). Dodecylbenzene, which was used as micellar marker, was obtained from Alfa Aesar (Lancashire, UK). All reagents were used without further purification. The water used to prepare stock solutions and running buffers was purified by a Milli-Q apparatus (Millipore, Molsheim, France). The stock solutions of aromatic sulfonates were individually prepared at a concentration of 100 mM in acetonitrile (ACN)/methanol (MeOH) (60:40) mixture, except for 1-methylpyridinium-3-sulfonate, which was prepared in water. Dodecylbezene, which was used as micellar marker was dissolved in MeOH at ca. 2 mg mL−1 . All stock solutions were kept at 4 ◦ C. Working standard solutions consisting of all the 13 sulfonates and a micellar marker were prepared by an appropriate dilution with the separation electrolytes. All buffer and standard solutions were filtered through a 0.45 m syringe filter (Sarstedt, N¨umbrecht, Germany) before analysis.
3. Results and discussion 3.1. Method development for analysis of aromatic hydrophobic sulfonates by MEKC Up to now, no simultaneous determination of water-insoluble aromatic sulfonates by MEKC has been reported. Thus, the aim of the present study was to provide an appropriate ana-
S. Ehala et al. / J. Chromatogr. A 1161 (2007) 322–326
lytical method for the separation of 12 aromatic sulfonates of hydrophobic nature. In the course of the method development, the electrolyte concentration, the effect of the nature and concentration of different surfactants – CTAB and SDS, the pH of the buffer, the amount of an organic solvent (ACN in the range of 10–20%, v/v) and the voltage applied on the migration time of the analytes under study were evaluated. 3.1.1. pH of separation buffer The pH of the buffer is an important parameter in controlling the electroosmotic flow (EOF). To improve the separation selectivity and/or the analysis time of the compounds under study, the effect of pH was evaluated. The pH of the separation buffer (10 mM phosphate) was varied between 4.6 and 7.0 with 10 mM CTAB and 10% ACN added to the separation media. EOF as well as electrophoretic mobility of the micellar phase was found to be almost constant throughout the pH range investigated. However, slightly better separation of the studied analytes was obtained at pH 6.0. At this pH eight of the studied compounds were baseline-resolved (Rs > 1.5). Employing the SDS buffer (50 mM SDS, 20 mM NaH2 PO4 and 10% ACN) the effect of two different pH values (7.4, 7.0) on the separation of the analytes under study was evaluated. At pH 7.4 only 10 peaks were obtained, as two pairs of analytes were not separated, namely ethyl benzenesulfonate (4) co-migrated with ␣-chloro-␣-hydroxy-o-toluenesulfonic acid ␥-sultone (5), and phenyl vinylsulfonate (6) with propargyl benzenesulfonate (7). At pH 7.0 the resolution of analytes was slightly improved and a good separation of 11 analytes was achieved. Only cyclohexyl p-toluenesulfonate (8) and methyl p-toluenesulfonate (9) migrated as one peak. 3.1.2. Surfactant composition and concentration The nature and the concentration of the surfactant are important parameters influencing analysis selectivity. During the optimisation of the method we used either cationic or anionic surfactants as a pseudo-stationary phase. A 10–30 mM concentration range of CTAB was prepared in a buffer consisting of 10 mM phosphate and 10% ACN at pH 6.0. The resolution of aromatic sulfonates continued to increase with increasing concentrations of CTAB. However, the separation efficiency decreased when concentrations higher than 25 mM were used. With increasing concentrations of CTAB in buffer also slightly increased the current, being −28 A at 10 mM and −36 A at 25 mM CTAB concentration, when the applied voltage was −25 kV. At higher CTAB concentrations the migration times of the analytes slightly increased. This may be explained by both the increase of the ionic strength of the separation buffer and the greater interaction between the analytes and micelles caused by the use of high CTAB concentrations. The surfactant concentration was also optimised in the aqueous SDS buffer. A 50–100 mM concentration range of SDS was prepared in a buffer consisting of 20 mM phosphate and 10% ACN at pH 7.0. With increase in the concentration of SDS decreased the mobilities and changed the migration order of the analytes. At both 50 and 60 mM SDS concentrations the separation of 11 analytes was achieved. However, the two
analytes that co-migrated were different (Fig. 2A and B). In 50 mM SDS buffer co-migrated cyclohexyl p-toluenesulfonate (8) with methyl p-toluenesulfonate (9), and in 60 mM SDS buffer phenyl vinylsulfonate (6) with propargyl benzenesulfonate (7). Besides, changed slightly the elution order of the analytes, in 60 mM SDS buffer methyl p-toluenesulfonate (9) migrated after methyl 4-nitrobenzenesulfonate (10). Further experiments revealed that the migration order of these analytes changes at 57.5 mM SDS concentration. While the migration velocities of all solutes decreased with increase in the concentration of SDS, the migration of methyl 4-nitrobenzenesulfonate (10) seemed to be slightly less influenced. Also, when the migration factors of cyclohexyl p-toluenesulfonate (8) and methyl p-toluenesulfonate (9) increased linearly with increasing SDS concentrations in the BGE solution, the dependence of migration factors of methyl 4-nitrobenzenesulfonate (10) on SDS concentration was found to be non-linear. All this indicates that methyl 4-nitrobenzenesulfonate (10) is not neutral under the studied conditions. Nevertheless, without the addition of SDS to the separation buffer only one, but tailing peak was obtained. As the interior cores of SDS micelles are highly hydrophobic, the selectivity of neutral solutes in MEKC is mainly governed by hydrophobic interaction. However, in contrast to uncharged compounds, the migration of a charged analyte is influenced not only by the interaction with micellar pseudophase (mainly electrostatic interactions), but also by its own electrophoretic mobility in the aqueous phase . This caused methyl 4-nitrobenzenesulfonate (10) to elute before methyl ptoluenesulfonate (9) at higher SDS concentrations. Nevertheless, 50 mM SDS was chosen as optimum as it enabled the separation of the analytes under study within 35 min to be performed compared to 50 min when 60 mM SDS buffer was used. A further increase of the SDS concentration increased the migration time of the analytes and decreased the separation efficiency. 3.1.3. Inﬂuence of organic solvent concentration on the separation The aromatic sulfonates analysed are highly hydrophobic compounds, except for 1-methylpyridinium-3-sulfonate used as EOF marker. In order to achieve the partition of analytes between the running buffer and a pseudo-stationary phase, and to avoid the precipitation of analytes in the capillary, ACN was added to the running buffer. It is generally known that adding too much organic solvent may destroy the micellar structure. However, the presence of CTAB micelles has been observed in a surfactant system consisting of 40 mM CTAB–40% ACN–5 mM phosphate , and the critical micelle concentration for CTAB in acetonitrile–water mixed solvent (10:90% v/v) has been reported to be 2.18 mM . In this study, 10% ACN containing the running buffer solution (10 mM phosphate, 25 mM CTAB) was found to be optimal for the separation. Increasing the ACN content to 20% in the CTAB buffer decreased the electrophoretic mobilities and caused the peak broadening, which in turn deteriorated the resolution of the analytes. This could be brought about by a decrease in the difference in polarity between the mobile and pseudo-stationary phases, which in turn decreases the affinity of hydrophobic solutes for the micellar phase. In the
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SDS buffer the amount of the organic solvent added was not optimized. 3.1.4. Electrolyte concentration and applied voltage The electrolyte concentration was optimized by varying it in the range of 5–20 mM at pH 6.0 with 25 mM CTAB and 10% ACN added to the buffer. The best ratio between good separation and reasonable analysis time was achieved using 10 mM phosphate buffer. The optimal voltage for the separation of sulfonates with a CTAB containing buffer was found to be −20 kV. At higher voltages the resolution of some of the compounds decreased as the separation window became narrower. In the SDS buffer, the electrolyte concentration and the voltage applied were not optimized. In all experiments, 20 kV was used. 3.2. Comparison of the methods
Fig. 2. Comparison of electropherograms of 12 hydrophobic sulfonates (analytes concentration 1 mM each) separated under various electrophoretic conditions. Electrophoretic conditions: 75 m I.D. × 82 cm (73 cm effective) fused-silica capillary, 25 ◦ C capillary temperature, 214 nm UV detection and injection at 50 mbar for 2 s. (A) 50 mM SDS in 20 mM sodium phosphate buffer (pH 7.0), 10% ACN. 20 kV applied voltage (58 A). (B) 60 mM SDS in 20 mM phosphate buffer (pH 7.0), 10% ACN. 20 kV applied voltage (63 A). (C) 25 mM CTAB in 10 mM sodium phosphate buffer (pH 6.0), 10% ACN. −20 kV applied voltage (−25 A). The number of each peak is correspondent to the compound number in Fig. 1; mc: micellar marker.
In MEKC the electroosmotic velocity and the velocity of the micelles determine the migration window that characterizes each system and has strong influence on resolution. The so-called analytical window can be expressed by the difference between the highest and lowest values of μeff . Effective mobilities of hydrophobic sulfonates in different buffers under optimized conditions are presented in Table 1. For methyl 4nitrobenzenesulfonate (10), which under the studied conditions was charged, the effective mobility was not calculated. As seen from Table 1, a buffer containing SDS had much larger migration window compared to a CTAB containing buffer. The migration window has an important impact on resolution as can be seen in Fig. 2A and C, where the separation of hydrophobic aromatic sulfonates with two different running electrolytes under optimized conditions is shown. It is obvious from the presented results that system with SDS micelles had higher separation efficiency than CTAB-based MEKC. Although, with the CTAB containing buffer the shortest analysis times were obtained, it allowed the baseline separation of only eight sulfonates (Fig. 2C), ␣-chloro-␣-hydroxy-o-toluenesulfonic acid ␥-sultone (5) and phenyl vinylsulfonate (6); cyclohexyl ptoluenesulfonate (8) and methyl p-toluenesulfonate (9); as well as methyl 4-nitrobenzenesulfonate (10) and phenyl trifluoromethanesulfonate (13) co-migrated; and resolution of the cyanomethyl benzenesulfonate (3) from the ethyl benzenesulfonate (4) was only 0.57. The analysis time was nearly two times longer when the SDS containing buffer was employed. Nevertheless, of the running electrolytes used, this buffer proved to be the most effective for the separation of hydrophobic sulfonates, enabling the baseline separation of nine analytes (Fig. 2A). Additionally, the separation differences of ethyl benzenesulfonate (4) and ␣-chloro-␣-hydroxy-o-toluene-sulfonic acid ␥-sultone (5), and phenyl vinylsulfonate (6) and propargyl benzenesulfonate (7) are readily apparent from the electropherogram in Fig. 2A, although they were not baseline-resolved (Rs of 0.80 and 1.25, respectively). Only analytes (8) and (9) migrated as one peak in SDS containing buffer. This indicates that SDS micelles possess a greater hydrophobic environment for solute partitioning than CTAB micelles. By comparing the electro-
S. Ehala et al. / J. Chromatogr. A 1161 (2007) 322–326
Table 1 Mean effective mobilities and standard deviation of 11 aromatic sulfonates in various buffer systems (n = 5) Analyte
Effective electrophoretic mobility (×10−9 m2 V−1 s−1 )a 25 mM CTAB
(1) EOF marker (2) Phenyl methanesulfonate (3) Cyanomethyl benzenesulfonate (4) Ethyl benzenesulfonate (5) ␣-Chloro-␣-hydroxy-o-toluenesulfonic acid ␥-sultone (6) Phenyl vinylsulfonate (7) Propargyl benzenesulfonate (8) Cyclohexyl p-toluenesulfonate (9) Methyl p-toluenesulfonate (11) 3-Butynyl p-toluenesulfonate (12) 2-Cholroethyl p-toluenesulfonate (13) Phenyl trifluoromethanesulfonate Micellar marker a
50 mM SDS
−60.80 16.26 19.76 19.97 22.51 22.51 27.23 31.60 31.60 28.87 29.55 32.24 35.20
1.6 2.1 1.6 1.8 1.6 1.6 2.0 1.6 1.6 1.5 1.5 1.5 1.3
51.08 −19.85 −22.19 −24.74 −24.90 −25.86 −26.14 −27.63 −27.63 −33.81 −34.53 −36.97 −41.02
0.7 1.2 1.1 1.1 0.9 0.9 0.9 0.9 0.9 0.6 0.6 0.8 1.0
Experimental conditions as in Fig. 2.
pherograms obtained with the different optimised buffer systems (Fig. 2A and C), some interesting effects were observed. In fact, whereas for most of the compounds the elution order in both buffers was the same, for analytes (11) and (12) the elution order changed and they eluted before analytes (8), (9) and (10) in a CTAB containing buffer. The change in the elution order is an indication of an additional separation mechanism other than hydrophobic interactions, possibly based on a change in dipolar interactions and/or hydrogen bonding. 4. Conclusions In this study, a set of hydrophobic sulfonates with two different buffer systems has been analysed. The positively charged CTAB and negatively charged SDS were added to an aqueous phosphate buffer containing 10% ACN, and the sulfonates under study were separated within 16 and 35 min, respectively. Although a good electrophoretic profile was obtained with a buffer containing CTAB, the best resolution was observed when a buffer containing SDS was used. The migration time, the elution order, the baseline separation and the separation selectivity of the sulfonates investigated could be influenced by altering the concentration of the electrolyte, the species and the concentration of the surfactant, the amount of the added organic solvent, the separation voltage and the pH of the buffer. Considering the above-presented results, MEKC has proved to be a relatively fast
and easily applicable method for the separation of hydrophobic aromatic sulfonates. Acknowledgement The support of the Estonian Science Foundation (Grant 5145) is acknowledged. References              
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