Anal. Chem. 2006, 78, 3772-3779
Coated Wire Potentiometric Detection for Capillary Electrophoresis Studied Using Organic Amines, Drugs, and Biogenic Amines Justyna Sekula,† Joseph Everaert,† Hugo Bohets,† Bert Vissers,† Marek Pietraszkiewicz,‡ Oksana Pietraszkiewicz,‡ Filip Du Prez,§ Koen Vanhoutte,| Piotr Prus,‡ and Luc J. Nagels*,†
Chemistry Department, Antwerp University, Groenenborgerlaan 171, B-2020 Antwerp, Belgium, Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01224 Warsaw, Poland, Polymer Chemistry Research Group, Ghent University, Krijgslaan 281 S4-bis, B-9000 Ghent, Belgium, and Global Analytical Development, Janssen Pharmaceutica, Turnhoutseweg 30, 2340 Beerse, Belgium
Capillary electrophoresis was coupled successfully and reliably to potentiometric sensors, which are based on an ionically conductive rubber phase coating, applied on a 250 µm diameter metal substrate. The membrane components included potassium tetrakis(p-chlorophenyl)borate (TCPB), bis(2-ethylhexyl)sebacate (DOS), and high molecular mass poly(vinyl chloride) (PVC). Potentiometry reveals a very sensitive CE detection mode, with submicromolar detection limits for amines and the randomly chosen drugs quinine, clozapine, cocaine, heroine, noscapine, papaverine, and ritodrine. The lowest detection limit, 1 × 10-8 M injected concentration, was obtained for the quaternary ammonium compound tetrahexylammonium chloride. The more polar lower aliphatic amines and the biogenic amines dopamine, adrenaline, and cadaverine have much higher detection limits. The detection limits are log P dependent. Addition of a commercially available calixarene molecule or a synthetic macrocyclic amphiphilic receptor molecule to the electrode coatings enhanced the sensitivity respectively for the lower aliphatic amines and for the biogenic amines. A transpose of the Nikolskii-Eisenman-type function was suggested and used to convert the signal of the detector to a concentration-dependent signal. As stated in a review article on biomedical applications of capillary electrophoresis (CE),1 CE remains less popular than liquid chromatography (LC), which is the most commonly used method in life sciences. One of the main drawbacks of CE is the limited sensitivity of the available detection systems. Spectroscopic detection systems are by far the most preferred, even if the dependence of the response on the length of the light paths (in, for example, UV) disfavor them in miniaturized methods. Many attempts are made now to combine electrochemical methods of detection with miniaturized separation methods, such as CE * Corresponding author e-mail:
[email protected]. † Antwerp University. ‡ Polish Academy of Sciences. § Ghent University. | Janssen Pharmaceutica. (1) Hempel, G. Clin. Chem. Lab. Med. 2003, 41 (6), 720-723.
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integrated in microchip devices: see recent reviews on the topic (i.e.,2 conductivity3 and conductivity, amperometry, and potentiometry). The use of electrochemistry based detectors in CE has not yet yielded really successful commercial products.4 Amazingly, one of the most sensitive electrochemical techniques (i.e., potentiometry) has hardly been used in CE or other miniaturized techniques or even in conventional LC systems! The latest published applications in CE were from the groups of Haddad5,6 and Manz.7 The first experiments from our group on the CE/ potentiometry combination started in 1996.8-10 Our work done in this direction was summarized in 2000.11 Although the results were encouraging, they were still not competitive to spectroscopic modes of detection. We therefore concentrated on the potentiometry/LC combination first, as we realized that a lot of problems had to be solved first using millimeter-sized electrodes. A review of the LC/potentiometry situation was published in 2004 by Nagels.12 We showed that potentiometric LC detection has the potential to play a major role, as well for the detection of small organic molecules, and as for the detection of important larger biomolecules such as, for example, oligonucleotides.13 Because of its nature (the response is only dependent on the analyte concentration, not on flow rates and cell dimensions) and its sensitivity, potentiometry lends itself to application in miniaturized (as well as in non-miniaturized) techniques. The present publication will demonstrate the potential of the CE/potentiometry coupling applied to simple organic amines and to more complex (2) Guijt, R. M.; Evenhuis, C. J.; Macka, M.; Haddad, P. R. Electrophoresis 2004, 25 (23-24), 4032-4057. (3) Vandaveer, W. R.; Pasas-Farmer, S. A.; Fischer, D. J.; Frankenfeld, C. N.; Lunte, S. M. Electrophoresis 2004, 25 (21-22), 3528-3549. (4) Polesello, S.; Valsecchi, S. M. J. Chromatogr. A 1999, 834, 103-116. (5) Macka, M.; Gerhardt, G.; Andersson, P.; Bogan, D.; Cassidy, R. M.; Haddad, P. R. Electrophoresis 1999, 20 (12), 2539-2546. (6) Zakaria, P.; Macka, M.; Gerhardt, G.; Haddad, P. R. Analyst 2000, 125 (9), 1519-1523. (7) Tantra, R.; Manz, A. Anal. Chem. 2000, 72 (13), 2875-2878. (8) DeBacker, B. L.; Nagels, L. J. Anal. Chem. 1996, 68 (24), 4441-4445. (9) Poels, I.; Nagels, L. J. Anal. Chim. Acta 1999, 385 (1-3), 417-422. (10) Poels, I.; Nagels, L. J. Anal. Chim. Acta 1999, 401 (1-2), 21-27. (11) Nagels, L. J.; Poels, I. Trends Anal. Chem. 2000, 19 (7), 410-417. (12) Nagels, L. J. Pure Appl. Chem. 2004, 76 (4), 839-845. (13) Bao, Y.; Everaert, J.; Pietraszkiewicz, M.; Pietraszkiewicz, O.; Bohets, H.; Geise, H. J.; Peng, B. X.; Nagels, L. J. Anal. Chim. Acta 2005, 550, 130136. 10.1021/ac060066y CCC: $33.50
© 2006 American Chemical Society Published on Web 04/26/2006
ones such as drugs of abuse and biogenic amines. The biggest market for CE is still in the area of small molecules such as pharmaceuticals,1 but CE is known to be suited for large (bio)molecules too. Many papers are published each year dealing with the potentiometric batch analysis of pharmaceuticals. A review on this topic goes back to 1993.14 We recently tested new electrode materials in this respect and compared them to the most popular materials and electrode constructions used in the literature.15 The most widely used electrode coatings for the determination of basic drugs are of the so-called “liquid membrane” type. These membranes develop a surface potential when contacting a drug in solution. They are materials in the “rubber” state, made up of a polymer (mostly PVC) and a plasticizer. They are made ionically conductive by the addition of a large lipophilic (immobile) anion and a mobile cation. The present study shows the possibilities of the CE/potentiometry combination for simple amines, drugs of abuse, and biogenic amines. Among the drugs chosen as analytes are some tropane alkaloids of interest to pharmaceutical quality control, clinical drug monitoring and toxicology,16 or in the forensic field.17 Mixtures of drugs were separated in a co-electroosmotic mode. The electrode coating materials used are polymers in the rubber phase, with ionic conductance. EXPERIMENTAL SECTION Reagents, Products, and Electrode Preparation. Eluents were prepared weekly. They were filtered though a 0.2 µm cellulose acetate filter (Alltech Associates), and degassed with helium before use. Sample solutions were prepared by dilution of 10-3 M concentrations to the desired concentration with the eluent used for the separation. All membrane components were of the highest quality grade available from Fluka. These membrane components included potassium tetrakis(p-chlorophenyl)borate (TCPB), plasticizer bis(2-ethylhexyl)sebacate (DOS), high molecular mass poly(vinyl chloride) (PVC), tetrahydrofurane (THF), and calix[6]arene-hexaethyl acetate. Analyte molecules: amines and quaternary ammonium compounds were from Fluka (purum), dopamine was from Fluka (puriss.), cocaine and heroine were from Cerillant (France); cadaverine, ritodrine, and adrenaline were from Sigma. Two types of membranes were used in this study. For type 1 (“borate” electrode), a membrane was composed of 2% (wt %) TCPB, 32% PVC, and 66% DOS. Type 2 electrodes had an extra calixarene receptor molecule and contained 2% TCPB, 5% calixarene, 31% PVC, and 62% DOS. A total of 3 mL of THF was added to a 300 mg amount of these mixtures. This THF solution was applied on an insulated copper wire of 250 µm diameter by dipping the top of the wire in the solution, inverting the wire, and letting the solvent evaporate for 1 min. This dipping and evaporation (solvent casting) operation is repeated three times. A film of around 100 µm thickness is formed, covering the copper electrode and the surrounding plastic isolation (see Figure 1). The film thickness and shape were studied by stereomicroscopy with a (14) Cosofret, V. V.; Buck, R. P. Crit. Rev. Anal. Chem. 1993, 24 (1) 1-58. (15) Vissers, B.; Bohets, H.; Everaert, J.; Cool, P.; Vansant, E. F.; Du Prez, F.; Kauffmann, J. M.; Nagels, L. J. Electrochim. Acta/Bioelectrochem. (in press). (16) Boone, C. M.; Douma, J. W.; Frankle, J. P.; de Zeeuw, R. A.; Ensing, K. Forensic Sci. Int. 2001, 121 (1-2), 89-96. (17) Lurie, I. S.; Bethea, M. J.; Mc Kibben, T. D.; Hays, P. A.; Pellegrini, P.; Sahai, R.; Garcia, A. D.; Weinberger, R. J. Forensic Sci. 2001, 46 (5), 10251032.
Figure 1. CE and detector setup. See the Experimental Section for more details. A detail of the sensor electrode and coating and its positioning vs the capillary exit are shown on the right.
Zeiss SV11 stereomicroscope equipped with a Sony video camera HHD-930. Before application of the film, the insulated copper wire was prepared by cutting it with a stainless steel tube cutter, used in HPLC (Alltech Associates). It was then polished on a chromium oxide film disk (0.5 µm, 3M, St Paul, MN). For this polishing operation, the insulated copper wire was placed in a plastic holder to keep it in a vertical position versus the chromium oxide film disk. Synthesis of a Calixarene Phosphoric Acid Ester. The structure of the macrocycle is shown in the insert in Figure 9. From 0.005 M (5.5 g) of n-undecylcalix[4]resorcinarene, water was removed by azeotropic distillation with toluene. To the toluene (50 mL) solution, THF was added (30 mL) after cooling the mixture to room temperature. Then 0.02 M (2.74 g) triethylamine and 0.02 M (1.02 g) phosphorus trichloride was added dropwise. The reaction mixture was stirred at room temperature for 1 h, diluted with 100 mL of THF, and treated with 3 mL of 30% aqueous hydrogen peroxide at 0 °C. After 2 h the solvents were removed under reduced pressure, and the residue was treated with cold water (50 mL). The white product was filtered off, washed with 2 × 30 mL of cold water, and dried in a vacuum. Yield 80%. 1H NMR (DMFd,7 TMS): δ 0.89 (t, 12 H, 4 × CH CH ), 1.29 3 2 (bs, 80 H, 4 × (CH2)10), 4.31 (m, 4 H, CH(CH2)10), 7.57 (m, 8 H, resorcinol unit). Anal. Calcd.: C 63.89%, H 8.04%. Found: 63.92%, H 8.16%. MS (m/z) calcd: 1353, found: 1352 (M-). Apparatus. The CE system was home-built (see Figure 1). A high-voltage power supply (Spectraphoresis 100, Thermo Separation Products) with a maximum voltage output of +30 kV/-30 kV was used. An uncoated fused silica capillary of 75 µm i.d., 363 µm o.d., and 30 cm length (Supelco) was dipped with the injection end into a sample with electrolyte buffer, together with the platinum anode for the high-voltage field. The whole injection system was placed in a Plexiglas box, equipped with safety switches. The detection end of the capillary was dipped into a 250 mL Pyrex beaker containing the electrolyte buffer, reference electrode, sensing microelectrode, and platinum cathode. The microelectrode (250 µm i.d.,) and the capillary were aligned using a lab-made transparent (poly(methyl methacrylate) x,y,z positioning device. This positioning was done using eye control with a magnifying glass. The electrode-capillary distance was kept at 100 µm. This was obtained by first placing the electrode and Analytical Chemistry, Vol. 78, No. 11, June 1, 2006
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Table 1. Detection Limits (DL), Measured for the Compounds Used in This Study (first column), as a Function of Log P (second column)a
Figure 2. (A) Injection of a homologous series of linear aliphatic amines, from methylamine (C1) up to hexylamine (C6). The sensor contained potassium tetrakis(p-chlorophenyl)borate. The injected concentrations for all amines was 10-3 M. (B) Injection of tetrabutylammoniumchloride (TBACl, 10-4 M) and tetrahexylammoniumchloride (THACl, 3 × 10-4 M). The mobile phase was 30 mM NaH2PO4 + 10% acetonitrile. The applied voltage was 12.5 kV. Electrokinetic injection: 10 s, 12.5 kV.
capillary against each other. A screwing system was then used to increase the capillary-electrode distance. The exact distance was obtained by relating the number of screw rotations to this distance. The accuracy was better than 10 µm. The potential difference between the sensor and the reference electrode was monitored with a high impedance voltmeter PHYWE (World Precision Instruments, 1013 Ω) and amplified with a homemade amplifier. The signal was recorded by a PC-based data station equipped with Shimadzu Class-VP integration software. The sampled data were transferred to and handled in Excel. The cell assembly was placed in a faraday cage. The electrodes were conditioned with electrolyte under separation voltage until a stable baseline was obtained (about 20 min). Samples were injected electrokinetically by applying a voltage of 5-15 kV for 10 s. RESULTS AND DISCUSSION Obtained Detection Limits and Dependence on Compound Characteristics. A mixture of six aliphatic primary amines was first examined, containing methylamine, ethylamine, propylamine, butylamine, pentylamine, and hexylamine. An electropherogram is shown in Figure 2A. The quaternary ammonium compounds (“quats”) tetrabutylammoniumchloride (TBACl) and tetrahexylammoniumchloride (THACl) were injected separately in analogous conditions (see Figure 2B). The applied potential was 12.5 kV, which is lower than the 25 kV mostly applied in CE, resulting in higher retention times. This lower applied potential was used as it yielded somewhat better signal-to-noise ratios for the potentiometric detector. The potentiometric sensor contained a TCPB-based electrode material (see the Experimental Section). The response of a potentiometric sensor is a well-studied but complex function of canalyte.18 For peaks exceeding 60 mV peak heights, the detector response becomes dependent on log canalyte (see later in the text). Therefore, the peak shapes of the electropherograms of Figure 2B start to look unfamiliar to CE and to separation methods specialists. The log canalyte dependence of the signal at higher concentrations yields non-Gaussian (18) Bakker, E.; Bu ¨ hlmann, P.; Pretsch, E. Talanta 2004, 63, 3-20.
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compound
log P
DL (no ionophore) (M)
DL (ionophore) (M)
methylamine ethylamine propylamine butylamine pentylamine hexylamine TBACl THACl quinine clozapine cocaine noscapine papaverine heroine ritodrine dopamine adrenaline cadaverine
-0.65 -0.18 0.28 0.76 1.25 1.76 2.86 4.21 4.33 2.24 2 2.55 3.56 1.91 1.22 -0.48 -0.91 -0.49
3.62 × 10-5 1.26 × 10-5 7.58 × 10-6 3.35 × 10-6 1.60 × 10-6 7.10 × 10-7 1.00 × 10-7 1.00 × 10-8 2.9 × 10-7 1.46 × 10-7 6.63 × 10-8 1.04 × 10-7 7.65 × 10-8 1.35 × 10-7 3.73 × 10-7 >10-3 >10-3 >10-3
5.66 × 10-6 1.67 × 10-5 2.23 × 10-5 1.78 × 10-5 1.19 × 10-5 5.88 × 10-6
1.34 × 10-5 1.09 × 10-5 2.18 × 10-5
a Log P values were calculate via the http://www.logP.com website from ChemSilico. The two coatings used contained either no ionophore (third column) or the ionophore with synthesis described in the Experimental Section and structures shown in Figure 9 (fourth column). DL values are given as injected molar concentrations.
“compressed” peak shapes. This gives the false impression of abnormal peak broadening (see especially the TBACl peak in Figure 2B). In Figure 2A, peak shapes are noncompressed as the signal is smaller than 60 mV, and we work in the part of the calibration curve having a linear relationship between signal and concentration (see later in the text). Also baseline noise is relatively accentuated in these non-transformed recordings, as it is situated at the low mV (noncompressed) signals side. To have a practically useful detector, the detector output should be transformed to a canalyte-dependent signal. This will be discussed and demonstrated later in this text. It is quite clear from Figure 2 that, for example, for the homologous series of aliphatic amines the molar responses (peak heights obtained for equimolar injections) are in order of the alkyl group chain length. All compounds were injected in the same (10-3 M) concentration, and the C6 amine (hexylamine) gives the highest response, followed by C5, C4, C3, C2, and C1 (methylamine). It is well-known that potentiometric sensors without builtin selective component tend to have a “Hofmeister series” behavior. They respond better to lipophilic organic compounds. We have shown in earlier work,15 that log P is an interesting physicochemical property that can be used to estimate the response behavior of organic cationic substances on most potentiometric sensors used in the literature. Quite a few of such sensors have been described for batch measurements of basic pharmaceutical drugs.14 For batch measurements, the rubber phase membranes usually contain the drug to be measured ionpaired with a negatively charged lipophilic counterion. Table 1 lists the log P values of all compounds used in the present study together with the detection limits that were measured experimentally. Besides amines and basic drugs, the table also contains the biogenic amines dopamine, adrenaline, and cadaverine. The detection limits were measured by injecting decreasing concentrations of the substances until the peak height approaches the detection limit (DL). DL is then calculated as the injected
Figure 3. Log P dependence of the detection limit (DL) for the molecules listed in Table 1 (except dopamine, adrenaline, and cadaverine). Distinction was made between linear aliphatic amines (b) and basic drugs (×). Log P values were calculated using freeware from ChemSilico at http://www.logP.com.
concentration for which the peak height equals 12 × σnoise. This is mostly used in chromatographic situations: 4 × σnoise corresponds to what is generally considered to be the “baseline noise”. This means that we take 3× baseline noise as the detection limit. σnoise, the standard deviation of a section of the baseline noise, was calculated in Excel after transfer of all measured data to Excel. A high correlation is observed between the logarithm of the measured detection limits and the calculated log P values (see Figure 3). The correlation is highest for the subgroup containing the linear aliphatic amines and the quaternary ammonium compounds. These compounds are represented by a b in Figure 3. If this group is taken apart, the correlation coefficient R2 is as high as 0.9984. More complicated basic nitrogen-containing substances such as the drugs quinine, clozapine, cocaine, noscapine, papaverine, heroine, and ritodrine (see next paragraph) are represented by × in Figure 3. They showed a more complex response behavior, as their response was less log P related. In another study15 on the response behavior of a much larger set of basic drugs in batch FIA conditions however, also a very high log P dependence of the response was noted. Biogenic amines dopamine, adrenaline, and cadaverine had such low log P values that they could hardly be detected (see Table 1) unless special “ionophores” were used (see later in the text). Data from biogenic amines were not used in Figure 3. Table 1 and Figure 3 give a good estimation of which sensitivity can be expected at this moment for borate-type electrodes (no ionophore added). Compounds with log P values above 1 can be determined quite comfortably (detection limits better than 10-6 M). The response behavior of organic cationic compounds (log P related) is different from the response behavior of organic anionic compounds (no relation to log P) (see refs 8 and 12). It is not clear yet to us why this is the case. Making the Detector Output Concentration-Dependent. As mentioned above, the output signal of the potentiometric detector (mV) is a complex function of the analyte concentration, canalyte. Therefore, electropherograms as shown in Figure 2 are not an ideal representation of the variation of the molar concentrations of the analytes in the eluent. The function that is often taken in potentiometry to describe the relation between signal (in mV)
Figure 4. Calibration curve for cocaine in CE conditions of Figure 6. (9) Measured peak heights. The curve was obtained via a Nikolskii-Eisenman-type function that was fit to the measured data points (see text).
and molar concentration of the analyte, canalyte, is the NikolskiiEisenman equation:
E(mV) ) E0 +
RT pot ln(canalyte + ki,j cj ) nF
(1)
wherein the analyte ion is usually labeled i, and j is the interfering ion. The Nikolskii-Eisenman equation is used here for a monovalent ion, as all the compounds used in the present study have a pot +1 charge at the pH used (pH 3.8). In CE conditions, the ki,j cj (interfering ion) term is determined by the eluent buffer.8 It is constant. We have shown theoretically earlier8 that if canalyte , pot ki,j cj, the peak signal becomes linear to canalyte. This was often verified experimentally by our group in CE and LC conditions. It can also be checked by simulating the behavior of eq 1 in Excel. At higher analyte concentrations, the canalyte term is dominant, and the signal (mV) generated by the detector is log canalyte dependent. The experimentally recorded peak height (in mV) versus log canalyte curve is shown in Figure 4 for cocaine in CE conditions. In practice, one will seldom need all the information contained in Figure 4 (i.e., the total calibration graph over several decades) to do an accurate determination. An analyst will be more concerned about working in either the log c dependent range (10-3-10-5 M injected concentrations) or in the lowest concentrations ( 1), that is, peaks exceeding 60 mV peak heights after baseline zeroing. This is demonstrated in Figure 5, where an electropherogram of the above mixture of alkylamines, spiked with heroine and cocaine (electropherogram 5A), was transformed via a transpose Nikolskii-Eisenman-type 10E/S - 1 transformation. The injected concentrations for all amines were 10-3 M, and the concentrations of cocaine and heroine were 3 × 10-4 M. Cyclodextrin was added to the eluent to improve the separation. Electropherogram A in Figure 5 shows the original recording; electropherogram B shows the transformed data. The latter electropherogram gives a realistic view of how the analytes’ concentrations vary in time. In electropherogram A, peaks look broader as the signal at the peak bottom is relatively wide as compared to the log c dependent (middle) part of the signal (E > 60 mV). Non-transformed recordings also (19) Bu ¨ hlmann, P.; Pretsch, E.; Bakker, E. Chem. Rev. 1998, 98 (4), 15931687. (20) Bencini, A.; Bernardo, M. A.; Bianchi, A. Garcia-Espana, E.; Giorgi, C.; Luis, S.; Pina, F.; Valtancoli, B. Adv. Supramol. Chem. 2002, 8, 79-130. (21) Sessler, J. L.; Camiolo, S.; Gale, P. A. Coord. Chem. Rev. 2003, 240, 1755. (22) Zielinska, D.; Gil, A.; Pietraszkiewicz, M.; Pietraszkiewicz, O.; Van de Vijver, D.; Nagels, L. J. Anal. Chim. Acta 2004, 523, 177-184. (23) Harland, C. E. Ion Exchange: Theory and Practice; Royal Society of Chemistry: London, 1994. (24) Submitted.
Figure 5. Electropherogram of aliphatic amines (1-6), heroin (7), and cocaine (8). Curve A is the non-transformed recording; curve B is the 10E/S - 1 transformation of curve A. Sensor: potassium tetrakis(p-chlorophenyl)borate. Injected concentrations were 10-3 M (amines), 3 × 10-4 M drugs. Cyclodextrin (15 mM) was added to the eluent, which contained 50 mM NaH2PO4 with 10% acetonitrile. The applied voltage was 12.5 kV. Electrokinetic injection: 10 s, 12.5 kV.
give a wrong impression of the low mV signals (they are relatively exaggerated) yielding high signal/noise ratios in a recording like the electropherogram from Figure 5A. The same observations are made when mixtures of drugs of abuse are injected (see Figure 6). Electropherogram 6A shows the non-transformed detector output for an injection of quinine, clozapine, cocaine, and noscapine. In the transformed data of this recording, shown in Figure 6B, the observed peaks are sharper, and the signal/noise ratio is excellent. The electropherogram in Figure 6C was recorded using another electrode, with the same composition as the one used in the recording in Figure 6A,B. In the recording from Figure 6C, the four drugs were injected at 10-5 M concentrations. Only our best electrodes reached the good signal/noise ratios as obtained in the electropherograms of Figure 6. Inter-electrode variations were still operator dependent and varied from highly sensitive to unsensitive. Inspection (photography) of the electrodes with the light microscope did not give information on any relationship between the physical appearance of the coatings and the sensor’s sensitivity. It is also not yet clear whether the broader peaks in the electropherogram from Figure 6C are due to detector-induced peak broadening or to the condition of the capillary at the time of recording. We strongly felt the need for better visualization equipment to inspect the electrode coating before applying it in the CE system. Also, better methods should be found to obtain reproducible coatings in the sub-250 µm electrode diameter range. The present electrodes were still too large as compared to the capillary dimensions, which sometimes resulted in extra peak broadening. Peak broadening, induced by diffusion kinetics or by slow adsorption-desorption phenomena was not yet observed (this was also not observed in LC applications, where many different families of organic chemical compounds are under study in our group).
Figure 6. Electropherograms of mixtures of 10-4 M quinine (1), clozapine (2), cocaine (3), and noscapine (4). Curve A is the untransformed recording, curve is B the 10E/S - 1 transformation of curve A. Curve C is an electropherogram of the same mixture at 10-5 M. The eluent contained 50 mM NaH2PO4 with 10% acetonitrile. The applied voltage was 12.5 kV. Electrokinetic injection: 10 s, 12.5 kV.
The peaks in the electropherogram shown in Figure 6C are not yet accurately 10× smaller than the peaks in the electropherogram shown in Figure 6B. The two electropherograms were recorded however using different capillaries and different electrodes. A thorough quantitative study of the injected concentration versus transformed signal (peak height or area) is being done by our group. Another characteristic of the 10E/S - 1 transformation is visible in Figure 7, where we separated quinine (1), clozapine (2), papaverine (3), and ritodrine (4) in 10-4 M concentrations. Whereas the original non-transformed recording (Figure 7A) can give a better view of the smaller peaks such as peaks 1, 2, and 4, the transformed electropherogram 7B shows that the compounds are in reality better separated as one may conclude from visual inspection of the log c dependent recording 7A. This is an interesting characteristic of the potentiometric detector. Because of its highly logc dependent output, one can observe a window of concentrations covering several decades. The obtained detection limits (Table 1) are already quite interesting, especially taking into account the present primitive coating technology used for obtaining the (still too large) 250 µm size electrodes. Miniaturization does not seem to affect the sensitivity of potentiometric detection. Also, the influence of the large electric field needed for separation is very reasonable as no special precautions were taken as yet to minimize this influence. Application of Receptor Molecules and Determination of Biogenic Amines. As shown in Table 1, lower amines and socalled biogenic amines are the toughest amines to be determined with the potentiometric detection system in its present state Analytical Chemistry, Vol. 78, No. 11, June 1, 2006
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Figure 9. Electropherogram of biogenic amines cadaverine (1), dopamine (2), and adrenaline (3), injected at 10-3 M concentrations. Other conditions as in Figure 6. The receptor molecule shown in the insert was used in the electrode coating. R ) -(CH2)10CH3.
Figure 7. Electropherograms of a mixture of quinine (1), clozapine (2), papaverine (3), and ritodrine (4). Injected concentrations were 10-4 M quinine (1), clozapine (2), papaverine (3), and ritodrine (4). Curve A is the non-transformed electropherogram; curve B is the 10E/S - 1 transformation. Other conditions as in Figure 6.
Figure 8. Electropherograms of the linear aliphatic amines from methylamine (C1) up to hexylamine (C6). The sensor contained potassium tetrakis(p-chlorophenyl)borate plus calix[6]arene-hexaethyl acetate. The injected concentration for all amines was 10-3 M. Other conditions as in Figure 6.
because of their low log P values. Potentiometric sensors should also be able to do good determinations of these compounds if the correct receptor molecules (called “ionophores”) could be found. For the industrial lower amines, calixarene molecules proved to have a positive effect (see Figure 8). When compared to the electropherogram from Figure 2, it is clear that the lower amines methylamine (C1) and ethylamine (C2) show much improved responses. No such effects were noted 3778 Analytical Chemistry, Vol. 78, No. 11, June 1, 2006
however for the drugs or for the biogenic amines when the calixarene or many other commercial and noncommercial receptor candidates were tried as an ionophore. Making a good potentiometric sensor for biogenic amines still is a challenge. The biogenic amines are formed during normal metabolic processes in living organisms and occur in everyday food products. In Figure 9, we see the electropherograms obtained by injecting a mixture of cadaverine, dopamine, and adrenaline in a CE/sensor system. The sensor contained a selected receptor molecule (see Figure 9 insert and the Experimental Section), which was found to have an effect on the potentiometric response of the biogenic amines tested. The detection limit of cadaverine, dopamine and adrenaline improved significantly (with a factor over 100 for adrenaline) as compared to membranes that do not contain the receptor molecule (see Table 1). Inspection of Table 1 shows that the detection limits for these low log P compounds were much worse (even with ionophore added to the electrode material) than the detection limits obtained for compounds having log P values above, for example, 2. The lack of good receptor molecules for organic cationic substances is generally known.19 Positive developments in this (mostly macrocyclic chemistry) field would lead to lower detection limits for less lipophilic cationic organics in potentiometry. Synthetic receptors for anionic organic substances were developed with more success20,21 and could be used by our group to obtain sensitive potentiometric LC detection of small and large (multiply charged) molecules.12,22 Conclusions. One of the reasons of the slow application of potentiometry in separation methods may well be the uncertainty on the mechanism of the potential generating process in the case of rubber phase membrane materials. This discussion was recently addressed in an interesting review,18 from which it was also clear that electrochemists consider the use of potentiometry in separation methods as a niche application. The past decades, however, separation methods (especially LC) boosted UV/Vis spectrophotometry, mass spectrometry, and conductivity to highly successful techniques, and in our opinion, this may well be the case in the future for potentiometry. The coated wire systems used in our study have a classical ionically conducting rubber composition. They exchange cations with the eluent dynamically (the anionic TCPB sites are immobile), which is a quite slow process.15 Much
of their behavior can be learnt from the knowledge of ion exchange materials in chromatography and in other applications.23 Once the ion-exchange system is in equilibrium with the eluent, the surface potential of the ionically conducting rubber coatings is stable. In the absence of external electric fields, our electrodes have a steady drift of 1.3 mV per 24 h, which was thoroughly measured on large sets of electrodes in another recent study.24 The interaction of the analyte ion with the surface changes the surface potential. In our opinion, this surface potential generation is the sole source of signal of the above-described sensors. It is a rapid, non-faradaic process (no redox chemistry is taking place). The electrode coating behaves as an ionic capacitor, not as an ion-electron transducer. During contact, the chemical interaction energy of analyte and surface are converted into an electrical (Donnan) component, which we think is the basis of the sensor. In chromatographic conditions (hydrodynamics, continuous eluent renewal at the electrode surface), the analyte ion is rapidly displaced from the surface by the eluent buffer ions and the signal (25) Nagels, L. J.; Bazylak, G.; Zielinska, D. Electroanalysis 2003, 15 (5-6), 533-538.
returns to baseline. References 25 and 12 give a more thorough description of the above ideas, which are found very scattered in the electrochemistry literature. Yet, better models of sensor electrolytics, new ionically conductive membrane materials, and especially design methods for cationic organic receptor molecules seem hardly needed. Our immediate future goals for the CE/ potentiometry combination are directed to application to charged biomolecules. ACKNOWLEDGMENT The authors thank the Instituut voor de Aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen and the Ministerie van de Vlaamse Gemeenschap BIL01/63 for funding and Prof. Schepens and Prof. Haemers for providing drug substances.
Received for review January 10, 2006. Accepted March 29, 2006. AC060066Y
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