Anal. Chem. 1003, 65, 1662-1667
1662
Quantitative Analysis in Capillary Zone Electrophoresis Using Ion-Selective Microelectrodes as On-Column Detectors Andre Nann,’ Ivo Silvestri, and Wilhelm Simon’ Department of Organic Chemistry, Swiss Federal Institute of Technology, Universitiitstrasse 16, CH-8092Ziirich, Switzerland
Inorganic and organic cations can be quantified using capillary zone electrophoresis with an ionselective microelectrode as on-column detector. This is illustrated by determining the linear calibration plots for potassium,sodium, rubidium, calcium, and dopamine in the range of 10-h10-3 M and for histamine in the range of 10-L104 M, where imidazole and potassium were used as internal standards. Quantification of the potassium concentration in blood plasma is shown by use of both internal standard and external calibration plot. By delogarithmizing the emf values of the ISME response and integrating the resulting curve, a linear relationship between integral and analyte quantity is obtained. The linear function between this integral and the specific resistance of the electrokinetically injected sample solution is explained by theory based on transport numbers.By computer simulation,experimentalresponsetimes of the ISME detector are shown to be short enough to ensure undistorted peak shapes and reproducible integrals.
INTRODUCTION Since its introduction by Mikkers et al.’ and Jorgenson and Lukacs? capillary zone electrophoresis (CZE) has become a powerful tool for analyzing ionic species. Currently, up to 20 papers on capillary electrophoresis (CE) are published monthly, whereas in the whole year of 1983, only four appeared.3 The advantages of CE as compared to ion chromatography are obvious. Higher resolution, shorter analysis times, and ease of use, as well as simple maintainance of the apparatus, make CE a versatile supplementary or even replacement technique for ion chromatography and HPLC. For several reasons, the development of CE systems leads to increasingly narrower capillaries. The main advantages of using capillaries of radius r < 25 pm are higher resolution: smaller sample amounts (proportional to r2),and better heat dissipation due to higher surface to volume ratio. As the flow friction per unit volume is a function of this ratio, height differences of up to a few centimeters in the buffer levels at the injection and detection side of narrow capillaries (r < 10 pm) induce no significant flow.5 This implies that sample injection must be performed by pressure pulse or electrokinetically. In contrast to the former technique, the latter c a w s Author deceased November 17, 1992. (1) Mikkers, F. E. P.; Everaerts, F. M.; Verheggen, Th.P. E. M. J. Chromatogr. 1979,169. 1-10, (2) Jorgenson,J. W.; Lukaca, K. D. Anal. Chem. 1981,53,1298-1302. (3) Science Citation Index; Institute for Scientific Information: Philadelphia, PA 19104, 1983. (4) Lukaca, K. D.;Jorgenson, J. W. J. High Resolut. Chromatogr. Chrornatogr. Cornmun. 1986,8,407-411. (6)Grushka, E. J. Chromatogr. 1991,559,81-93. t
0003-2700/93/0365-1662$04.00/0
no zone broadening due to injection dispersion. On the other hand, the amount of analytes injected electrokinetically depends not only on their concentrations but also on their electrophoretic mobilities and the specific resistance of the sample solution.6 Unfortunately, on-column UV detectors cannot be used with narrow capillaries because, due to a shorter path length, the detection sensitivity drastically decreases. With fluorescent detectors, technical problems with focusing the laser beam (diffraction and refraction effects) arise. In contrast, with electrochemical and potentiometric electrodes as detectors, extremely small detection volumes are possible. As an example, Ewing and Olefirowicz were able to determine catecholaminesin single cells with an amperometric detector, using capillaries of 2-5-pm i.d. which allowed direct sampling.’ Contrary to amperometrical detectors, which are suitable for determining electroactive analytes only, potentiometric detectors can be used for analyzing inorganic and organic ions (e.g., neurotransmitters) by CZE in asingle run.8 This was f i t shown with ion-selective microelectrodes (ISMEs) in postcolumn position.gJ0 Later, the introduction of a conically etched capillary aperture made on-column detection with ISMEs possible without decoupling them from the electrophoretic field, yielding electropherograms of drastically reduced drift and noise.” The present work describes the quantification of inorganic and organic cations with ISMEs as on-column CZE detectors in fusedsilica capillaries of 10-pmi.d., obtaining a precision generally better than 5% in the range of 1V-109 M.
THEORY
Potentiometric Detection. The emf response, E, of an ion-selective electrode in a solution containing two cations (analyte ion I and discriminated background ion J, with activities ai and aj, and charge numbers zi and z j , respectively) is described with good approximation by the NicolskyEisenmanlz equation: E = Eio + s log[ai + KijP”t(aj)’J’jl
(1)
where Eio is the sum of the reference and liquid junction potentials (for the experimental setup used assumed to be constant, due to buffered background electrolyte and ai 0 ) (10) ofeqOiscomidered,themultiplierbeingcomtantforaspecific analyte. The peak integral, 6,then corresponds to JZJ dt, whereas the potentiometrically recorded peak area, A, is defined as J E dt. Equation 9 is valid only if the zone to he detected contains a single analyte and the background ion, i.e., for complete electrophoretic separations. When the sample solution contains two comigrating analyte ions with similar selectivity constants, accurate analysis with a single
D = 1 f l / a -1
(ID) W A C Recommendations for Nomenclature of lon.Selstive Elertrodcs. Pure Appl. C h m . 1976.48, 127-132. 1141 Chiea. R. L.: Burpl. D. S.AM!. Chem. 1992.64.489A496A.
Flgure 1. (Bottom)ElecbophaeHc zone containing anawe amount. mh (retention time. t,) detected wlth ISME In omlumn position (V, zone volume between a and b; wb zone width: L. total l e n m of CZE caplllary: r. Innerradlus). (Top)Delogarlthmlzedpeakof umespondlng emf cuwe for elution perlod of m,
ISME is not possible. On the other hand, if one analyte and the background ion are discriminated in the same degree, the other analyte ion can be quantified. As the activity, aj, of the background ion is assumed to be constant, the error due to thisapproximation(calculatedforthewomtcaseofunbuffered sample solutions) is 0.99)and precision (mean error < 5 % ) are reached in most cases. The experimental slopes for K+, Na+, Rb+, and the protonated forms of dopamine and imidazole do not heavily deviate from the theoretical slope of 58.94mV (corresponding to that for univalent ions at 24 "C, assuming a temperature rise in the capillary of -2 "C relative to room temperature due to Ohmic heatingm); Le., the ISME shows Nernstian behavior for these cations. ISMEs generally do not exhibit ideal Nernstian behavior for all ions over the same activity range21 and often show a flattening of the response curve at activity ranges
