Anal. Chem. 1998, 70, 360-365
Simultaneous Determination of Small Cations and Anions by Capillary Electrophoresis Petr Kuban and Bo Karlberg*
Department of Analytical Chemistry, Stockholm University, S-106 91, Stockholm, Sweden
A capillary electrophoresis system for the simultaneous determination of small cations and anions has been developed. Cations and anions in a sample are separated in one single electrophoretic run using one capillary and just one detector. The sample is injected into the first end of the separation capillary and subsequently into the second end. When the high voltage is applied, the cations and the anions in the two injected sample portions start to migrate against each other toward the center of the capillary. The detection window is placed approximately in the middle of the capillary. A suitable electrolyte consists of 6 mM 4-aminopyridine, 2.7 mM H2CrO4, and 30 µM cetyltrimethylammonium bromide at pH 8. The composition of the background electrolyte provides excellent separation of 22 small inorganic and organic anions and 1A and 2A class cations within 5 min. The overall repeatability of the migration times is less than 0.3%, and the repeatability of the peak area ranges from 1.7 to 5.5% (n ) 9) based on manual hydrodynamic injections with a duration of 40 s. The technique has been applied for the analysis of tap water, rainwater, and process water samples. The total time for an analysis of both cations and anions in a water sample is less than 3 min. Milk and mud samples were also analyzed after off-line dialysis pretreatment. During the last decade, capillary electrophoresis (CE) has attained great acceptance mostly due its high separation efficiency, low sample and electrolyte consumption, and short analysis times. Most applications involve separation and quantitation of large organic molecules and biomolecules. However, some attention has recently been paid to the determination of small ions as well. The advantages of using CE for this latter type of application are mainly the higher resolution and the shorter analysis times in comparison with ion chromatography (IC), which is the predominantly used routine technique in this case. Among the various available detection principles used for the determination of small anions and cations by CE, conductimetry is sometimes preferred since it is sensitive and universal. However, spectrophotometry is a viable detection alternative even if most small anions and cations lack the necessary intrinsic chromophore properties allowing a straightforward, sensitive detection at any wavelength above 200 nm. Consequently, the indirect UV detection principle is applied; commonly used 360 Analytical Chemistry, Vol. 70, No. 2, January 15, 1998
electrolytes for anion determination are chromate1 and pyrromellitate2 and, for cation determination, 4-aminopyridine,3 imidazole,4 and creatinine.5 Since the migration velocities of small, charged ions exceed the velocity of the electroosmotic flow (EOF), the coelectroosmotic separation principle is usually preferred. In uncoated capillaries, the direction of the EOF is from the anode to the cathode, which means that only cations can be separated without any modification of the capillary wall. The direction of the EOF has to be decreased or reversed to achieve reasonable analysis times for small anions. EOF modifiers such as cetyltrimethylammonium bromide (CTAB),6 tetradecyltrimethylammonium bromide (TTAB),7 or hexamethonium hydroxide (HMH)2 have been used for this purpose. The pH of the separation electrolyte has to be carefully selected. While cationic separations are mostly performed at pH 3-5, anionic separations are seldom carried out using an electrolyte with a pH value below 7.5. This fact can be attributed to the different behavior and properties of cations and anions in aqueous solutions. Cations, especially transition metal and some of the 2A group cations, readily precipitate at high pH, explaining why such separations are predominantly carried out at a low pH. In addition, the electrolyte constituents, used to provide UV absorbance background for cation separations, have pKa values in the acidic pH range, which means that they are protonated and charged at low pH values. To match the mobility of the sample cations, the pH has to be sufficiently low so that almost all the electrolyte constituents are present as cations. Certain applications, e.g., separation of transition metal cations, require addition of a weak complexing agent to the background electrolyte such as hydroxyisobutyric acid (HIBA)5 or citric8 or lactic9 acid. These compounds form weak complexes with most of the transition metal cations, and enhanced selectivity is obtained. For the determination of anions, the electrolyte pH is of minor concern, especially when only anions of strong acids are present in the sample. If, however, anions of weak acids are present, e.g., acetate and carbonate, serious tailing and unacceptably long (1) Jandik, P.; Jones, W. R. J. Chromatogr. 1991, 546, 431-443. (2) Harrold, M. P.; Wojtusik, M. J.; Riviello, J.; Henson, P. J. Chromatogr. 1993, 640, 463-471. (3) Beck, W.; Engelhardt, H. Fresenius J. Anal. Chem. 1993, 346, 618-621. (4) Beck, W.; Engelhardt, H. Chromatographia 1992, 33, 313-316. (5) Foret, F.; Fanali, S.; Nardi, A.; Bocek, P. Electrophoresis 1990, 11, 780783. (6) Altria, K.; Simpson, C. Anal. Proc. 1986, 23, 453-454. (7) Huang, X.; Luckey, J. A.; Gordon, M. J.; Zare, R. Z. Anal. Chem. 1989, 61, 766-770. (8) Lin, T. I.; Lee, Y. H.; Chen, Y. C. J. Chromatogr. 1993, 654, 167-176. (9) Shi, Y.; Fritz, J. S. J. Chromatogr. 1993, 640, 473-479. S0003-2700(97)00613-6 CCC: $15.00
© 1998 American Chemical Society Published on Web 01/15/1998
Figure 1. Effect of the CTAB concentration on the migration times of some anions and cations. Electrolyte: 6 mM 4-aminopyridine, 2.7 mM H2CrO4, pH 8. Injection conditions: hydrodynamic injection; capillary ends elevated to a height of 10 cm for 40 s (cathode end) and to 5 cm for 20 s (anodic end). Time between the injections, 0 s.
migration times occur when electrolytes with pH values below 7.5 are used. The separation conditions for small anions and cations by CE are quite different and it seems that a compromise electrolyte composition may be difficult to find although some progress has been reported recently. It has been possible to separate anions and cations of large organic molecules and biomolecules in a reasonable time10,11 when the migration velocities are low for both categories of ions. On the basis of the same principle, slowly migrating organic anions and inorganic cations (Li+, K+) could be separated in a system with a high EOF.12 Some small chloroanions and inorganic cations were simultaneously determined by Ba¨chman et al.13 using the indirect fluorescence detection principle. Their system employed one common electrolyte solution but two capillaries and two detectors, making this approach experimentally complicated. This paper describes a new CE application using one common electrolyte for separation of the most common small inorganic/ organic anions and cations. The system uses one capillary and just one detector. The sample is first injected into one end of the separation capillary and subsequently into the other end. The detector is placed in the center of the capillary. The technique has successfully been applied to the simultaneous determination (10) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302. (11) Shamsi, S. A.; Danielson, N. D. Anal. Chem. 1995, 67, 4210-4216. (12) Foret, F.; Fanali, S.; Ossicini, L.; Bocek, J. Chromatogr. 1989, 470, 299308. (13) P. Ba¨chmann, K.; Haumann, I.; Groh, T. Fresenius J. Anal. Chem. 1992, 343, 901-902.
of anions and cations in natural water samples. Milk and mud samples pretreated by dialysis have also been analyzed. EXPERIMENTAL SECTION Reagents and Samples. All chemicals were of analytical grade; 18-crown-6 was provided by ICN (ICN Pharmaceuticals Inc., Costa Mesa, CA). The stock solutions of anions and cations, 10 000 ppm each, were prepared by dissolving the respective salts in deionized water. Na+ and Cl- salts were used for anion and cation stock solutions, respectively, except for Mg2+, which was prepared from MgSO4. The background electrolyte was prepared daily by diluting stock solutions of 50 mM 4-aminopyridine, 1 M CrO3, and 50 mM CTAB (5% v/v acetonitrile) up to the final concentrations of 6 mM, 2.7 mM, and 30 µM, respectively. The pH value of this electrolyte was 8. The electrolyte was degassed in an ultrasonic bath for 5 min prior to introduction into the capillary. Samples were introduced without any pretreatment, except for milk and mud samples which were subjected to offline dialysis. Instrumental Procedures. The separations were carried out in fused-silica capillaries, 50 µm i.d., 375 µm o.d., 50 cm total length (Polymicro Technologies, Phoenix, AZ). The detection window was burnt out at a distance of 20 cm from the anodic capillary end. The high-voltage supply (Bertan Associates Inc., Hicksville, NY) was operated at 20 kV during all separations. The samples and the electrolyte solution were sequentially introduced into both capillary ends by elevating each end to a specified height (5-15 cm) and keeping it there for a given time period. The indirect detection was carried out at 262 nm using an Isco CV4 spectroAnalytical Chemistry, Vol. 70, No. 2, January 15, 1998
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Figure 2. Influence of 18-crown-6 on the migration behavior of some anions and cations. Electrolyte: 6 mM 4-aminopyridine, 2.7 mM H2CrO4, 30 µM CTAB, pH 8. Injection conditions and time, the same as in Figure 1.
photometer (Isco, Lincoln, NE). All peaks in the electropherograms were reversed upon visual presentation. The data were processed in an ELDS Professional data system (Chromatography Data Systems, Kungsho¨g, Sweden). Dialysis. The dialysis device has been described elsewhere.14 It consisted of a Gilson peristaltic pump (Gilson, Villiers le Bel, France) which delivered the donor and the acceptor solutions at flow rates of 3.0 and 0.5 mL/min, respectively, to a Tecator 5050 dialysis module (Foss Tecator, Ho¨gana¨s, Sweden). The samples were introduced as donor solutions and were not subjected to any other pretreatment. The acceptor phase was collected in 1.5mL PE vials and then manually injected into the CE system. Capillary Rinsing Procedures. New capillaries were initially rinsed with 0.1 M NaOH for 30 min and then with water for 10 min. The daily starting up procedure entailed the following rinsing steps: 1 M HCl for 30 min, water for 5 min, air for 5 min, and running electrolyte for 15 min. This procedure proved to be efficient in avoiding Ca2+ and Mg2+ precipitation on the capillary walls when the electrolyte was used at pH 8. The daily closing down procedure was comprised of the following rinsing steps: water for 5 min, 1 M HCl for 5 min, water for 5 min, air for 5 min. These rinsing procedures enabled long-term stable performance of the capillaries during a period of at least one month. RESULTS AND DISCUSSION Choice of the Background Electrolyte. The electrolyte should contain cationic and anionic species that individually can (14) Kuban, P.; Karlberg, B. Anal. Chem. 1997, 69, 1169-1173.
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provide a UV absorbance background. The cationic form of 4-aminopyridine (pKa ) 9.16), introduced by Beck and Engelhart,3 is UV absorbing and was selected since pH must be raised in order to deprotonize weak acids in the sample. Chromate was added as H2CrO4 to provide background UV absorbance for anion separation; sodium or potassium chromate could not be used since these cations are potential analyte ions. Finally, addition of CTAB was found to fulfill two purposes: first, to modify the EOF, and, second, to improve the peak shapes of Ca2+ and Mg2+ at the selected pH value of 8. Figure 1 shows the effect of the CTAB concentration in the electrolyte on the migration times for some selected anions and cations. It can be seen that, at zero concentration of CTAB, the migration times for weak acid anions such as phosphate and carbonate are extremely long. When the CTAB concentration is increased, the migration times of the anions decrease drastically while an increase is observed for the cations. A CTAB concentration of 30 µM seems to be a suitable compromise. Thus, a background electrolyte with the following composition was chosen for all subsequent studies: 6 mM 4-aminopyridine, 2.7 mM CrO42-, and 30 µM CTAB. This electrolyte solution has a pH value of 8. Note that K+ and NH4+ ions can be easily separated without addition of any selector compound. This is explained by the partial deionization of the NH4+ ion which occurs at pH values above 8, causing its retardation.3 The separation selectivity of some of 1A and 2A group cations can be altered by addition of macrocyclic compounds such as crown ethers.15 Figure 2 illustrates the influence of addition of a
Figure 3. Separation of 14 common anions and cations with addition of crown ether to the electrolyte. Electrolyte: 6 mM 4-aminopyridine, 2.7 mM H2CrO4, 30 µM CTAB, 2 mM 18-crown-6, pH 8. Injection conditions and time, the same as in Figure 1. Detection principle: indirect UV at 262 nm, peak reversal.
crown ether, 18-crown-6, to the electrolyte having constant concentrations of 4-aminopyridine, CTAB, and CrO42- at pH 8. The crown ether concentration was varied in the range 0-3 mM. The migration times of the anions and some of the cations are not significantly changed. However, the migration times of K+ and Sr2+ are greatly influenced. Complete separation of Sr2+ and Ca2+ can be accomplished. Further, a reversal of the migration order for K+ and NH4+ can be observed. A typical electropherogram demonstrating the separation of 14 common cations and anions is shown in Figure 3. The electrolyte contained a 2 mM 18-crown-6 concentration which gives complete baseline separation of NH4+, K+, and Sr2+. Aspects of the Injection. As mentioned, hydrodynamic injection of the sample was carried out into both ends of the capillary, which means that the second injection may expel the first injected sample portion. This problem can easily be solved in two ways: (1) by hydrodynamic injection of the electrolyte solution after the first injection of sample or (2) by applying the high voltage during a short period of time while the capillary end is immersed in the electrolyte solution. The first approach was adopted. When the second injection of sample is made into the opposite capillary end, only the electrolyte plug is expelled. As a result, the two injected sample portions are maintained inside the capillary and the high voltage can be applied. Alternatively, electrokinetic injection could be applied throughout, thereby avoiding problems with flow induced dispersion. It can be concluded by examining the graphs shown in the Figure 1 that baseline separation of all selected anions and cations cannot be achieved when the two sample portions are being injected at the same time. Consequently, we have investigated the possibility to displace the two injections in time. Figure 4 shows three electropherograms obtained for the same ion mixture but with time-displaced injections. The time intervals between the two injections were 0, 30, and 60 s. Registration of the electropherograms was started after the second injection had been performed. As can be seen, excellent separations can be achieved and overlap of anion and cation peaks can be avoided. We have (15) Francois, C.; Morin, P.; Dreux, M. J. Chromatogr. 1995, 706, 535-553.
Figure 4. Electropherograms for standard anion and cation mixture with time displaced injections. Time elapse between the two injections: (a) 0, (b) 30, and (c) 60 s. Electrolyte and injection conditions are the same as in Figure 2. Detection principle: indirect UV at 262 nm, peak reversal. The linear baseline drift observed during the first 2 min was corrected for. Peak identification: (1) K+, (2) NH4+, (3) Cl-, (4) SO42-, (5) NO3-, (6) Na+, (7) F-, (8) HPO42-, (9) Ca2+, (10) Mg2+, (11) HCO3-, and (12) Li+.
Figure 5. Separation of 22 anions and cations. Electrolyte and injection conditions, the same as in Figure 2. Time between the injections, 80 s. Detection principle: indirect UV at 262 nm, peak reversal. The linear baseline drift observed during the first 3 min was corrected for. Peak identification: (1) S2O32-, (2) Br-, (3) Cl-, (4) SO42-, (5) NO2-, (6) NO3-, (7) WO42-, (8) MoO42-, (9) citrate, (10) maleate, (11) fumarate, (12) F-, (13) HPO42-, (14) Cs+, (15) K+, (16) NH4+, (17) HCO3-, (18) acetate, (19) Na+, (20) Ca2+, (21) Mg2+, and (22) Li+.
also investigated to what extent the stopping of the separation run influences band broadening. The high voltage was interrupted after 30 s, and electrolyte was introduced instead of sample. The high voltage was then applied again. No significant change in the theoretical plate number was observed. Separation of 22 Anions and Cations in One Run. An electropherogram of 22 anions and cations, separated in less than 5 min, is shown in Figure 5. It should be noted that only 1A and 2A group cations can be separated by using the described electrolyte since transition metal cations as well as Ba2+ form hydroxides or are precipitated by the chromate ion. Alternative electrolytes can be used for samples containing these ions, and Analytical Chemistry, Vol. 70, No. 2, January 15, 1998
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Figure 6. Analysis of tap water (A) and rainwater (B) samples. Electrolyte, the same as in Figure 2. Injection conditions: hydrodynamic injection; capillary ends elevated (A) to a height of 10 cm for 10 s (cathode end) and to 5 cm for 10 s (anodic end); (B) to a height of 10 cm for 40 s (cathode end) and to 10 cm for 40 s (anodic end). Time between the injections, 0 s. Detection principle: indirect UV at 262 nm, peak reversal. Table 1. Repeatability of Migration Times (tM) and Peak Areas (PA) (rsd, n ) 9) ion
tM (%)
PA (%)
ion
tM (%)
PA (%)
K+ NH4+ Na+ Ca2+ Mg2+ Li+
0.30 0.26 0.26 0.20 0.21 0.23
4.1 3.4 5.5 2.5 2.4 1.7
ClSO42NO3FHPO42HCO3-
0.15 0.16 0.16 0.18 0.16 0.18
3.1 4.9 4.3 1.8 3.9 3.8
development of other electrolyte compositions is in progress. However, transition metal cations are not present in real samples such as tap water and rainwater at concentration levels jeopardizing the determination of the main ion constituents. Validation. The method parameters that were validated are summarized in Table 1. The repeatability was evaluated as the relative standard deviation of migration times and peak areas for nine consecutive injections. The rsd values for the migration times indicate a very good repeatability (less than 0.3%), while the rsd values for the peak areas are in the range 1.7-5.5%. This can be attributed to the imprecision in the manual injection technique; rsd values of up to 10% are frequently reported.16 The repeatability 364 Analytical Chemistry, Vol. 70, No. 2, January 15, 1998
Figure 7. Electropherograms of milk (A) and mud (B) samples. The samples were injected into the capillary after off-line dialysis. Electrolyte, the same as in Figure 2. Injection conditions: hydrodynamic injection; capillary ends elevated (A) to a height of 5 cm for 10 s (cathode end) and to 5 cm for 10 s (anodic end); (B) to a height of 5 cm for 20 s (cathode end) and to 10 cm for 40 s (anodic end). Time between the injections: (A) 60 and (B) 30 s. Detection principle: indirect UV at 262 nm, peak reversal. (*) unidentified peaks.
can be improved by using an internal standard, e.g., Li+. The detection limits (LOD) were comparable to those obtained for separate anion and cation determination systems. Real Samples, Quantitative Analysis. The system was applied to the analysis of real samples, and typical electropherograms for tap water and rainwater samples are depicted in Figure 6. All cations and anions can be separated in less than 3 min. Reported values agree with those obtained by using alternative techniques (IC, wet chemistry methods). Some wastewater samples were also injected without any pretreatment other than sedimentation. Nutrient ions such as phosphate, nitrate, and ammonium could be quantified. We have shown previously14 that dialysis is an efficient sample pretreatment technique which can be used to separate small analyte ions from a complex matrix before introduction into a CE system. Milk samples, for instance, contain proteins and large molecules that easily adsorb on the capillary walls, thereby deteriorating the separation. Mud samples contain solid particles (16) Jandik, P.; Bonn, G. Capillary electrophoresis of small molecules and ions; VCH: New York, 1993.
and cannot be injected directly into the CE system since capillary blocking can occur. Milk and mud samples were therefore dialyzed. The dialyzate was collected in PE vials and injected into the CE separation system. Typical electropherograms are shown in Figure 7. The total analysis time for a milk sample was less than 5 min. Neither adsorption nor blocking effects were observed. CONCLUSIONS The developed CE system for the simultaneous determination of small anions and cations has proven to be an efficient analytical tool for “total ion analysis” of various aqueous samples. The two injections of the sample can be temporally displaced to accomplish complete baseline separation of both anions and cations. The
system is simple since just one detector and one capillary are used; automation of the described analytical procedure should thus be easy to realize. The sampling frequency can reach 10/h, capillary rinsing included, and in each run up to 22 different analyte ions can be determined. Alternative electrolyte compositions would be possible to develop for specific samples containing transition metal ions.
Received for review June 16, 1997. Accepted October 30, 1997.X AC9706133 X
Abstract published in Advance ACS Abstracts, December 15, 1997.
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