Anal. Chem. 1996, 68, 2366-2369
On-Line Chemiluminescence Detection for Capillary Ion Analysis Bo Huang, Jian-jun Li, Le Zhang, and Jie-ke Cheng*
Department of Chemistry, Wuhan University, Wuhan 430072, China
Highly sensitive chemiluminescence detection for capillary ion analysis has been developed. The new idea lies in using the chemiluminescence reagent luminol as a component of the separation electrolyte, thus preventing loss of the light signal where luminol and H2O2 are mixed in advance in conventional flow injection analysis or ion chromatography chemiluminescence detection. Signal enhancement is achieved by sample stacking injection in the electrophoresis process. The detection limit (S/N ) 3) of 20 zmol (5 × 10-13 mol/L) for cobalt(II) is ∼2 orders of magnitude better than the most sensitive results (1.7 × 10-11 mol/L) reported so far. Detection limits are 2 amol, 80 amol, 740 amol, and 100 fmol for copper(II), nickel(II), iron(III), and manganese(II), respectively. The sensitivity is significantly better than that in the electrochemical detection and the commonly used UV detection in capillary ion analysis. The capillary electrophoresischemiluminescence detector has been used to separate five metal ions, cobalt(II), copper(II), nickel(II), iron(III), and manganese(II), within 8 min with an average theoretical plate number of 4.6 × 105. Recently, several interesting approaches to the separation of low molecular weight ions have been developed with the help of the newly emerging capillary electrophoresis (CE) methodology. Jandik et al.1 proposed a name for this new areascapillary ion analysis (CIA). Although CE as a powerful separation method has the virues of high resolution, rapid separation, and small analyte consumption, its weakness is thought to be its detection capabilities.2,3 By its capillary nature, the technique typically requires only nanoliter volumes of analyte, a characteristic that motivates the search for detection methods of high sensitivities. So far, UV absorption detection has been the most commonly used method for CIA, with sensitivities typically of 10-5-10-6 mol/L. An alternative detection scheme is chemiluminescence (CL), which has been applied to liquid and gas chromatography, immunoassay, and flow injection analysis.4-7 CL can be anticipated to provide excellent sensitivity because of its low background nature. To combine the high separation ability of CE with the high sensitivity of CL is a brand-new subject.8-12 (1) Jandik, P.; Jones, W. R.; Weston, A.; Brown, P. R. LC-GC 1994, 5, 20-27. (2) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302. (3) Gordon, M. J.; Huang, X.; Pentoney, S. L., Jr.; Zare, R. N. Science 1988, 242, 224-228. (4) Kawasaki, T.; Maeda, M.; Tsuji, A. J. Chromatogr. 1985, 328, 121-126. (5) Worsfold, P. J.; Nabi, A. Anal. Chim. Acta 1985, 171, 333-336. (6) Bronstein, I.; McGrath, P. Nature 1989, 338, 599-600. (7) Lewis, S. W.; Prince, D.; Worsfold, P. J. J. Biolumin. Chemilumin. 1993, 8, 183-199. (8) Dadoo, R.; Colon, L. A.; Zare, R. N. J. High. Resolut. Chromatogr. 1992, 15, 133-135. (9) Ruberto, M. A.; Grayeski, M. L. Anal. Chem. 1992, 64, 2758-2762.
2366 Analytical Chemistry, Vol. 68, No. 14, July 15, 1996
A variety of metal ions are sensitive to the chemiluminescence system due to their catalytic effect on the luminol-hydrogen peroxide reaction, but serious interference and lack of selectivity limit its application. Although ion chromatography (IC)13,14 has been developed as a separation means to enhance the selectivity, problems of complicated performance, limited analytes, and low separation efficiency make it unsatisfactory. Here, we utilize a self-constructed capillary electrophoresis on-line CL detector to realize rapid separation and sensitive detection of five metal ions. Since CL detection demands introducing chemiluminescent reagent, this scheme is similar to the postcapillary reactor originally developed by Rose and Jorgenson15 to carry out derivatization reactions before laser-induced fluorence (LIF) detection. The aim of this study is to develop a sensitive and novel means for CIA. To enhance the sensitivity, sample stacking injection16 is used in the electrophoresis process. EXPERIMENTAL SECTION Reagents and Solutions. Distilled, deionized 18 MΩ water was used to prepare all solutions. Unless stated otherwise, all the chemicals were of analytical-reagent grade or better (Shanghai Reagent Factory). Solutions of luminol (A.R. grade, Sanxi Formal University) and hydrogen peroxide in 2.5 × 10-2 mol/L sodium acetate buffer were prepared daily from stock solutions of 1 × 10-2 mol/L luminol and 1.0 mol/L hydrogen peroxide. The electrophoretic buffer and reagent solution were adjusted to pH 4.54 with 0.1 mol/L acetic acid and pH 11.6 with 1.0 mol/L sodium hydroxide solution. Metal sample standard solutions were prepared daily by serial dilution of 1 × 10-3 mol/L stock standards of cobalt(II) nitrate, copper(II) sulfate, nickel(II) nitrate, iron(III) sulfate, and manganese(II) sulfate. Before use, all utensils (sample containers, pipet tips, etc.) were soaked in 3.6 mol/L nitric acid for 48 h, rinsed thoroughly in deionized water, and dried. All solutions were filtered through 0.22 µm membrane filters prior to use. Instrumentation and Procedures. The capillary electrophoresis-chemiluminescence detector was laboratory-built17 based on a photomultiplier tube (PMT, Hamamatsu Photonics k. k., Iwata-Gun, Japan) and a signal magnifier (Institute of Chemistry, Chinese Academy of Sciences, Beijing). The CE separations were carried out using a high voltage power (0-30 kV, Peking University). An analog signal was monitored by means of a chart (10) Wu, N.; Huie, C. W. J. Chromatogr. 1993, 634, 309-315. (11) Zhao, J.-Y.; Labbe, J.; Dovichi, N. J. J. Microcolumn Sep. 1993, 5, 331-339. (12) Hara, T.; Okamura, S.; Katou, S.; Yokogi, J.; Nakajima, R. Anal. Sci. (Suppl.) 1991, 7, 261-264. (13) Yan, B.; Worsfold, P. J. Anal. Chim. Acta 1990, 236, 287-292. (14) Gammelgaard, B.; Jons, O.; Nielsen, B. Analyst 1992, 117, 637-640. (15) Rose, D. J., Jr.; Jorgenson, J. W. J. Chromatogr. 1988, 447, 117-131. (16) Chein, R. L.; Burgi, D. S. Anal. Chem. 1992, 64, 489A-496A. (17) Huang, B.; Li, J.-J.; Cheng, J.-K. Chem. J. Chin. Univ. 1996, 17, 528-530. S0003-2700(95)01125-5 CCC: $12.00
© 1996 American Chemical Society
Chemiluminescence Intensity
Time (min)
Figure 1. Chemiluminescence intensity-time profile for luminol and hydrogen peroxide performed at LKB 1251 luminometry: 0.2 mL of 1 × 10-5 mol/L luminol + 1.0 × 10-2 mol/L Na2CO3 (pH 10.6); 0.2 mL of 2.5 × 10-2 mol/L H2O2 + 1.0 × 10-2 mol/L Na2CO3 (pH 10.6).
3066 recorder (The Fourth Instrumental Factory of Sichuan). Experimental conditions were explored at an LKB 1251 luminometry (Pharmacia LKB Co.). Three sizes of fused silica capillaries (Hebei Optical Fiber Factory) were used for the separation and detection. A 5 cm section at the end of an electrophoresis capillary (50 µm i.d.) was etched and carefully inserted into a reaction capillary (530 µm i.d.). A 1 cm detection window was made on the reaction capillary by burning off the coating and was situated just in front of the PMT. Reagent capillary (120 µm i.d.) was used to deliver reagents by gravity. All the capillaries were fixed with a tee connector. The buffer reserviors and the PMT were enclosed within a self-made light-tight box. The new electrophoresis capillaries were washed with 0.1 mol/L NaOH and 0.1 mol/L HCl, rinsed with water, and then equilibrated overnight with the electrolyte. After each working day, the capillary was washed with 0.1 mol/L HCl and water. Injections were performed by electromigration at 10 or 15 kV for various time intervals. RESULTS AND DISCUSSION Mixing Mode of the Analyte and the Chemiluminescent Reagent. Since CL detection requires chemiluminescent reagent, the mixing mode of the analyte and the chemiluminescent reagent influences the light intensity significantly. Different mixing modes might give rise to light quench, even under the same conditions. In conventional flow injection analysis (FIA) and IC chemiluminescence analysis,7,13 luminol stream and hydrogen peroxide stream mix in advance and then react with analyte. A chemiluminescence intensity-time profile study was attempted in order to see the intensity upon this mixing mode. We found that at pH 10.6, the light intensity reached a maximum value in 1-3 s, decayed very quickly in 13 s, and then kept constant after 1 min (Figure 1). Since FIA and IC analysis usually require several hundred microliter volumes of sample consumption, light loss resulting from the mixing of luminol and hydrogen peroxide in advance has a slight effect on the sensitivity of the detection. However, when sample injection is very small (i.e., nanoliters), such a mixing mode might lead to a significant decrease in sensitivity. A 0.05 mol/L Na2B4O7 solution (pH 9.1) was used as the electrophoretic buffer, and 10-4 mol/L luminol + 0.05 mol/L Na2B4O7 (pH 11.0) and 0.05 mol/L H2O2 + 0.05 mol/L Na2B4O7 (pH 11.0) solutions were simultaneously delivered through reagent
capillaries. A steady background signal was produced when the mixed streams flowed through the reaction capillary. Although Co2+, Cu2+, Ni2+, and Fe3+ ions could all produce electrophoretic peaks, the sensitivity was not satisfactory. Due to the apparatus limitation, the observed CL emission intensity showed about 60% light loss by the time the mixed stream of luminol and H2O2 in the tee had reached the detection window after flowing through a 4 cm distance. The detection limit of the most efficient catalyst, Co2+ ion, was only 10-6 mol/L. To improve the sensitivity, we think it is better to use luminol as one of the electrophoretic components and the H2O2 solution as the only reagent to be introduced postcapillary. This mixing mode can be effective because luminol, H2O2, and the metal ions will meet at the detection window simultaneously, while the light signal produced due to the fast kinetics can be detected at the same time. Electrophoretic Separation of Metal Ions in Basic Medium. Metal ions catalyze the oxidation of luminol by H2O2 in alkaline solution; the optimum pH is 11.0-11.8.13 Therefore, it is better to match the pH condition of the electrophoretic medium with that of the reaction zone. In the separation process, the net mobility of an ion is the summation of electroosmotic flow (EOF) and electrophoretic mobility. Since metal ions belonging to groups such as the transition and lanthanide groups have similar electrophoretic mobilities, it is necessary to selectively alter the mobilities via some process such as complexation. Chelating reagents such as EDTA,18 4-(2-pyridylazo)resorcinol (PAR),19 8-hydroxyquinoline-5-sulfonic acid (HQS),20 sodium cyanide (NaCN),21 and R-hydroxyisobutyric acid (HIBA)22,23 have been used as reagents for electrophoretic separation of metal ions. Since PAR is a nonaqueous reagent, NaCN is poisonous, and EDTA inhibits CL intensity due to its strong complexation with metal ions, we chose the weak chelate reagent HQS and HIBA. A 0.025 mol/L Na2B4O7 + 10-3 mol/L luminol + 10-4 mol/L HQS or HIBA (pH 9.1) solution as electrophoretic electrolyte and 0.025 mol/L Na2B4O7 + 0.05 mol/L H2O2 (pH 11.0) were introduced postcapillary. Although the sensitivity can reach a 10-6 mol/L order of magnitude, it is not easy to identify the metal ions from the mixture electropherogram since the background signal is sometimes complicated (Figure 2). Electrophoretic Separation of Metal Ions in Acidic Medium. The optimum pH for metal ions is 3-4 in conventional stationary CL analysis;24 therefore, separating metal ions in acidic medium can avoid the complications due to the hydrolysis effect. But low pH is not suitable for CL reaction. The volume of the sample zone flowing in the 50 µm i.d. electrophoretic capillary is by far small enough compared to the volume of the reagent flowing in the 530 µm i.d. reaction capillary; therefore, the optimum pH environment for separation and detection can be obtained by maintaining acidic conditions to the point of detection, because the latter is mainly dependent on the reagent pH. (18) Motomizu, S.; Oshima, M.; Matsuda, S.; Obata, Y.; Tanaka, H. Anal. Sci. 1992, 8, 619-625. (19) Iki, N.; Hoshino, H.; Yotsuyanagi, T. Chem. Lett. 1993, No. 4, 701-704. (20) Timerbaev, A. R.; Buchberger, W.; Semenova, O. P.; Bonn, G. K. J. Chromatogr. 1992, 630, 379-389. (21) Buchberger, W.; Semenova, O. P.; Timerbaev, A. R. J. High Resolut. Chromatogr. 1993, 16, 153-156. (22) Huggins, T. G.; Henion, J. D. Electrophoresis 1993, 14, 531-539. (23) Chen, M.; Cassidy, R. M. J. Chromatogr. 1993, 640, 425-431. (24) Liu, X.-H.; Lu, M.-G.; Li, X.-Q. Shengwuhuaxue Yu Shengwuwuli Jinzhan 1992, 19, 464-466.
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Figure 2. Separation of 1.0 × 10-6 mol/L Cu2+, Ni2+, Zn2+, and Fe3+ ions mixture. Peaks: 1, Cu2+; 2, Ni2+; 3, Zn2+; 4, Fe3+. Electrophoretic medium, 1 × 10-3 mol/L luminol + 1.0 × 10-4 mol/L HIBA + 5.0 × 10-2 mol/L Na2B4O7 (pH 9.1); postcapillary reagent: 5.0 × 10-2 mol/L H2O2 + 5.0 × 10-2 mol/L Na2B4O7 (pH 10.75); electroinjection, 10 s at 15 kV; operating voltage, 22 kV.
The weak complexing agent HIBA is used to enhance differences in the electrophoretic mobilities, which are the weighted averages of the mobilities of the free metal ions and their various complexes. The pH range from 3.5 to 5.0 (pKa of HIBA ) 3.79)25 was studied since both the EOF and the speciation of the metal ions are influenced by pH. Low pH leads to an increase in the baseline noise and lower signals, which may have been caused by excessive heating due to an increase in the current as a result of the greater mobility of the hydronium ions. At a high pH of 5.0, the peaks of metal ions began to broaden or tail slightly, possibly due to interaction between the cations and the capillary silica surface. Based on the above, a pH of 4.54 was chosen to optimize the sensitivity and resolution. The net electrophoretic mobility of metal ions depends on the degree of complex formation, and thus the separation of the ions is influenced by the concentration of HIBA. The concentration of HIBA was investigated over the range of 0-10 mmol/L at pH 4.54 with an electrolyte solution of 0.01 mol/L NaAc-HAc buffer containing 1 × 10-3 mol/L luminol. The separation was impossible in the absence of HIBA. At high concentration of 10 mmol/L HIBA, the electrophoretic peaks tailed and the sensitivity decreased due to the inhibition of light intensity as a result of the strong complexation of HIBA and the metal ions. The best separation was achieved when the concentration of HIBA was 6.0 mmol/L. The relevant electropherogram is shown in Figure 3. Co2+, Cu2+, Ni2+, Fe3+, and Mn2+ ions were completely separated with very sharp and symmetric peaks in less than 8 min. A peak width at half-height of about 0.25 s obtained with this interface and CL detection was much sharper compared to that for conventional UV detection1 in capillary ion analysis. The decrease in peak width has been previously observed and can be explained in terms of the “chemical band narrowing” effect.26-28 The CL reaction kinetics allow for an intense, rapidly decaying signal to be produced when the metal ions enter the reaction zone of the interface. Unlike conventional photometric detectors, a signal is (25) Perrin, D. D. Stability Constants of Metal Ion Complexes, Part B; IUPAC Chemical Data Series 22; Pergamon Press: Oxford, 1982; p 187. (26) Rule, G.; Seitz, W. R. Clin. Chem. 1979, 25, 1635-1638. (27) Grayeski, M. L.; Weber, A. J. Anal. Lett. 1984, 17, 1539-1552. (28) DeJong, G. J.; Lammers, N.; Sprint, F. J.; Brinkman, U. A. Th.; Frei, R. W. Chromatographia 1984, 18, 129-133.
2368 Analytical Chemistry, Vol. 68, No. 14, July 15, 1996
Figure 3. Separation of metal ions. Peaks: 1, Co2+ (1 × 10-11 mol/L); 2, Cu2+ (5 × 10-8 mol/L); 3, Ni2+ (1 × 10-7 mol/L); 4, Fe3+ (1 × 10-6 mol/L); 5, Mn2+ (3.3 × 10-4 mol/L). Electrophoretic electrolyte, 1 × 10-3 mol/L luminol + 6 × 10-3 mol/L HIBA + 2.5 × 10-2 mol/L NaAc-HAc buffer (pH 4.54); reagent, 5 × 10-2 mol/L H2O2 + 2.5 × 10-2 mol/L NaAc (pH 11.6); sample injection, 5 s at 15 kV; separation voltage, 22 kV. Table 1. Effect of Mixing Time (Tm) of Co(II) Ion and 0.01 mol/L HAc-NaAc Buffer (pH 4.54) on Maximum Chemiluminescence Signal (Max CL) Performed at LKB 1251 Luminometry Tm (min) max CL
0
0
5
10
15
20
40
110
923.0a 1080 1378 665.3 616.0 579.3 547.2 468.3
a 0.1 mL of 10-7 mg/mL Co2+ ion in 0.2 mL of water. 1, 0.2 mL of 1 × 10-5 mol/L luminol + 1.0 × 10-2 mol/L Na2CO3 (pH 10.6); 2, 0.2 mL of 1.0 × 10-2 mol/L HAc-NaAc (pH 4.54) + 0.1 mL of 10-7 mg/ mL Co2+ solution; 3, 0.2 mL of 1.0 × 10-2 mol/L H2O2. Adding order: 1, 2, 3.
produced only when the analyte is in contact with “fresh” reagents. Once the reagents are spent, the signal stops, thus reducing the effective volume of the flow cell to a smaller reaction zone, because a signal is not recorded for the entire residence time in the flow cell.9 Some reports11,29 on CE-CL have mentioned the main shortcoming of the interface, a relatively low number of theoretical plates due to the band broadening as a result of the postcapillary mixing of analyte and reagent and slow CL reaction kinetics. In this experiment, the average theoretical plate number reaches 4.6 × 105, which is superior to other CE-CL results reported so far. This suggests that the metal ion-luminol-H2O2 chemiluminescence system is quite suitable for the CE-CL system. Enhancement of Sensitivity. The detection sensitivity of this apparatus has been improved with some design changes compared with prior CE-CL detectors.10 The reaction capillary is situated just in front of the PMT without using optical fibers to transport light, hence avoiding light loss by 10%.10 Good ground connection and the dry, insulated status of the high-voltage part help to keep the electrophoretic base line low and steady. An electric capacitor between the high and low positions of the chart recorder can effectively filter the system noise and hence improve the detection sensitivity. (29) Dadoo, R.; Seto, A. G.; Colon, L. A.; Zare, R. N. Anal. Chem. 1994, 66, 303-306.
Table 2. Linear Ranges and a Comparison of Detection Limits for the Metal Ions in CL Detection with That in UV and Electrochemical Detection (ECD) detection limit cations
linear range (mol/L)
CL (mol/L)
UVa (10-6 mol/L)
ECDb (10-6 mol/L)
Co(II) Cu(II) Ni(II) Fe(III) Mn(II)
1.0 × 10-11-5.0 × 10-6 5.0 × 10-9-2.0 × 10-4 2.0 × 10-8-5.0 × 10-4 8.0 × 10-7-4.0 × 10-4 5.0 × 10-5-1.0 × 10-3
5.0 × 10-13 (20 zmol) 1.0 × 10-10 (2 amol) 5.0 × 10-9 (80 amol) 5.0 × 10-8 (740 amol) 8.0 × 10-6 (100 fmol)
1.8 3.2 2.0 2.4 2.2
5.1 5.0 2.2
a Experimental conditions: 20 kV over a 75 µm × 52 cm capillary; hydrostatic injection, 30 s.35 b Experimental conditions: 30 kV over a 25 µm × 90 cm capillary; injection, 20 kV for 1 s.36
Several techniques have been reported for sample preconcentration in CZE. Although isotachophoresis (ITP)30,31 can produce a very sharp band, its major disadvantage is the need to use several different types of support buffers in a single capillary column, a process known as discontinuous support buffer. In addition, only one type of ion can be determined in a particular separation. The most widely used concentration technique, oncolumn sample concentration in a single, continuous support buffer, was first used in CZE by Mikkers et al.32 It takes advantage of conductivity differences between the sample zone and the background electrolyte.16,32,33 When a sample dissolved in water or dilute buffer is applied, a higher electrical field strength in the sample zone leads to concentration of the ionic analyte at the front of the zone. As a result, the sample volume can be increased by a factor of 10 without contributing to band broadening. The concentration effect occurs with both hydrodynamic injection and electrokinetic injection: the lower the buffer concentration in the sample, the higher the analyte concentration that is injected. The effect of mixing time of cobalt(II) and 0.01 mol/L HAc-NaAc buffer (pH 4.54) on the light intensity was studied at LKB 1251 luminometry (Table 1). The experiment suggests that the 0.01 mol/L HAc-NaAc support electrolyte interacts with cobalt(II) ion and leads to the decrease in light intensity as time elapses; hence, we simply dissolved the sample metal ions in water. In the meantime, stacking sample injection is used in the electrophoresis process for enhancement of sensitivity and reproducible results. The minimum detectable concentrations for the metal ions are based on a peak height of 3 times the baseline noise. The detection limit of Co2+ reaches 5 × 10-13 mol/L (20 zmol), which is ∼2 orders of magnitude lower than the best results reported so far13,34 (1.7 × 10-11 mol/L). Figure 4 shows the electropherogram of Co(II) ion at a concentration of 2.5 × 10-12 mol/L. Detection limits for Cu2+, Ni2+, Fe3+, and Mn2+ are 2 amol, 80 amol, 740 amol, and 100 fmol, respectively. The sensitivity is significantly better than those of electrochemical detection and the commonly used UV detection in CIA (Table 2). The relative standard deviations (RSDs) in migration time and peak height for each of the metal ions are shown in Table 3. (30) Everaerts, F. M.; Verheggen, T. P. E. M.; Mikkers, F. E. P. J. Chromatogr. 1979, 169, 21-38. (31) Jandik, P.; Jones, W. R. J. Chromatogr. 1991, 546, 431-443. (32) Mikkers, F. E. P.; Everaerts, F. M.; Verheggen, T. P. E. M. J. Chromatogr. 1979, 169, 11-20. (33) Burgi, D. S.; Chien, R.-L. Anal. Chem. 1991, 63, 2042-2047. (34) Yan, B.; Lewis, S. W.; Worsfold, P. J.; Lancaster, J. S.; Gachanja, A. Anal. Chim. Acta 1991, 250, 145-155. (35) Weston, A.; Brown, P. R.; Jandik, P.; Jones, W. R.; Heckenberg, A. L. J. Chromatogr. 1992, 593, 289-295. (36) Lu, W. Z.; Cassidy, R. M. Anal. Chem. 1993, 65, 1649-1653.
Figure 4. Electropherogram of 2.5 × 10-12 mol/L Co(II) ion. Electrophoretic electrolyte, 1 × 10-3 mol/L luminol + 5 × 10-3 mol/L HIBA + 2.5 × 10-2 mol/L NaAc-HAc buffer (pH 4.3); other conditions as in Figure 3. Table 3. Reproducibility in Peak Height and Migration Time of Five Metal Ions RSD (%) metal ion
migration time (min)
migration time (n ) 6)
peak height (n ) 4)
Co(II) Cu(II) Ni(II) Fe(III) Mn(II)
3.0 4.8 5.7 6.2 7.4
0.18 0.32 0.47 0.26 0.43
5.6 4.1 3.5 4.6 7.2
Conventional chemiluminescence analysis requires a certain amount of analyte; usually the less analyte, the lower the senstivity. Our experiment obtains an excellent detection limit based on less than 20 nL volumes of sample injection. This study has not only developed a way to solve the problem of interference in chemiluminescence detection but also built a new and sensitive means for capillary ion analysis. ACKNOWLEDGMENT The authors gratefully acknowledge support for this research by a doctoral special grant from the State Commission of Education of the People’s Republic of China.
Received for review November 16, 1995. Accepted March 28, 1996.X AC9511253 X
Abstract published in Advance ACS Abstracts, May 15, 1996.
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