On-column Indirect Photothermal Interference Detection for Capillary Zone Electrophoresis Yonggang Hua, Jieke Chenga and Yanzhuo Deng*b a Department of Chemistry, Wuhan University, Hubei 430072, China b Center of Analysis and Testing, Wuhan University, Hubei 430072, China An indirect photothermal interference detection method, which is based on the diffraction of a probe laser beam by a capillary tube, for detecting metal cations separated by capillary zone electrophoresis (CZE) is described.In this capillary photothermal interference detector, a 2 mW He–Ne probe laser is employed to provide the probe beam and an 18 mW He–Ne pump laser is used to supply the pump beam. Factors that contribute to the noise and signal in indirect photothermal interference detection in CZE, such as the noise from the scattered light caused by both the probe beam and the pump laser beam, and effects of the concentration of ethanol and NaCl on the separation efficiency, were studied.The effect of scattered light can be reduced effectively with the configuration constraints investigated for photothermal capillary absorption measurements. With Methylene Blue added to carrier electrolyte as an absorber, three typical metal cations (KI, CuII and AlIII) were separated using this system. For CuII, a 2.1 3 1027 mol l21 concentration detection limit (S/N = 2) without preconcentration and a 1.02 3 10217 mol detection limit as absolute amount were measured, considering the optical sampling volume estimated to be 50 pl, and 1.99 3 106 theoretical plates were observed on the laboratory-made CE system.It was demonstrated that indirect photothermal interference detection is suitable for small capillaries and a large ionic strength range for CE analysis. Keywords: Indirect photothermal interference detection; capillary zone electrophoresis; metal cations; Methylene Blue As a powerful technique for separations, the number of applications of capillary zone electrophoresis (CZE) has increased greatly in recent years.In principle, CZE is very suitable for small ionic compounds. However, the application of CZE to inorganic species, especially metal cations, is rare, in contrast to the separation of biological macromolecules.1,2 The reason is probably that many of these classes of compounds lack chromophores at useful wavelengths or have such low molar absorptivities as to preclude adequate sensitivity with absorption detection.Cation determinations by CZE are also hampered by coulombic interactions of the cations with the surfaces of the fused-silica capillaries commonly employed in CZE. These interactions can result in band broadening, which reduces both detectability and resolution. Methods adopting color reaction techniques to improve the LODs of metal cations with absorbance detection or laserinduced fluorescence detection can be easily devised. For example, 8-hydroxyquinoline-5-sulfonic acid (HQS2) was used for the determination of metal ions by CZE with laser-induced fluorescence.3 However, the main disadvantage is that the chemical properties of these complexes are usually unstable during the process of separation and it is difficult to optimize the separation and detection conditions for these complexes.4 The second disadvantage is that multiple complexes may exist in solution and multiple peaks would appear, making it very difficult to distinguish the signal peaks.The third disadvantage is that the analytes are chemically altered and collection and further studies are almost impossible. The fourth disadvantage is that derivatization is time consuming and inefficient. Hence there is a need for an all-purpose detector for CZE. Indirect detection is the most common method for metal cations which lack chromophores in CZE. A comprehensive account of indirect detection methods has been given by Yeung.5 Several reasons for developing indirect detection schemes have been mentioned.First, indirect detection is universal, and can be used for compounds which lack chromophores or fluorophores. Second, it is possible to broaden the applicability of highly sensitive but selective detectors by implementing indirect detection. Third, quantification is easier with indirect detection since tedious chemical derivatization procedures can be avoided. Fourth, indirect detection is nondestructive since no chemical manipulation is necessary and collection and further studies are facilitated.The indirect signal (displacement peaks) is a minor perturbation on the background signal. The concentration limit of detection, Clim, is given by6 C C TR DR lim = � M (1) where CM is the concentration of the relevant component added to the mobile phase, TR is the displacement ratio and DR is the dynamic reserve and is equal to the signal-to-noise ratio (S/N). It indicates that, for a given system, the more stable the background signal (larger DR), the more effective the displacement process (larger TR) and the lower is CM, the lower are the detection limits that can be obtained.Because DR and TR possibly decrease as one decreases CM, on the other hand, in order to improve the concentration LOD, detection methods with a capability for ultratrace detection and a larger range of detection are necessary to maintain a larger and stable background signal so as to allow the use of CM values that are as low as possible.Conductivity detection is an important indirect method for metal cations in CZE,7 which also functions in a displacement mode. The signal arises from the difference in equivalent conductance or mobility of the charge carrier electrolyte ion and the analyte ion. Several problems are usually encountered with conductivity detection. (1) It is advantageous to use a low ionic strength, poorly ionized buffer system to produce a low conductivity background.If good separation is desired, the concentration of the background electrolyte must be high relative to the concentration of the sample. However, this condition (high electrolyte concentration) results in a decrease in sensitivity due to the elevated background conductivity. (2) Background noise from the high voltage prevents highsensitivity detection. (3) A large difference between the mobility of the carrier electrolyte ion and that of the analyte ion leads to excessive peak tailing/fronting in CE. (4) Cell design is difficult since there must be no significant voltage drop between the cell electrodes; small dimensions of the capillary, high ionic strengths and a high separation electric field are challenges for the use of conductivity detection in CZE.Analyst, October 1997, Vol. 122 (1089–1093) 1089Indirect photometric detection, in which the signal arises from the difference in equivalent absorptivities between the carrier/eluent and the analyte ion, can offer a significant advantage over conductimetric detection in that ionic mobility and optical absorption are unrelated parameters.It is possible to choose carrier ions of suitable mobility and with desired optical absorption characteristics. Hence it is not necessary to compromise between separation efficiency and detection sensitivity. Since DR for photometry will degrade as optical pathlength, CM, or the absorption strengths decrease because incoherent light sources are used with low intensity and the presence of stray light in UV detection, indirect UV detection, commonly adopted in commercial CE instruments, is limited to the detection of inorganic ions in capillaries of @50 mm id without preconcentration.8 In indirect laser fluorimetry efficient rejection of stray light is made possible by the high collimated beam, so indirect laser fluorimetry can be used for capillaries of < 25 mm id with ultratrace detection. The advantages of indirect laser fluorimetry become apparent as one tries to decrease CM to improve the concentration LOD or mass LOD.9 However, fluorescence gives rise to a noisier baseline than does absorption, the end result being that the LOD is not any better than those for indirect UV detection and conductivity detection.The common problem encountered with the indirect detection methods mentioned above is that a low ionic strength of the buffer is needed r sensitive detection.However, several disadvantages arise under this condition. (1) The surface of the column wall will begin to affect detection because retention (e.g., via ion exchange or absorption on the column wall) can significantly alter the background electrolyte concentration. This surface effect can further reduce TR as CM is lowered and lead to a higher LOD, and the advantage of improving the concentration LOD by decreasing CM would be offset.10 (2) Low ionic strengths can result in distortion of the analyte zones and reduce the resolution.11 (3) It is impossible to maintain a stable baseline following an injection.(4) Interaction between the ions in the injected sample and the surface of the fused-silica capillary may be responsible for this effect at low ionic strengths. This effect would lead to considerable column-tocolumn variability.12 (5) Low ionic strengths preclude the use of low or high pH conditions, as H+ and OH2 ions, respectively, will dominate the displacement. This results in a decrease in TR and must be balanced to obtain the best sensitivity possible for the system.13 Therefore, CZE is applicable to the separation of inorganic ions, but their detection with high detection sensitivity, a low detection volume and a high ionic strength matrix remains a challenge.Lasers, with their unique spatial coherence, are ideally suited as light sources for capillary detection. Applications of lasers in CZE include fluorescence,14 refractive index,15 Raman16 and photothermal absorbance detection.17 The applications of photothermal absorbance detection for CE reported in recent years have been used mainly in analyses for amino acids18 and nucleosides and nucleotides19 with derivatization.The sensitivity of photothermal absorption techniques is 2–3 orders of magnitude higher than those of conventional detection methods. Since the absorption wavelength of analytes is required to match the emission wavelength of the pump beam well and the sensitivity of photothermal detection is related to the molar absorptivity of the analytes, these detectors are therefore not suitable for detecting non-absorbing analytes.This is one important reason why crossed-beam photothermal (CBPT) techniques have not been employed in CE for determinations of metal ions. In this paper, an indirect photothermal interference detection for detecting metal cations without derivatization after CE separation is described.Methylene Blue was chosen as a background absorber. The addition of ethanol and sodium chloride to the background electrolyte solution (BGES) can reduce the absorption of Methylene Blue and metal cations on the capillary wall, enhance the detector signal and improve the separation efficiency. Experimental Apparatus The indirect photothermal interference detector is shown in Fig. 1. A 2 mW He–Ne laser (Wuhan Institute of Laser Technology, Wuhan, China) produces a probe beam with a wavelength of 632.8 nm.A 46 mm focal length biconvex lens is used to focus the probe beam into the capillary tube. After traversing the capillary tube, the beam propagates to a photodiode (Semiconductor Factory, Wuhan University, Wuhan, China) with a 0.5 mm slit width. An M1000 He–Ne laser (Shanghai Institute of Laser Technology, Shanghai, China) produces an 18 mW pump laser beam. A model 194A mechanical chopper (EG&G, Princeton Applied Research, Princeton, NJ, USA) modulates the pump beam in a square wave.This beam is focused with a 10 mm focal length biconvex lens into the capillary tube. A three-axis translation stage holds the pump beam lens to provide complete freedom in the location of the pump beam waist, a two-axis stage is used to move the capillary, which is fixed firm on a special installation with a pinhole along the beam path of the probe laser, in the plane formed by the two laser beams, and a two-axis stage moves the detector across the probe beam profile.The interaction of the pump laser beam with the samples results in a refractive index change within the sample. This refractive index change causes a movement of the interference fringes and a change in the probe beam intensity which is synchronized with the pump beam modulation function. The modulated component of the probe beam intensity is measured by using an ND240 lock-in amplifier (Nanjing University Instrument Factory, Nanjing, China).A reference signal for the lock-in amplifier is generated within the chopper head. During the process of the electrophoresis, the signals from the lock-in amplifier are recorded by our SE 790 recorder (Australia). The capillary electrophoresis system was constructed in the laboratory. A high-voltage dc power supply (0–30 kV) (Shanghai, China) is employed. Electrical connections to the capillary are enclosed within a plastic box equipped with a safety interlock that acts to prevent accidental contact with the high voltage.A 60 cm long polymide-coated fused-silica capillary (Yongnian Optical Fiber Factory, Hebei, China) of 50 mm id and 320 mm od is used for electrophoresis. The polymer coating of the detection region is scraped away carefully. The distance from the injector to the detector is 50 cm. During the experiment, the optical breadboard is held at ground potential. Fig. 1 Schematic diagram of the detection system.PL, probe laser; M1 and M2, mirrors; L1 and L2, lenses; Ph1 and Ph2, pinholes; Ch, chopper; C, capillary; HL, heat laser; D, photodiode; LI, lock-in amplifier; R, recorder. 1090 Analyst, October 1997, Vol. 122Reagents All reagents were of analytical-reagent grade and solutions were prepared by dissolution in doubly distilled, de-ionized water. Oxides of metals were obtained from Shanghai Chemical (Shanghai, China). Samples were diluted by using the carrier electrolyte to avoid stacking and introduced into the capillary by using hydrostatic injection from a height of 10 cm.BGES were diluted with the same carrier electrolyte. All solutions and the buffer were filtered with 0.22 mm cut-off cellulose acetate filters before use. This filtration greatly reduces noise spikes and prevents blockage of the small capillaries. All data were obtained at room temperature (25 ± 1 °C). Results and Discussion Choice of Background Absorber The characteristics of the background absorber are very important for detection and separation with the utilization of indirect absorption detection in CZE.A higher molar absorptivity (ev) at the detection wavelength and a low CM will give a lower detection limit, as described by Wang and Hartwick.20 Five color reagents were obtained in order to assess their suitability as indirect background absorbers before separation. Experiments showed that the absorption wavelengths of pyrocatechol and Eriochrome Black T could match the pump laser beam (632.8 nm).However, these color reagents were unstable during the separation process and faded easily, because phenolic hydroxyl radicals are easily oxidized in an air environment. Victoria Blue B and Coomassie Brilliant Blue G 250 are not easily dissolved in aqueous solution. The solubility could be improved by adding ethanol, acetone or dimethylformamide to the electrolyte solution, but it was found that some small solid grains appeared on evaporation of the solvent, and these blocked the capillaries.Therefore, the color reagents mentioned above are not suitable for detection or separation in CZE. The molar absorptivity of Methylene Blue at 633 nm is about 4.5 3 104 l mol21 cm21. Experiments showed that characteristics of Methylene Blue are suitable for indirect interference detection and CZE separation. Noise Analysis It was found that the noise in indirect photothermal interference detection includes at least three sources: the detector, the color reagent and Joule heating. A significant effort was made to minimize the vibration of optical components. For example, the optical components are held with massive fixtures to a wall-damped optical table.When a laser beam strikes a capillary, a scattered light beam with many light and dark fringes can be observed, as described by Folestad et al.21 and Kerker and Matijewc22 and this scattered light beam is an important noise source in detection.First, portions of this scattered light, coming from the scattered light beam produced by the probe laser and the pump laser, could retroreflect back to both the probe laser source and pump beam laser source. This kind of retroreflection could cause fluctuations of the laser source and increase the noise level. Second, it was found that the noise level increases when the portion of scattered light coming from the scattered light beam produced by the pump laser hits the photodiode in the far-field after the capillary.This part of the scattered light is modulated by a mechanical chopper, and both the pump laser beam and probe laser beam are at the same wavelength in this system. In the present work, the probe laser beam was focused into the capillary at a right-angle, which tilted up to the capillary tube axis (a Å 89°). After being focused by a biconvex lens, the modulated pump laser beam was tilted slightly down into the capillary tube axis (b Å 89°).Further, the pump laser beam and the probe beam crossed obliquely at the capillary at a rightangle (g Å 89°) and two pinholes were used to extinguish the scattered light from other optical components, as shown in Fig. 1. It was demonstrated that the noise caused by the scattered light could be minimized successfully in this configuration. The reason is that the beams of scattered light in the plane of the interaction of the laser beam and the capillary were tilted to the capillary tube axis and not easy to retroreflect to the laser source and photodiode. As noted by Higashijima et al.23 the use of Methylene Blue in an acidic buffer leads to peak tailing characteristic of absorption of the positively charged dye on the capillary surface and would lead to higher noise.As shown later in Fig. 4, this type of absorption on the capillary surface could be minimized by adding ethanol to the electrolyte solutions and using an electrolyte solution of higher ionic strength.This phenomenon can be attributed to the fact that ethanol and a higher ionic strength can limit the dissociation of silanol on the capillary surface and the absorption of Methylene Blue. Further, ethanol and a higher ionic strength also enhanced the Methylene Blue detection signal, because the dn/dT of solutions containing ethanol and a higher ionic strength electrolyte is larger than that of aqueous solutions.24 This was beneficial for improving the sensitivity of indirect photothermal interference detection. It was also found that an excessively low concentration of the color reagent would lead to higher noise, as the ratio of the background absorbance to the noise somehow depends on the concentration of the color reagent and decreases simultaneously. 25 Excessive Joule heating should be avoided in CE as it prevents efficient heat dissipation to attain high efficiencies and causes thermal instability in the detection region.In general, as described in the Introduction, a low ionic strength of the buffers is required in indirect detection. However, an important effect was found in our experiments, namely that, in a certain range, an increase in the separation voltage in CZE did not decrease but actually improved the sensitivity of indirect photothermal interference detection. The reason is that the temperature rise in the detection region inside the capillary would maintain a stable level under the conditions of a stable voltage,26 and this kind of temperature increase of the background electrolyte could lead to an increase in the dn/dT value, as demonstrated by Franko and Tran.27 The contribution to noise from the Joule heating was found to be small under the condition of a stable voltage.Indirect Detection Before detection for CZE separation, several alignment constraints were qualitatively investigated for photothermal capillary absorption measurements.As expected, the signal was highest when the pump laser beam waist was centered in the capillary. Since a tightly focused pump beam also minimizes the system volume, all measurements reported in this paper were obtained with the pump waist located in the capillary. For a given interference pattern, the fringes that were found to be more sensitive were those that appear near the optical axis of the probe beam but still retain high intensity and contrast.When the interference pattern and the slit width of detector were fixed, an optimum distance for detection from the sample cuvette to the photodiode was observed. Agreeing well with a lower noise level and higher sensitivity, in the experiment the photodiode was set at a position of 60 cm in the far-field after the capillary. Under these conditions, the capillary was located at a position of 19 mm before the probe beam waist to obtain higher sensitivity.When the capillary containing the solutions was drawn gradually closer to the probe beam waist, it was found that the signal decreased quickly. The reason may be that the contrast of the interference fringes also decreased quickly. Analyst, October 1997, Vol. 122 1091A finite time is required to reach thermal equilibrium in photothermal absorbance measurements. As a general rule of thumb, a frequency of 6.5 Hz was used in these experiments. A short time constant is of value since very fast phenomena may be studied without degradation due to instrumental artifacts, and the detector time constant was set at 1 s.An absorbance calibration curve of Methylene Blue was obtained in the static state for the 50 mm id capillaries. The calibration curve was linear (r = 0.995) over more than three orders of magnitude of concentration. Linearity extended from the detection limit, A = 6.1 31027, to the highest concentration sample studied, A = 0.023.Water–ethanol solution was chosen as the solvent because it produces greater sensitivity than water for photothermal absorbance measurements. As shown in Fig. 2, for Methylene Blue solution, the concentration LOD was about 3.1 3 1028 mol l21 (S/N = 2). With a highly collimated beam, the detection volume of this detector, the intersection volume of the pump and probe beam, is small, slightly less than 1 pl. We use a much more conservative definition of the detection volume, as described by Bornhop and Dovichi,28 as a cylinder whose radius and height are given by the capillary radius.For the 50 mm id tube, the detector volume was about 50 pl. Then, the detection limit as absolute amount was 1.5 3 10218 mol. Of course, a significantly smaller amount of analyte was present within the small intersection volume of the laser beam than the result estimated above. It has been demonstrated that, for absorption measurement in narrow-bore capillaries, crossed-beam photothermal (CBPT) absorption techniques can offer definite advantages: the sensitivities are not linear with respect to pathlength, and with a decrease in the capillary diameter the pump beam is more efficiently quenched at the walls.Therefore, according to the assumption described in ref. 28, this detector could also be used with a capillary tube as small as 5 mm id. The results in Fig. 3 show that the addition of ethanol to the BGES can reduce absorption on the capillary surface and lead to an improvement in the peak shape of the analyte.Ethanol also enhanced the Methylene Blue and analyte detection signal, because the dn/dT value of ethanolic solutions is larger than that of aqueous solutions, and this is beneficial for the improvement of sensitivity in indirect interference detection. Further, it could be observed that the elution time became longer with an increase of ethanol concentration. A probable cause is that the ethanol interacts strongly with the capillary wall, resulting in a higher apparent concentration of ethanol within the double layer as described in ref. 29. These interactions then result in higher apparent viscosities within the double layer and lead to a reduced electroosmotic flow. The effect of NaCl on CZE separation and detection was investigated. The results (Fig. 4) showed that efficiency of KI and the peak shape were improved at higher concentrations of NaCl without a decrease in detection sensitivity.These results and that of the Joule heating phenomenon (see above) indicated that indirect photothermal interference detection is suitable for CE separation systems with a high ionic strength buffer. As the sensitivity that CBPT provides was almost unrelated to the light path, this result implies that indirect photothermal interference detection may be used for detection in CZE with a larger ionic strength range and overcomes the problem of the decrease in sensitivity for small id capillaries and shot-noise limitation in photometry.The characteristics of photothermal absorbance detection and the results obtained above are, for example, beneficial for the analysis of samples with weak absorption, ultratrace detection and larger range of linearity, and permit a lower concentration of the background absorber (Methylene Blue) to be used to improve the LOD. As shown in Fig. 5, positive peaks are observed for KI and CuII and AlIII produces a negative peak.A positive peak indicates an increase in concentration of the background absorber present at the detector, whereas a negative peak indicates a decrease. The migration time of the system peak corresponds to the mobility of the Methylene Blue cation. An interesting phenomenon is observed in Fig. 5, namely that the migration order is KI > CuII > AlIII. In order to understand the elution order, it has to be taken into account that the electrophoretic mobility depends on the charge, shape and size of the analyte.This phenomenon could be explained by the fact that, under conditions of low pH and excess of chloride, Fig. 2 Electropherogram of Methylene Blue. Carrier electrolyte, 50 mmol l21 Na2HPO4–10% ethanol (pH 6.0); sample, 5 3 1027 mol l21 Methylene Blue; CE voltage, 16 kV; current, 14 mA; injection, 10 s. Fig. 3 Electropherograms of KI (1.0 3 1025 mol l21). Carrier electrolyte; (a) 109 mmol l21 acetic acid–5% ethanol; (b) 109 mmol l21 acetic acid 210% ethanol; and (c) 109 mmol l21 acetic acid–20% ethanol.CE voltage, 14 kV; injection, 15 s; Methylene Blue concentration, 5.0 3 1024 mol l21. Fig. 4 Electropherogram of KI (1.0 3 1025 mol l21). Carrier electrolyte, 109 mmol l21 acetic acid–20% ethanol–100 mmol l21 NaCl; CE voltage, 14 kV; current, 24 mA; other conditions as in Fig. 3. 1092 Analyst, October 1997, Vol. 122CuCl4 22 and AlCl42 complexes are formed in solution, but KI is incapable of forming chloride complexes.Therefore, the peak of KI appeared first. The elution order of CuCl4 22 and AlCl42 can be rationalized by the fact that AlCl42 seems to be a poorly hydrated anion compared with CuCl4 22. Hence this lower hydration will reduce the size of the moving ion, which may result in greater electrophoretic migration and, accordingly, retardation. The phenomenon is similar to the studies of Aguilar et al.30 and Baraj et al.,31 in which chloride and cyanide were used as ligands for the determination of metal cations.For CuII, the samples and carrier solution were prepared with almost the same conductivity, and consequently the stacking effect was not used; a 2.1 3 1027 mol l21 concentration detection limit (S/N = 2) without preconcentration and a 1.20 3 10217 mol detection limit as absolute amount, considering the optical sampling volume estimated to be 50 pl, were measured, and 1.99 3106 theoretical plates were observed with the laboratorymade CE system.The concentration LODs measured here were comparable to the best results obtained with other detection methods for ions,32–34 and the mass LOD of this detector could be decreased to that of an indirect fluorescence detector or conductivity detector (10217 mol) and at least two orders of magnitude lower than that of a commercial UV detector. The CE method offers several advantages over other indirect detection methods for ions. First the use of a larger range of ionic strengths permits the application dynamic range to be increased35,36 and extends the upper limit of sample ionic strength that still permits effective stacking and thus facilitates the determination of trace constituents in a high ionic strength matrix.Second, the lower mass LOD permits small id capillaries to be used to improve the resolution of samples and ultra trace components. Obviously, this characteristic of indirect photothermal interference detection is better than that of commercial detection.Even then the best mass LODs of conductimetric detection and indirect fluorescence detection are similar to those of this method; as one mode of absorption detection techniques, this method has all the advantages of photometric detection over conductimetric detection mentioned earlier, and maybe a less noisy baseline than that in indirect fluorescence detection, since the main disadvantage of indirect fluorescence detection is that fluorescence gives rise to a noisier baseline than does absorption and leads to a higher concentration LOD.Third, as the values of dn/dT for most non-aqueous media are larger than those of aqueous solutions, this detector will be of advantage for detection in non-aqueous medium capillary electrophoresis, an important field in the study of capillary electrophoresis. In future work we will investigate possible improvements to the detection sensitivity, including the design of an electronic noise canceller to reduce electronic noise, altering the injection mode and the use of highly stable lasers.The relatively low cost, simple construction, small volume, universal detection, larger range of ionic strengths and excellent detection limits of this method should prove attractive for a number of applications in capillary electrophoresis separation techniques. This work was supported by the National Nature Science Foundation of China. References 1 Stoker, F.S., Haymor, B. L., and McBeath, R., J. Chromatogr., 1989, 470, 241. 2 Burlity, N., and Jorgenson, J., J. Chromatogr., 1989, 480, 301. 3 Swaile, D. F., and Sepaniak, M. J., Anal. Chem., 1991, 63, 179. 4 Hu, Y., Deng, Y., and Cheng, J., Prog. Nat. Sci., 1996, 6, S-42. 5 Yeung, E. S., Acc. Chem. Res., 1989, 22, 125. 6 Yeung, E. S., and Kuhr, W. G., Anal. Chem., 1991, 63, 275A. 7 Kaniansky, D., Zelenska, V., and Baluchova, D., Electroporesis, 1996, 17, 1890. 8 Jandik, P., and Bonn, G., Capillary Electrophoresis of Small Ions, VCH, Weinheim, 1993. 9 Pfeffer, W. D., and Yeung, E. S., J. Chromatogr., 1990, 506, 401. 10 Wilson, S. A., and Yeung, E. S., Anal. Chim. Acta, 1984, 157, 53. 11 Mikkers, F. E. P., Everaerts, F. M., and Verheggen, Th. P. E. M., J. Chromatogr., 1979, 169, 11. 12 Kuhr, W. G., and Yeung, E. S., Anal. Chem., 1988, 60, 2642. 13 Takeuchi, T., and Yeung, E. S., J. Chromatogr., 1986, 370, 83. 14 Gussman, E., Kuo, R. N., and Zare, R. N., Science, 1985, 230, 813. 15 Pawliszyn, J., J. Liq. Chromatogr., 1987, 10, 3377. 16 Cheng, C. Y., and Morris, M. D., Appl. Spectrosc., 1988, 42, 515. 17 Yu, M., and Dovich, N. J., Mikrochimica III, 1988, 27. 18 Yu, M., and Dovich, N. J., Anal. Chem., 1989, 61, 37. 19 Krattiger, B., Bruno, A. E., Widmer, H. M., and Pandliker, R., Anal. Chem., 1995, 67, 124. 20 Wang, T. S., and Hartwick, R. A., J. Chromatogr., 1992, 607, 119. 21 Folestad, S., Johnson, L., Josefsson, B., and Galle, B., Anal.Chem., 1982, 54, 925. 22 Kerker, M., and Matijewc, E., J. Opt. Soc. Am., 1961, 51, 506. 23 Higashijima, T., Fuchigami, T., Imasaka, T., and Ishibashi, N., Anal. Chem., 1992, 64, 711. 24 Fujiwara, K., Lei, W., Uckiki, H., Shimokoshi, F., Fuwa, K., and Kobayashi, T., Anal. Chem., 1982, 54, 2026. 25 Takenchi, T., and Yeung, E. S., J. Chromatogr., 1986, 370, 83. 26 Bruno, A. E., Krattiger, B., Maystre, F., and Widmer, H. M., Anal. Chem., 1991, 63, 2689. 27 Franko, M., and Tran, C.D., J. Phys. Chem., 1991, 95, 6688. 28 Bornhop, D. J., and Dovichi, N. J., Anal. Chem., 1987, 59, 1632. 29 VanOrman, B. B., Liversidge, G. G., and McIntire, G. L., J. Microcolumn Sep., 1991, 2, 176. 30 Aguilar, M., Farran, A., and Martinnez, M., J. Chromatogr., 1993, 635, 127. 31 Baraj, B., Sastre, A., Merkoci, A., and Martinnez, M., J. Chromatogr., 1995, 718, 227. 32 Weston, A., Broun, P. R., Jandik, P., Heckenberg, A. L., and Jones, W. R., J. Chromatogr., 1992, 608, 395. 33 Kuhr, W. G., and Yeung, E. S., Anal. Chem., 1988, 60, 2642. 34 Kaniansky, D., Zelenska, V., and Baluchova, D., Electrophoresis, 1996, 17, 1890. 35 Nielen, M. W. F., J. Chromatogr., 1991, 542, 173. 36 Green, J. S., and Jorgenson, J. W., J. Chromatogr., 1987, 478, 63. Paper 7/00119C Received January 6, 1997 Accepted June 18, 1997 Fig. 5 Electropherogram of a mixture of three metal cations. Methylene Blue concentration, 5.0 3 1026 mol l21. Other conditions as in Fig. 4. Peaks: 1, KI (2.5 3 1026 mol l21); 2, system peak; 3, CuII (1.56 3 1026 mol l21); and 4, AlIII (3.85 3 1026 mol l21). Analyst, October 1997, Vol. 122 1093 On-column Indirect Photothermal Interference Detection for Capillary Zone Electrophoresis Yonggang Hua, Jieke Chenga and Yanzhuo Deng*b a Department of Chemistry, Wuhan University, Hubei 430072, China b Center of Analysis and Testing, Wuhan University, Hubei 430072, China An indirect photothermal interference detection method, which is based on the diffraction of a probe laser beam by a capillary tube, for detecting metal cations separated by capillary zone electrophoresis (CZE) is described.In this capillary photothermal interference detector, a 2 mW He–Ne probe laser is employed to provide the probe beam and an 18 mW He–Ne pump laser is used to supply the pump beam. Factors that contribute to the noise and signal in indirect photothermal interference detection in CZE, such as the noise from the scattered light caused by both the probe beam and the pump laser beam, and effects of the concentration of ethanol and NaCl on the separation efficiency, were studied.The effect of scattered light can be reduced effectively with the configuration constraints investigated for photothermal capillary absorption measurements. With Methylene Blue added to carrier electrolyte as an absorber, three typical metal cations (KI, CuII and AlIII) were separated using this system.For CuII, a 2.1 3 1027 mol l21 concentration detection limit (S/N = 2) without preconcentration and a 1.02 3 10217 mol detection limit as absolute amount were measured, considering the optical sampling volume estimated to be 50 pl, and 1.99 3 106 theoretical plates were observed on the laboratory-made CE system. It was demonstrated that indirect photothermal interference detection is suitable for small capillaries and a large ionic strength range for CE analysis. Keywords: Indirect photothermal interference detection; capillary zone electrophoresis; metal cations; Methylene Blue As a powerful technique for separations, the number of applications of capillary zone electrophoresis (CZE) has increased greatly in recent years.In principle, CZE is very suitable for small ionic compounds. However, the application of CZE to inorganic species, especially metal cations, is rare, in contrast to the separation of biological macromolecules.1,2 The reason is probably that many of these classes of compounds lack chromophores at useful wavelengths or have such low molar absorptivities as to preclude adequate sensitivity with absorption detection. Cation determinations by CZE are also hampered by coulombic interactions of the cations with the surfaces of the fused-silica capillaries commonly employed in CZE.These interactions can result in band broadening, which reduces both detectability and resolution. Methods adopting color reaction techniques to improve the LODs of metal cations with absorbance detection or laserinduced fluorescence detection can be easily devised.For example, 8-hydroxyquinoline-5-sulfonic acid (HQS2) was used for the determination of metal ions by CZE with laser-induced fluorescence.3 However, the main disadvantage is that the chemical properties of these complexes are usually unstable during the process of separation and it is difficult to optimize the separation and detection conditions for these complexes.4 The second disadvantage is that multiple complexes may exist in solution and multiple peaks would appear, making it very difficult to distinguish the signal peaks.The third disadvantage is that the analytes are chemically altered and collection and further studies are almost impossible. The fourth disadvantage is that derivatization is time consuming and inefficient. Hence there is a need for an all-purpose detector for CZE. Indirect detection is the most common method for metal cations which lack chromophores in CZE.A comprehensive account of indirect detection methods has been given by Yeung.5 Several reasons for developing indirect detection schemes have been mentioned. First, indirect detection is universal, and can be used for compounds which lack chromophores or fluorophores. Second, it is possible to broaden the applicability of highly sensitive but selective detectors by implementing indirect detection.Third, quantification is easier with indirect detection since tedious chemical derivatization procedures can be avoided. Fourth, indirect detection is nondestructive since no chemical manipulation is necessary and collection and further studies are facilitated. The indirect signal (displacement peaks) is a minor perturbation on the background signal. The concentration limit of detection, Clim, is given by6 C C TR DR lim = � M (1) where CM is the concentration of the relevant component added to the mobile phase, TR is the displacement ratio and DR is the dynamic reserve and is equal to the signal-to-noise ratio (S/N).It indicates that, for a given system, the more stable the background signal (larger DR), the more effective the displacement process (larger TR) and the lower is CM, the lower are the detection limits that can be obtained. Because DR and TR possibly decrease as one decreases CM, on the other hand, in order to improve the concentration LOD, detection methods with a capability for ultratrace detection and a larger range of detection are necessary to maintain a larger and stable background signal so as to allow the use of CM values that are as low as possible.Conductivity detection is an important indirect method for metal cations in CZE,7 which also functions in a displacement mode. The signal arises from the difference in equivalent conductance or mobility of the charge carrier electrolyte ion and the analyte ion.Several problems are usually encountered with conductivity detection. (1) It is advantageous to use a low ionic strength, poorly ionized buffer system to produce a low conductivity background. If good separation is desired, the concentration of the background electrolyte must be high relative to the concentration of the sample. However, this condition (high electrolyte concentration) results in a decrease in sensitivity due to the elevated background conductivity.(2) Background noise from the high voltage prevents highsensitivity detection. (3) A large difference between the mobility of the carrier electrolyte ion and that of the analyte ion leads to excessive peak tailing/fronting in CE. (4) Cell design is difficult since there must be no significant voltage drop between the cell electrodes; small dimensions of the capillary, high ionic strengths and a high separation electric field are challenges for the use of conductivity detection in CZE.Analyst, October 1997, Vol. 122 (1089–1093) 1089Indirect photometric detection, in which the signal arises from the difference in equivalent absorptivities between the carrier/eluent and the analyte ion, can offer a significant advantage over conductimetric detection in that ionic mobility and optical absorption are unrelated parameters. It is possible to choose carrier ions of suitable mobility and with desired optical absorption characteristics. Hence it is not necessary to compromise between separation efficiency and detection sensitivity.Since DR for photometry will degrade as optical pathlength, CM, or the absorption strengths decrease because incoherent light sources are used with low intensity and the presence of stray light in UV detection, indirect UV detection, commonly adopted in commercial CE instruments, is limited to the detection of inorganic ions in capillaries of @50 mm id without preconcentration.8 In indirect laser fluorimetry efficient rection of stray light is made possible by the high collimated beam, so indirect laser fluorimetry can be used for capillaries of < 25 mm id with ultratrace detection.The advantages of indirect laser fluorimetry become apparent as one tries to decrease CM to improve the concentration LOD or mass LOD.9 However, fluorescence gives rise to a noisier baseline than does absorption, the end result being that the LOD is not any better than those for indirect UV detection and conductivity detection.The common problem encountered with the indirect detection methods mentioned above is that a low ionic strength of the buffer is needed for sensitive detection. However, several disadvantages arise under this condition. (1) The surface of the column wall will begin to affect detection because retention (e.g., via ion exchange or absorption on the column wall) can significantly alter the background electrolyte concentration. This surface effect can further reduce TR as CM is lowered and lead to a higher LOD, and the advantage of improving the concentration LOD by decreasing CM would be offset.10 (2) Low ionic strengths can result in distortion of the analyte zones and reduce the resolution.11 (3) It is impossible to maintain a stable baseline following an injection.(4) Interaction between the ions in the injected sample and the surface of the fused-silica capillary may be responsible for this effect at low ionic strengths.This effect would lead to considerable column-tocolumn variability.12 (5) Low ionic strengths preclude the use of low or high pH conditions, as H+ and OH2 ions, respectively, will dominate the displacement. This results in a decrease in TR and must be balanced to obtain the best sensitivity possible for the system.13 Therefore, CZE is applicable to the separation of inorganic ions, but their detection with high detection sensitivity, a low detection volume and a high ionic strength matrix remains a challenge.Lasers, with their unique spatial coherence, are ideally suited as light sources for capillary detection. Applications of lasers in CZE include fluorescence,14 refractive index,15 Raman16 and photothermal absorbance detection.17 The applications of photothermal absorbance detection for CE reported in recent years have been used mainly in analyses for amino acids18 and nucleosides and nucleotides19 with derivatization.The sensitivity of photothermal absorption techniques is 2–3 orders of magnitude higher than those of conventional detection methods. Since the absorption wavelength of analytes is required to match the emission wavelength of the pump beam well and the sensitivity of photothermal detection is related to the molar absorptivity of the analytes, these detectors are therefore not suitable for detecting non-absorbing analytes. This is one important reason why crossed-beam photothermal (CBPT) techniques have not been employed in CE for determinations of metal ions.In this paper, an indirect photothermal interference detection for detecting metal cations without derivatization after CE separation is described. Methylene Blue was chosen as a background absorber. The addition of ethanol and sodium chloride to the background electrolyte solution (BGES) can reduce the absorption of Methylene Blue and metal cations on the capillary wall, enhance the detector signal and improve the separation efficiency.Experimental Apparatus The indirect photothermal interference detector is shown in Fig. 1. A 2 mW He–Ne laser (Wuhan Institute of Laser Technology, Wuhan, China) produces a probe beam with a wavelength of 632.8 nm. A 46 mm focal length biconvex lens is used to focus the probe beam into the capillary tube. After traversing the capillary tube, the beam propagates to a photodiode (Semiconductor Factory, Wuhan University, Wuhan, China) with a 0.5 mm slit width.An M1000 He–Ne laser (Shanghai Institute of Laser Technology, Shanghai, China) produces an 18 mW pump laser beam. A model 194A mechanical chopper (EG&G, Princeton Applied Research, Princeton, NJ, USA) modulates the pump beam in a square wave. This beam is focused with a 10 mm focal length biconvex lens into the capillary tube. A three-axis translation stage holds the pump beam lens to provide complete freedom in the location of the pump beam waist, a two-axis stage is used to move the capillary, which is fixed firm on a special installation with a pinhole along the beam path of the probe laser, in the plane formed by the two laser beams, and a two-axis stage moves the detector across the probe beam profile.The interaction of the pump laser beam with the samples results in a refractive index change within the sample. This refractive index change causes a movement of the interference fringes and a change in the probe beam intensity which is synchronized with the pump beam modulation function. The modulated component of the probe beam intensity is measured by using an ND240 lock-in amplifier (Nanjing University Instrument Factory, Nanjing, China). A reference signal for the lock-in amplifier is generated within the chopper head.During the process of the electrophoresis, the signals from the lock-in amplifier are recorded by our SE 790 recorder (Australia). The capillary electrophoresis system was constructed in the laboratory.A high-voltage dc power supply (0–30 kV) (Shanghai, China) is employed. Electrical connections to the capillary are enclosed within a plastic box equipped with a safety interlock that acts to prevent accidental contact with the high voltage. A 60 cm long polymide-coated fused-silica capillary (Yongnian Optical Fiber Factory, Hebei, China) of 50 mm id and 320 mm od is used for electrophoresis. The polymer coating of the detection region is scraped away carefully.The distance from the injector to the detector is 50 cm. During the experiment, the optical breadboard is held at ground potential. Fig. 1 Schematic diagram of the detection system. PL, probe laser; M1 and M2, mirrors; L1 and L2, lenses; Ph1 and Ph2, pinholes; Ch, chopper; C, capillary; HL, heat laser; D, photodiode; LI, lock-in amplifier; R, recorder. 1090 Analyst, October 1997, Vol. 122Reagents All reagents were of analytical-reagent grade and solutions were prepared by dissolution in doubly distilled, de-ionized water.Oxides of metals were obtained from Shanghai Chemical (Shanghai, China). Samples were diluted by using the carrier electrolyte to avoid stacking and introduced into the capillary by using hydrostatic injection from a height of 10 cm. BGES were diluted with the same carrier electrolyte. All solutions and the buffer were filtered with 0.22 mm cut-off cellulose acetate filters before use. This filtration greatly reduces noise spikes and prevents blockage of the small capillaries.All data were obtained at room temperature (25 ± 1 °C). Results and Discussion Choice of Background Absorber The characteristics of the background absorber are very important for detection and separation with the utilization of indirect absorption detection in CZE. A higher molar absorptivity (ev) at the detection wavelength and a low CM will give a lower detection limit, as described by Wang and Hartwick.20 Five color reagents were obtained in order to assess their suitability as indirect background absorbers before separation.Experiments showed that the absorption wavelengths of pyrocatechol and Eriochrome Black T could match the pump laser beam (632.8 nm). However, these color reagents were unstable during the separation process and faded easily, because phenolic hydroxyl radicals are easily oxidized in an air environment. Victoria Blue B and Coomassie Brilliant Blue G 250 are not easily dissolved in aqueous solution.The solubility could be improved by adding ethanol, acetone or dimethylformamide to the electrolyte solution, but it was found that some small solid grains appeared on evaporation of the solvent, and these blocked the capillaries. Therefore, the color reagents mentioned above are not suitable for detection or separation in CZE. The molar absorptivity of Methylene Blue at 633 nm is about 4.5 3 104 l mol21 cm21. Experiments showed that characteristics of Methylene Blue are suitable for indirect interference detection and CZE separation.Noise Analysis It was found that the noise in indirect photothermal interference detection includes at least three sources: the detector, the color reagent and Joule heating. A significant effort was made to minimize the vibration of optical components. For example, the optical components are held with massive fixtures to a wall-damped optical table. When a laser beam strikes a capillary, a scattered light beam with many light and dark fringes can be observed, as described by Folestad et al.21 and Kerker and Matijewc22 and this scattered light beam is an important noise source in detection.First, portions of this scattered light, coming from the scattered light beam produced by the probe laser and the pump laser, could retroreflect back to both the probe laser source and pump beam laser source. This kind of retroreflection could cause fluctuations of the laser source and increase the noise level.Second, it was found that the noise level increases when the portion of scattered light coming from the scattered light beam produced by the pump laser hits the photodiode in the far-field after the capillary. This part of the scattered light is modulated by a mechanical chopper, and both the pump laser beam and probe laser beam are at the same wavelength in this system. In the present work, the probe laser beam was focused into the capillary at a right-angle, which tilted up to the capillary tube axis (a Å 89°). After being focused by a biconvex lens, the modulated pump laser beam was tilted slightly down into the capillary tube axis (b Å 89°).Further, the pump laser beam and the probe beam crossed obliquely at the capillary at a rightangle (g Å 89°) and two pinholes were used to extinguish the scattered light from other optical components, as shown in Fig. 1. It was demonstrated that the noise caused by the scattered light could be minimized successfully in this configuration. The reason is that the beams of scattered light in the plane of the interaction of the laser beam and the capillary were tilted to the capillary tube axis and not easy to retroreflect to the laser source and photodiode.As noted by Higashijima et al.23 the use of Methylene Blue in an acidic buffer leads to peak tailing characteristic of absorption of the positively charged dye on the capillary surface and would lead to higher noise.As shown later in Fig. 4, this type of absorption on the capillary surface could be minimized by adding ethanol to the electrolyte solutions and using an electrolyte solution of higher ionic strength. This phenomenon can be attributed to the fact that ethanol and a higher ionic strength can limit the dissociation of silanol on the capillary surface and the absorption of Methylene Blue. Further, ethanol and a higher ionic strength also enhanced the Methylene Blue detection signal, because the dn/dT of solutions containing ethanol and a higher ionic strength electrolyte is larger than that of aqueous solutions.24 This was beneficial for improving the sensitivity of indirect photothermal interference detection.It was also found that an excessively low concentration of the color reagent would lead to higher noise, as the ratio of the background absorbance to the noise somehow depends on the concentration of the color reagent and decreases simultaneously. 25 Excessive Joule heating should be avoided in CE as it prevents efficient heat dissipation to attain high efficiencies and causes thermal instability in the detection region.In general, as described in the Introduction, a low ionic strength of the buffers is required in indirect detection. However, an important effect was found in our experiments, namely that, in a certain range, an increase in the separation voltage in CZE did not decrease but actually improved the sensitivity of indirect photothermal interference detection. The reason is that the temperature rise in the detection region inside the capillary would maintain a stable level under the conditions of a stable voltage,26 and this kind of temperature increase of the background electrolyte could lead to an increase in the dn/dT value, as demonstrated by Franko and Tran.27 The contribution to noise from the Joule heating was found to be small under the condition of a stable voltage.Indirect Detection Before detection for CZE separation, several alignment constraints were qualitatively investigated for photothermal capillary absorption measurements. As expected, the signal was highest when the pump laser beam waist was centered in the capillary. Since a tightly focused pump beam also minimizes the system volume, all measurements reported in this paper were obtained with the pump waist located in the capillary. For a given interference pattern, the fringes that were found to be more sensitive were those that appear near the optical axis of the probe beam but still retain high intensity and contrast.When the interference pattern and the slit width of detector were fixed, an optimum distance for detection from the sample cuvette to the photodiode was observed. Agreeing well with a lower noise level and higher sensitivity, in the experiment the photodiode was set at a position of 60 cm in the far-field after the capillary.Under these conditions, the capillary was located at a position of 19 mm before the probe beam waist to obtain higher sensitivity. When the capillary containing the solutions was drawn gradually closer to the probe beam waist, it was found that the signal decreased quickly. The reason may be that the contrast of the interference fringes also decreased quickly. Analyst, October 1997, Vol. 122 1091A finite time is required to reach thermal equilibrium in photothermal absorbance measurements. As a general rule of thumb, a frequency of 6.5 Hz was used in these experiments. A short time constant is of value since very fast phenomena may be studied without degradation due to instrumental artifacts, and the detector time constant was set at 1 s.An absorbance calibration curve of Methylene Blue was obtained in the static state for the 50 mm id capillaries. The calibration curve was linear (r = 0.995) over more than three orders of magnitude of concentration. Linearity extended from the detection limit, A = 6.1 31027, to the highest concentration sample studied, A = 0.023.Water–ethanol solution was chosen as the solvent because it produces greater sensitivity than water for photothermal absorbance measurements. As shown in Fig. 2, for Methylene Blue solution, the concentration LOD was about 3.1 3 1028 mol l21 (S/N = 2). With a highly collimated beam, the detection volume of this detector, the intersection volume of the pump and probe beam, is small, slightly less than 1 pl.We use a much more conservative definition of the detection volume, as described by Bornhop and Dovichi,28 as a cylinder whose radius and height are given by the capillary radius. For the 50 mm id tube, the detector volume was about 50 pl. Then, the detection limit as absolute amount was 1.5 3 10218 mol. Of course, a significantly smaller amount of analyte was present within the small intersection volume of the laser beam than the result estimated above.It has been demonstrated that, for absorption measurement in narrow-bore capillaries, crossed-beam photothermal (CBPT) absorption techniques can offer definite advantages: the sensitivities are not linear with respect to pathlength, and with a decrease in the capillary diameter the pump beam is more efficiently quenched at the walls. Therefore, according to the assumption described in ref. 28, this detector could also be used with a capillary tube as small as 5 mm id. The results in Fig. 3 show that the addition of ethanol to the BGES can reduce absorption on the capillary surface and lead to an improvement in the peak shape of the analyte. Ethanol also enhanced the Methylene Blue and analyte detection signal, because the dn/dT value of ethanolic solutions is larger than that of aqueous solutions, and this is beneficial for the improvement of sensitivity in indirect interference detection.Further, it could be observed that the elution time became longer with an increase of ethanol concentration. A probable cause is that the ethanol interacts strongly with the capillary wall, resulting in a higher apparent concentration of ethanol within the double layer as described in ref. 29. These interactions then result in higher apparent viscosities within the double layer and lead to a reduced electroosmotic flow. The effect of NaCl on CZE separation and detection was investigated.The results (Fig. 4) showed that efficiency of KI and the peak shape were improved at higher concentrations of NaCl without a decrease in detection sensitivity. These results and that of the Joule heating phenomenon (see above) indicated that indirect photothermal interference detection is suitable for CE separation systems with a high ionic strength buffer. As the sensitivity that CBPT provides was almost unrelated to the light path, this result implies that indirect photothermal interference detection may be used for detection in CZE with a larger ionic strength range and overcomes the problem of the decrease in sensitivity for small id capillaries and shot-noise limitation in photometry.The characteristics of photothermal absorbance detection and the results obtained above are, for example, beneficial for the analysis of samples with weak absorption, ultratrace detection and larger range of linearity, and permit a lower concentration of the background absorber (Methylene Blue) to be used to improve the LOD.As shown in Fig. 5, positive peaks are observed for KI and CuII and AlIII produces a negative peak. A positive peak indicates an increase in concentration of the background absorber present at the detector, whereas a negative peak indicates a decrease. The migration time of the system peak corresponds to the mobility of the Methylene Blue cation. An interesting phenomenon is observed in Fig. 5, namely that the migration order is KI > CuII > AlIII. In order to understand the elution order, it has to be taken into account that the electrophoretic mobility depends on the charge, shape and size of the analyte. This phenomenon could be explained by the fact that, under conditions of low pH and excess of chloride, Fig. 2 Electropherogram of Methylene Blue. Carrier electrolyte, 50 mmol l21 Na2HPO4–10% ethanol (pH 6.0); sample, 5 3 1027 mol l21 Methylene Blue; CE voltage, 16 kV; current, 14 mA; injection, 10 s.Fig. 3 Electropherograms of KI (1.0 3 1025 mol l21). Carrier electrolyte; (a) 109 mmol l21 acetic acid–5% ethanol; (b) 109 mmol l21 acetic acid 210% ethanol; and (c) 109 mmol l21 acetic acid–20% ethanol. CE voltage, 14 kV; injection, 15 s; Methylene Blue concentration, 5.0 3 1024 mol l21. Fig. 4 Electropherogram of KI (1.0 3 1025 mol l21). Carrier electrolyte, 109 mmol l21 acetic acid–20% ethanol–100 mmol l21 NaCl; CE voltage, 14 kV; current, 24 mA; other conditions as in Fig. 3. 1092 Analyst, October 1997, Vol. 122CuCl4 22 and AlCl42 complexes are formed in solution, but KI is incapable of forming chloride complexes. Therefore, the peak of KI appeared first. The elution order of CuCl4 22 and AlCl42 can be rationalized by the fact that AlCl42 seems to be a poorly hydrated anion compared with CuCl4 22. Hence this lower hydration will reduce the size of the moving ion, which may result in greater electrophoretic migration and, accordingly, retardation. The phenomenon is similar to the studies of Aguilar et al.30 and Baraj et al.,31 in which chloride and cyanide were used as ligands for the determination of metal cations.For CuII, the samples and carrier solution were prepared with almost the same conductivity, and consequently the stacking effect was not used; a 2.1 3 1027 mol l21 concentration detection limit (S/N = 2) without preconcentration and a 1.20 3 10217 mol detection limit as absolute amount, considering the optical sampling volume estimated to be 50 pl, were measured, and 1.99 3106 theoretical plates were observed with the laboratorymade CE system. The concentration LODs measured here were comparable to the best results obtained with other detection methods for ions,32–34 and the mass LOD of this detector could be decreased to that of an indirect fluorescence detector or conductivity detector (10217 mol) and at least two orders of magnitude lower than that of a commercial UV detector.The CE method offers several advantages over other indirect detection methods for ions. First the use of a larger range of ionic strengths permits the application dynamic range to be increased35,36 and extends the upper limit of sample ionic strength that still permits effective stacking and thus facilitates the determination of trace constituents in a high ionic strength matrix. Second, the lower mass LOD permits small id capillaries to be used to improve the resolution of samples and ultra trace components. Obviously, this characteristic of indirect photothermal interference detection is better than that of commercial detection.Even then the best mass LODs of conductimetric detection and indirect fluorescence detection are similar to those of this method; as one mode of absorption detection techniques, this method has all the advantages of photometric detection over conductimetric detection mentioned earlier, and maybe a less noisy baseline than that in indirect fluorescence detection, since the main disadvantage of indirect fluorescence detection is that fluorescence gives rise to a noisier baseline than does absorption and leads to a higher concentration LOD.Third, as the values of dn/dT for most non-aqueous media are larger than those of aqueous solutions, this detector will be of advantage for detection in non-aqueous medium capillary electrophoresis, an important field in the study of capillary electrophoresis.In future work we will investigate possible improvements to the detection sensitivity, including the design of an electronic noise canceller to reduce electronic noise, altering the injection mode and the use of highly stable lasers. The relatively low cost, simple construction, small volume, universal detection, larger range of ionic strengths and excellent detection limits of this method should prove attractive for a number of applications in capillary electrophoresis separation techniques. This work was supported by the National Nature Science Foundation of China. References 1 Stoker, F. S., Haymor, B. L., and McBeath, R., J. Chromatogr., 1989, 470, 241. 2 Burlity, N., and Jorgenson, J., J. Chromatogr., 1989, 480, 301. 3 Swaile, D. F., and Sepaniak, M. J., Anal. Chem., 1991, 63, 179. 4 Hu, Y., Deng, Y., and Cheng, J., Prog. Nat. Sci., 1996, 6, S-42. 5 Yeung, E. S., Acc. Chem. Res., 1989, 22, 125. 6 Yeung, E. S., and Kuhr, W. G., Anal. Chem., 1991, 63, 275A. 7 Kaniansky, D., Zelenska, V., and Baluchova, D., Electroporesis, 1996, 17, 1890. 8 Jandik, P., and Bonn, G., Capillary Electrophoresis of Small Ions, VCH, Weinheim, 1993. 9 Pfeffer, W. D., and Yeung, E. S., J. Chromatogr., 1990, 506, 401. 10 Wilson, S. 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Chromatogr., 1991, 542, 173. 36 Green, J. S., and Jorgenson, J. W., J. Chromatogr., 1987, 478, 63. Paper 7/00119C Received January 6, 1997 Accepted June 18, 1997 Fig. 5 Electropherogram of a mixture of three metal cations. Methylene Blue concentration, 5.0 3 1026 mol l21. Other conditions as in Fig. 4. Peaks: 1, KI (2.5 3 1026 mol l21); 2, system peak; 3, CuII (1.56 3 1026 mol l21); and 4, AlIII (3.85 3 1026 mol l21). Analyst, October 1997, Vol. 122 1093