Development of an Aerosol Chemiluminescent Detector Coupled to

This CL detector is composed of a postcapillary nebulizer and porous alumina as catalyzer in quartz tube. The CL emission could be generated due to th...
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Anal. Chem. 2005, 77, 7356-7365

Development of an Aerosol Chemiluminescent Detector Coupled to Capillary Electrophoresis for Saccharide Analysis Guangming Huang, Yi Lv, Sichun Zhang, Chengdui Yang, and Xinrong Zhang*

Department of Chemistry, Key Laboratory for Atomic and Molecular Nanosciences of Education Ministry, Tsinghua University, 100084, Beijing, P. R. China

A novel aerosol chemiluminescent (CL) detector coupling to capillary electrophoresis (CE) for the detection of saccharides is reported. This CL detector is composed of a postcapillary nebulizer and porous alumina as catalyzer in quartz tube. The CL emission could be generated due to the catalyzing oxidization of saccharides on the surface of porous alumina. The saccharides such as sucrose, r-lactose, maltose, raffinose, galactose, xylose, and glucose with only weak UV absorbance can be successfully detected. The linear ranges of those saccharides are from 30-2000 to 50-2000 mg/L; relative standard deviations range from 2.1 to 3.7% (200 mg/L, n ) 11). Compared with the traditional UV detector currently used in CE, this novel detector shows the advantage of high sensitivity to the compounds with only weak UV absorption. Thus, it could be an important supplement of CE detectors for UVlacking compounds. Capillary electrophoresis (CE) is a powerful separation technique that can provide high resolution and is becoming a popular tool for the analysis of many compounds. CE plays an important role in the determination of saccharides in the fields of biochemistry, biotechnology, clinical chemistry, pharmacy, and food science. Up to now, the types of detector utilized for CE determination of saccharides are indirect UV, direct UV absorbance of derivatized sugars, electrochemical detection, refractive index detection, and electrospray ionization mass spectrometry.1,2 Since most of the saccharides lack UV adsorption, only a few studies were reported using direct UV detector.3,4 To improve the sensitivity of the UV detection of the saccharides, an indirect UV detection mode has been most widely used after CE separation. Ion chromophores such as sorbate5,6 and 2,6-pyridinedicarboxylic acid7,8 are added to the electrophoresis buffer to generate a * To whom correspondence should be addressed: (e-mail) zrzhang@ chem.tsinghua.edu.cn; (tel) +86-10-6278-7678; (fax) +86-10-6277-0327. (1) Montero, C. M.; Dodero, M. C. R.; Sanchez, D. A. G.; Barroso, C. G. Chromatographia 2004, 59 (1-2), 15-30. (2) El Rassi, Z. Electrophoresis 1999, 20 (15-16), 3134-3144. (3) Hoffstetter-Kuhm, S.; Paulus, A.; Gassmann, E.; Widmer, H. M. Anal. Chem. 1991, 63, 1541-1547. (4) Kakehi, K.; Susami, A.; Taga, A.; Suzuki, S.; Honda, S. J. Chromatogr., A 1994, 680 (1), 209-215. (5) Klockow, A.; Paulus, A.; Figueiredo, V.; Amado, R.; Widmer, H. M. J. Chromatogr., A 1994, 680, 187-200. (6) Zemann, A.; Nguyen, D. T.; Bonn, G. Electrophoresis 1997, 18, 1142-1147.

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constant signal due to the presence of those substances. Saccharides replace some of the ions, which caused a constant signal decrease when a band of the saccharides passed through the detector cell. But the application of indirect UV detection was limited to the narrow linear range and buffer contaminants.5-8 Furthermore, various derivatization reagents such as phenylethylamine (200 nm)9,10 and 6-aminoquinoline (245 nm)11 were used to increase the sensitivity of direct UV detection. While those reagents improved the detection, the complexity of derivatization procedures and the reaction time needed for derivatization greatly limited its applications. Refractive index detection and electrospray ionization mass spectrometry are both universal detection approaches for CE studies. Recently, several studies about the utility of saccharide analysis with CE refractive index detection have been reported.12,13 The relative poor detection limit and the shift of baseline limited the refractive index detection as a potential detector in CE of saccharides. Moreover, CE together with electrospray ionization mass spectrometry provides an optimum separation and detection of underivatized saccharides.14,15 However, the complexity of the instruments greatly defines the application of mass spectrometry detection in the field of CE. During the past decade, electrochemical detectors such as enzyme electrodes and amperometric detection have been intensively reported.16-19 In particular, electrochemical methods are among the most sensitive approaches currently available for the detection of underivatized saccharides. Unfortunately, at the present time, electrochemical detectors are not available from commercial sources because they suffer from (1) the limitations imposed by the alkaline conditions needed for the sensitive detection and (7) Soga, T.; Serwe, M. Food Chem. 2000, 69, 339-344. (8) Soga, T.; Heiger, D. N. Anal. Biochem. 1998, 261, 73-78. (9) Noe, C. R.; Lachmann, B.; Mollenbeck, S.; Richter, P. Z. Psychosomatic Med. Psychother. 1999, 208 (2), 148-152. (10) Noe, C. R.; Freissmuth, J. J. Chromatogr., A 1995, 704, 503-512. (11) Rydlund, A.; Dahlman, O. J. Chromatogr., A 1996, 738, 129-140. (12) Swinney, K.; Pennington, J.; Bornhop, D. J. Analyst 1999, 124 (3), 221225. (13) Burggraf, N.; Krattiger, B.; De Mello, A. J.; De Rooij, N. F.; Manz, A. Analyst 1998, 123 (7), 1443-1447. (14) Auriola, S.; Thibault, P.; Sadovskaya, I.; Altman, E. Electrophoresis 1998, 19 (15), 2665-2676. (15) Klampfl, C. W.; Buchberger, W. Electrophoresis 2001, 22, 2737-2742. (16) Updike S. J.; Hicks, G. P. Nature 1967, 214, 986-988. (17) Ye, J.; Baldwin, R. P. J. Chromatogr., A 1994, 687, 141-148, (18) Fermier, A. M.; Gostkowski, M. L.; Colon, L. A. Anal. Chem. 1996, 68, 1661-1664. (19) Baldwin, R. P. J. Pharm. Biomed. Anal. 1999, 19 (1-2), 69-81. 10.1021/ac0511290 CCC: $30.25

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differential electromigration, which restrict the useful pH to a very narrow range, i.e., pH >12, and (2) the reproducibility of electrochemical detection, which is relative poor because of the difficulty associated with the electrode/capillary alignment during an electrophoresis run and from run to run.20 Thus, the development of an easy, rapid, and sensitive detection of CE for those analytes that lack UV adsorption shows great importance. Recently, chemiluminescent (CL) emission based on the catalytic oxidation of organic analytes has been reported.21-31 McCord observed the CL emission on the surface of solid porous materials when porous silicon and nitric acid or persulfate were mixed together.21 Breysse reported a weak CL emission based on the catalytic oxidation of carbon monoxide on the surface of TiO2, which is known as “cataluminescence”.22 Previously, we observed the CL emission on porous materials and designed several gas sensors based on the phenomena.23-30 We have also developed a HPLC detector coupled with a spray system for the detection of the analytes that lack UV adsorption.31 This detector takes advantage of simple instrument setup (no radiation source was required for the detection of light scattering from particles), good quality of response to the nonvolatile and volatile organic compounds, and freedom from the interference of inorganic modifiers over evaporative light scattering detector.32,33 Above these advantages, the aerosol CL detector offers additional merits while coupling it to CE instrument. First, it is durable to high concentrations of salt in running buffers, where the strong scattering background at a high-salt concentration of running buffer greatly confines the application of ELSI in the field of CE. Second, the aqueous medium used as CE running buffers would shield the present detector from the high background CL emission. In CE separation, electrophoresis buffer is mostly composed of water, which would favor the detection of the analytes. Therefore, the high background CL emission caused by some organic solvents such as methanol was avoided. Moreover, this detector can be easily miniaturized, so as to be coupled with CE. In this paper, an aerosol CL detector coupled to CE for saccharide analysis has been proposed. With this CE detector, we have carried out the determination of R-lactose, sucrose, maltose, raffinose, galactose, xylose, and glucose. The results obtained in this study demonstrated that the detector could be (20) Patrick, C., Ed. Capillary Electrophoresis Theory And Practice, 2nd ed.; CRC Press LLC: Boca Raton, FL, 1998; pp 286-290. (21) Mccord, P.; Yau, S. L.; Bard, A. J. Science 1992, 257, 68-69. (22) Breysse, M.; Claudel, B.; Faure, L.; Guenin M.; Williams, R. J. J. J. Catal. 1976, 45, 137-144. (23) Zhu, Y. F.; Shi, J. J.; Zhang, Z. Y.; Zhang, C.; Zhang, X. R. Anal. Chem. 2002, 74 (1), 120-124. (24) Zhang, Z. Y.; Zhang, C.; Zhang, X. R. Analyst 2002, 127 (6), 792-796. (25) Cao, X. O.; Zhang, Z. Y.; Zhang, X. R. Sens. Actuator B 2004, 99 (1), 3035. (26) Zhang, Z. Y.; Jiang, H. J.; Xing, Z.; Zhang, X. R. Sens. Actuators B-Chem. 2004, 102 (1), 155-161. (27) Zhang, Z. Y.; Xu, K.; Xing, Z.; Zhang, X. R. Talanta 2005, 65 (4), 913-917. (28) Zhang, Z. Y.; Xu, K.; Baeyens, W. R. G.; Zhang, X. R. Anal. Chim. Acta 2005, 535 (1-2), 145-152. (29) Shi, J. J.; Li, J. J.; Zhu, Y. F.; Wei, F.; Zhang, X. R. Anal. Chim. Acta 2002, 466 (1), 69-78. (30) Shi, J. J.; Yan, R. X.; Zhu, Y. F.; Zhang, X. R. Talanta 2003, 61 (2), 157164. (31) Lv, Y.; Zhang, S. C.; Liu, G. H.; Huang, M. W.; Zhang, X. R. Anal. Chem. 2005, 77 (5), 1518-1525. (32) Charlesworth, J. M. Anal. Chem. 1978, 50, 1414-1420. (33) Mourey, T. H.; Oppenheimer, L. E. Anal. Chem. 1984, 56, 2427-2434.

Figure 1. Schematic diagram of the aerosol cataluminescence detection system.

successfully applied to detect saccharides by coupling it to CE separation. In comparison with a direct UV detector, this detector offers the advantage of direct detection of the analytes without involving a derivatization procedure. The addition of ionic chromophore into the electrophoresis buffer is therefore no longer needed. The aerosol CL detector is also robust over a relative wide range of pH, while a high pH value was requested by using amperometric detection. The low baseline shift, low interference, relative wide linear range, and long lifetime also make the CE aerosol chemiluminescent detector a potential means for routine analysis of saccharides. EXPERIMENTAL SECTION Aerosol CL Detector Coupled to CE. A schematic diagram illustrating the main features of aerosol CL detector as used in this study is shown in Figure 1. The aerosol CL detector is composed of three main components that individually govern three successive processes: nebulization of CE running buffer together with sheath liquid, CL reaction on the surface of porous alumina, and optical detection by PMT. A fused-silica capillary (75-µm i.d., 0.18-mm o.d.) is used for the continuous sampler, through which CE running buffer together with sheath liquid is introduced into the aerosol CL detector. The capillary is surrounded by a larger tube (0.35-mm i.d., 1.2-mm o.d.), through which filtered and pressurized air is forced. An air pump (GA5000A, Beijing Zhongxin Huili Co. Ltd., Beijing, China) is used for the air supply. A precision flowmeter (Beijing Keyi Lab Instrument Co. Ltd., Beijing, China) was employed for the detection of the gas flow rate. The CL system was made by sintering a 0.5-mm-thick layer of porous alumina powder on a cylindrical ceramic heater 6 mm in diameter. The ceramic heater was operated at the required temperature by a digital temperature controller. The homemade nebulizer and CL system were set in a straight quartz tube. The angle between the nebulizer and the porous alumina was set as 15° to achieve higher sensitivity. The experiments were performed on a laboratory-built instrument with a 20-kV high-voltage power supply and a 70-cm length (effective length) of 50-µm-i.d. fused-silica capillary. Before first use, a new capillary was pretreated with 0.2 M sodium hydroxide for 30 min, followed by water for 10 min. Before each run, the capillary was washed for 5 min with 0.2 M sodium hydroxide and preconditioned with running buffer for 5 min. The capillaries were filled with water for overnight storage. In addition, the outlet end of the capillary was always maintained at ground. Analytical Chemistry, Vol. 77, No. 22, November 15, 2005

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Separation voltage was 10 kV. The sample was injected by gravity injection for 10 s (10-cm height). A T-piece was used to mix CE running buffer together with the sheath liquid. Immediately after the two flows mixed together, and passed through the nebulizing capillary, the continuous and stable aerosol was produced. Then the aerosol was sprayed on the porous alumina, the solution droplets were initially dried, and the solvent was evaporated. The CL signal was emitted due to the presence of the analytes. The CL signal at a certain wavelength was detected and recorded with a computerized BPCL ultraweak chemiluminescence analyzer (Institute of Biophysics, Academia Sinica), equipped with a CR105 photomultiplier tube (Hamamatsu), through variable optical filters that can be changed from 400 to 640 nm. Data acquisition and treatment were performed with BPCL software running under Microsoft Windows 2000. Chemicals. All the chemicals were of analytical grade or better and used as purchased. Saccharides including sucrose, R-lactose, maltose, raffinose, galactose, xylose, and glucose were purchased from Tianjin Chemical Co. Ltd. (Tianjin, China). Poly(ethylene glycol) (400, 2000, 10 000), aluminum nitrate, ammonia solution (25%), ethanol, and other chemicals were obtained from Beijing Chemical Co. Ltd. (Beijing, China). Preparation of Porous Alumina. The preparation procedure of porous alumina was developed as following: poly(ethylene glycol) 400 (10 g), poly(ethylene glycol) 2000 (10 g), and poly(ethylene glycol) 10 000 (10 g) were added into 500-mL aqueous solutions of Al(NO3)3 (0.1 mol/L) in sequence and stirred for 10 min, and then 50 mL of ammonia solution (8%) was added at an isocratic rate of 5 mL/min. After the procedures mentioned, a gelatinized precipitate was obtained. Then the sol precipitate was coated on the surface of the cylindrical ceramic heater by using the dip-coating method with a drawing speed of 8.0 cm/min. The wet sol precipitate was then dried naturally (the room temperature was ∼26 °C) for 40 min. After repeating the procedure five times, the precipitate with the ceramic was calcined. The temperature was raised to 550 °C at 6-8 °C/min and maintained for 10 h. The produced alumina was cooled to room temperature in air, which took 1 h. The thickness of the alumina on the surface of the ceramic heater was ∼0.5 mm. RESULTS AND DISCUSSION Our previous work has demonstrated that the aerosol CL detector was a promising detector for HPLC.31 Whether it could be coupled to CE as a sensitive detector was in doubt because there are several challenges that must be faced: first, the low speed of running buffer moving in CE would cause instability of the aerosol formation, which would lead to a poor repeatability of the detection; second, the limitation of the CE injection volume would be an obstacle to achieve high sensitivity; third, the inorganic salt in the electrophoresis buffer might ruin the alumina film (the catalyzer), shortening the lifetime of the detector, and the various pH values of the running buffer required for the separation would influence the CL emission, causing inconsistant response of the detector. Therefore, a preliminary experiment was designed to evaluate the potential of the CL detector coupled to CE for saccharide detection. Characteristics of the Aerosol CL Detector. As the most common used CE detector, UV detection suffers poor sensitivity for the analytes with weak optical absorption in the UV-visible 7358 Analytical Chemistry, Vol. 77, No. 22, November 15, 2005

Figure 2. Typical recording of CL response for saccharides with weak UV-visible absorbance. Aerosol CL detector condition: sheath liquid, sodium bromide 25 mM, pH 7.5, and 200 µL/min; nebulization gas flow rate, 600 mL/min; nebulization capillary i.d., 75 µm; porous alumina temperature, 450 °C; detection wavelength, 460 nm. Sample information: glucose, 100 mg/L; R-lactose, sucrose, maltose, and raffinose, 200 mg/L.

region, such as saccharides. However, the saccharides could produce a strong CL emission on the surface of porous alumina. Five saccharides (glucose, R-lactose, sucrose, maltose, raffinose) were tested using the present detector, and the result is shown in Figure 2. From Figure 2, it can be seen that all the five saccharides tested could generate CL emission on the surface of porous alumina (glucose, 100 mg/L; R-lactose, sucrose, maltose, and raffinose, 200 mg/L), while most of these saccharides suffered low absorbance in UV-visible region.3,4,20 For example, the detection limit for glucose, which gains the highest UV absorbance among saccharides, based on UV detection, is ∼720 mg/L,20 but the detection limit for the same compounds by using the present CL detector is ∼10 mg/L at optimal conditions, 72 times lower than the UV method. The obtained result based on the present detector was even better than the method based on indirect UV detection (limit of detection, 30-60 mg/L) for R-lactose, sucrose, maltose, and raffinose, respectively, where the background electrolytes, such as 1-naphthylacetic acid and 2-naphthalenesulfonic acid, were added into the running buffer.34 The results above indicated that the detection ability of the present detector is much higher than UV detection for saccharide analysis. It is well known that high sensitivity for saccharides was achieved by using CE electrochemical detection.16-19 However, the requirements of a special pH value (above 11) to obtain detectable electrochemical signals seriously limited the optimization of CE separation. To demonstrate the ability of the present detector to tolerate a variety of pH conditions, an experiment was designed for the determination of saccharides in sodium tetrahydroborate, sodium hydroxide, and sodium phosphate buffers (pH ranged from 8 to 12). Figure 3A and B showed the CL responses of the 200 mg/L sucrose and 200 mg/L R-lactose in different buffers, respectively. The results indicate that there was no significant difference among the CL signals of saccharides when the pH value of the buffers changed from 8 to 12. It can be concluded that the present detector showed a relatively wide detection range, and the present detector could therefore be used at relatively low pH without decrease of sensitivity. The effect of the buffer concentrations on the present detector was also investigated. Experiments were carried out with different (34) Lee, Y. H.; Lin, T. I. J. Chromatogr., B 1996, 681 (1), 87-97.

Figure 3. Effect of the running buffer pH on CL response. Aerosol CL detector condition: sheath liquid, sodium bromide 25 mM, pH 7.5, and 200 µL/min; nebulization gas flow rate, 600 mL/min; nebulization capillary i.d., 75 µm; porous alumina temperature, 450 °C; detection wavelength, 460 nm. (A) Response of 50 mg/L sucrose; (B) response of 50 mg/L R-lactose.

Figure 4. Effect of the running buffer concentration on CL response. Aerosol CL detector condition: sheath liquid, sodium bromide 25 mM, pH 7.5, and 200 µL/min; nebulization gas flow rate, 600 mL/min; nebulization capillary i.d., 75 µm; porous alumina temperature, 450 °C; detection wavelength, 460 nm; running buffer, sodium hydroxide solution (25-75 mM).

concentrations of sodium hydroxide buffers at pH 10. Figure 4 shows the CL responses of saccharides in different concentrations of sodium hydroxide buffers. As indicated in Figure 4, there is no significant difference when the concentration of the running buffer solution changed from 25 to 75 mM for both saccharides. Based on the results above, we can conclude that the present detector can tolerate various pH values and different concentrations of running buffers. Thus, the present detector is superior to the electrochemical detector for the analysis of the saccharides, because the electrochemical detector can only be optimized at alkaline medium with the pH value above 11, seriously affecting the optimization of CE separation. Optimization of CE Coupled to Aerosol CL Detection. Optimization of the Sheath Liquid Parameters. In a normally designed CE system, the flow rate through the CE column (10100 nL/min) is too low to support a stable aerosol formation used in this study. Hence, the sheath liquid is introduced to stabilize the aerosol formation. In addition, similar to CE-ESI-MS,35 the sheath liquid for aerosol CL detector not only serves to establish an electrical connection between the outlet end of the CE column and the earthed electrode but also serves as a terminal pH to optimize the CL detection. Therefore, it is important to optimize the sheath liquid parameters to achieve high sensitivity for (35) Zheng, J.; Jann, M. W.; Hon, Y. Y.; Shamsi, S. A. Electrophoresis 2004, 25 (13), 2033-2043.

Figure 5. Effect of sheath liquid flow rate on the stability of the baseline. Sheath liquid: sodium bromide 25 mM, pH 7.5; sheath liquid flow rate, (a) 0, (b) 75, and (c) 200 µL/min.

saccharide detection. Therefore, the compositions and concentrations of sheath liquid were studied first, followed by pH and flow rate. Their effects on the detection sensitivity are discussed as follows. Effect of the Sheath Liquid Flow Rate. The sheath liquid flow rate was varied from 0 to 500 µL/min. When we directly connected the outlet of the CE capillary to a pneumatic nebulizer (with a sheath liquid flow rate of 0 mL/min), the flow rate through the CE column (10-100 nL/min) was too low to support a stable aerosol formation as described above. Consequently, the instable aerosol caused big noises as shown in Figure 5a. This result implied that the direct connection of the CE outlet to a pneumatic nebulizer was not suitable for analysis. This problem was solved subsequently by the introduction of a sheath liquid at optimal flow rate, with which the continuous and stable aerosol was formed (Figure 5c). However, it was observed that the CL response decreased while the flow rate increased due to the dilution effect (Figure 6). In contrast to the CL signal, a maximum signal-tonoise ratio was found at 200 µL/min (Figure 6). At the lower end of the sheath liquid flow rate (i.e., 75 µL/min), high noise was observed since this low flow rate is unable to support a stable aerosol formation (Figure 5b), although the highest CL response was provided at this flow rate. As a result, 200 µL/min was selected as the optimum sheath liquid flow rate. Effect of the Sheath Liquid Composition and pH Condition. The effects of sheath liquid composition and concentration were investigated. Several inorganic salts (sodium bromide, sodium chloride, sodium tetrahydroborate, sodium phosphate) with different concentrations were tested. As shown in Figure 7, the Analytical Chemistry, Vol. 77, No. 22, November 15, 2005

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Figure 6. Effect of sheath liquid flow rate on the CL signal. Sheath liquid, sodium bromide 25 mM, pH 7.5; sample, sucrose 200 mg/L.

Figure 7. Effect of sheath liquid composition on the CL signal. Aerosol CL detector condition: nebulization gas flow rate, 600 mL/ min; nebulization capillary i.d., 75 µm; porous alumina temperature, 450 °C; detection wavelength, 460 nm, sample, sucrose 300 mg/L.

sodium bromide in the sheath liquid gives the highest CL response probably due to the enhancement of sodium bromide on CL emission. This phenomenon has been observed in a traditional CL reaction based on luminol in our previous work.36 Therefore, sodium bromide solution was selected as the sheath liquid. Moreover, the effect of sheath liquid concentration was investigated. The sodium bromide concentration in sheath liquid was increased from 10 to 50 mM but keeping other conditions constant. As shown in Figure 7, 50 mM sodium bromide concentration in the sheath liquid provides the highest CL emissions, but a high concentration of sodium bromide in the sheath liquid would shorten the lifetime of the detector. To compromise both effects for the present detection, 25 mM sodium bromide in sheath liquid was finally chosen as the optimum. The effect of sheath liquid pH was studied by gradually increasing the sheath liquid pH from 4.0 to 10.0. As shown in Figure 8, a number of trends are noteworthy. First, the use of sheath liquid with a pH value of 6.0-8.0 provides 200% more intensity as compared to lower pH (under 6.0) or higher pH (above 8.0). This could be explained by the fact that the reaction between alumina and both acid and alkali solutions at high concentration would ruin the surface of porous alumina. Unlike the response of the analytes, where there is a plateau at the range of 6.0-8.0, the noise keeps increasing as the pH of the sheath liquid increases from 4.0 to 10.0. As a compromise of CL response and signal-tonoise ratio, the pH of the sheath liquid was set to a moderate value of 7.5. Although sheath liquid was successfully applied in the present detector, the sheath liquid would influence the peak boarding and 7360 Analytical Chemistry, Vol. 77, No. 22, November 15, 2005

Figure 8. Effect of sheath liquid pH on the CL signal. Sheath liquid: 25 mM sodium bromide solutions with pH from 4 to 10. Aerosol CL detector condition: nebulization gas flow rate, 600 mL/min; nebulization capillary i.d., 75 µm; porous alumina temperature, 450 °C; detection wavelength, 460 nm; sample, sucrose 200 mg/L.

detection limit. Recently, a sheathless technique was introduced into the field of the pneumatic sprayer to improve the detection limit.37,38 We have built up two sheathless pneumatic nebulizers (figure shown in Supporting Information) based on the literature.37,38 The experiments were designed to evaluate the new sheathless interface. The detection limits were greatly improved by using direct injection, since no dilution effects were observed without sheath liquid. Improvements of 150-200-fold were observed for sucrose, R-lactose, maltose, raffinose, galactose, xylose, and glucose by using a sheathless interface (data shown in Supporting Information). However, despite our efforts to design a sheathless interface according to the literature37 (figure shown in Supporting Information), we did not obtain a satisfactory result. We failed to establish a constant electrical connection to keep the CE capillary outlet grounded. That was because very small bubbles have always been generated at the abutment joint, which blocked the electrical current. Therefore, another homemade sheathless interface was fabricated (figure shown in Supporting Information) based on another study using the HF etching technique.38 With the HF etching technique, a constant electrical connection was established without the obstacle of small bubbles. Although the porous junction was firmly held in the buffer reservoir, it was still too fragile to carry out the CE operation; even the pressure caused by flushing the capillary with 0.2 M sodium hydroxide to precondition the capillary would ruin the joint. Therefore, the following investigations were still undertaken without using sheathless interface. Optimization of Nebulization. Effect of Nebulization Gas Flow Rate. The characteristics of the aerosol are strongly dependent on the flow rate of the nebulization gas. It is therefore important to examine the correlation between sensitivity and the flow rate. Figure 9 shows the effect of flow rate of nebulization gas on CL response over the ranges of 300-800 mL/min. As the gas flow rate was increased, the CL signal increased gradually because the droplets were getting smaller. The nebulization efficiency (36) Huang, G. M.; Ouyang, J.; Baeyens, W. R. G.; Yang, Y. P.; Tao, C. J. Anal. Chim. Acta 2002, 474 (1-2), 21-29. (37) Schaumloffel, D.; Encinar, J. R.; Lobinski, R. Anal. Chem. 2003, 75 (24), 6837-6842. (38) Janini, G. M.; Conrads, T. P.; Wilkens, K. L.; Issaq, H. J.; Veenstra, T. D. Anal. Chem. 2003, 75 (7), 1615-1619.

Figure 9. Effect of nebulization gas flow rate on CL response. Aerosol CL detector condition: sheath liquid, sodium bromide 25 mM, pH 7.5, and 200 µL/min; nebulization capillary i.d., 75 µm; porous alumina temperature, 450 °C; detection wavelength, 460 nm.

Figure 10. Effect of capillary dimensions on the CL intensity. Aerosol CL detector condition: sheath liquid, sodium bromide 25 mM, pH 7.5, and 200 µL/min; nebulization gas flow rate, 600 mL/min; porous alumina temperature, 450 °C; detection wavelength, 460 nm.

increased with increasing the nebulization gas flow rate, which has been approximated by a typical equation developed by Nukiyama et al.33 The maximum CL intensity was obtained at a flow rate of 600 mL/min. When the flow rate of the nebulization gas exceeded 600 mL/min, the CL responses decreased along the increase of the flow rate until 800 mL/min. This phenomenon could be explained by the fact that increasing the gas flow rate would bring a decrease of adsorbing analytes on the surface of alumina, which leads to the decrease of CL intensity. Like other pneumatic sprays connected to CE system,35 the nebulizer gas pressure has the potential to generate a suction force at the capillary outlet. As a result, a laminar flow is formed inside the capillary; thus, the separation efficiency may decrease and so does the Rs between two analytes. Although some measurements are affected such as using a nebulizer capillary with small dimensiona, feeding the sheath liquid to the interface by nebulizer self-aspiration, and using relative low nebulizer gas flow rates, we did not further optimize the nebulizer gas flow rate because the separation of the saccharides in the present study has already been achieved. Effect of Nebulization Capillary Dimension. In the pneumatic nebulization system, not only the nebulization gas flow rate but also the nebulization capillary dimension affected nebulization efficiency. Figure 10 shows a typical example of the effect of capillary dimensions on the CL intensity. Three inside diameters, 50, 75, and 150 µm, were tested. Figure 10 indicates that the CL

intensity of sucrose was linear with concentration in all three diameters tested. However, highest sensitivity was found when the inside diameter was set as 75 µm. It has been reported that a smaller nebulization capillary diameter leads to increased transfer efficiency;39 thus, the CL intensity increased when capillary diameter decreased. Although capillaries of even smaller inside diameters would improve sensitivity, a diameter of