Anal. Chem. 2005, 77, 1518-1525
Technical Notes
Development of a Detector for Liquid Chromatography Based on Aerosol Chemiluminescence on Porous Alumina Yi Lv,† Sichun Zhang,† Guohong Liu,† Minwen Huang,‡ and Xinrong Zhang*,†
Department of Chemistry, Key Laboratory for Atomic and Molecular Nanosciences of Education Ministry, Tsinghua University, 100084, Beijing, P. R. China, and Department of Chemistry, Jiaying University, 514071, Guangdong, P. R. China
This paper describes a novel aerosol chemiluminescencebased detector, which can be coupled to liquid chromatography for the determination of the chemicals with weak optical absorbance in the UV-visible region. This aerosol chemiluminescence (CL)-based detector, in which HPLC effluent is converted to aerosol and then generated CL emission on the surface of porous alumina, is composed of three main processes: nebuliztion of HPLC effluent, CL emission on surface of porous alumina material, and optical detection. To demonstrate the utility of the aerosol chemiluminescence detector, some compounds such saccharides, poly(ethylene glycol)s, amino acids, and steroid pharmaceuticals are determined by the present aerosol chemiluminescence detection method. Compared with an evaporative light scattering detector, the proposed detector shows the following features: (a) extensive CL emissions on porous alumina by many compounds tested, which leads to the potential application for the determination of volatile and nonvolatile chemicals with or without UV-visible absorbance; (b) a CL mechanism based on the catalytic oxidation of analytes, not on the light scattering, which suggests the present detector be free from the interference of the inorganic and nonvolatile mobile-phase modifiers. The CL characteristics and effect of different parameters, such as temperature and nebulizer gas flow rate, were also discussed in this paper. Furthermore, this aerosol chemiluminescence-based detector was successfully applied to the determination of raffinose, glucose, sucrose, maltose, and r-lactose. Detection for liquid chromatography can be accomplished by a variety of techniques. Commercially available spectrophotometric methods such as UV-visible and fluorescence detection are popularly and frequently employed in liquid chromatography (LC); however, these methods are not suitable for the direct measurement of chemicals without chromophores and fluorophores, such * To whom correspondence should be addressed: (e-mail) xrzhang@ chem.tsinghua.edu.cn; (tel) +86-10-6278-7678; (fax) +86-10-6277-0327. † Tsinghua University. ‡ Jiaying University.
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as carbohydrates, fatty acid esters, and poly(ethylene glycol)s. The refractive index detector provides more universal response, but it cannot be used with gradient elution precluding the analysis of complex mixtures.1 Since Ford and Kennard developed an evaporative light scattering detector (ELSD) for the first time in 1966, the application of ELSD is dramatically increasing in LC as the semiuniversal mass detection method.2,3 In LC combined with ELSD, the effluent from a chromatographic column is converted to an aerosol by a nebulizer with the aid of carrier gas. The aerosol is then carried into a heated drift tube where the solvent is evaporated to form small analyte particles that can arouse the light scattering. The scattered light is proportional to the amount of sample and is not dependent on a specific functional group or on chromophores, so the ELSD is suitable to detect nonvolatile compounds such as lipids,4 carbohydrates,5 and pharmaceutical compounds,6,7 which showed weak optical absorbance in the UVvisible region. As a consequence, there is no question about popularity and universality of the ELSD in liquid chromatography. However, ELSD also has several drawbacks.8 The most important limitation of the ELSD is that the nonvolatile mobile-phase modifiers would produce a constant background signal, which could lead to a loss of sensitivity. On the other hand, the ELSD could not be applied to the measurement of the volatile analytes. In recent years, porous materials are widely regarded as promising materials9,10 for applications in catalysis,11 separation,12 (1) Ewing, G. W., Ed. Analytical Instrumentation Handbook, 2nd ed.; Marcel Dekker: New York. 1997; Charpter 22. (2) Charlesworth, J. M. Anal. Chem. 1978, 50, 1414-1420. (3) Mourey, T. H.; Oppenheimer, L. E. Anal. Chem. 1984, 56, 2427-2434. (4) Moreau, R. A.; Powell, M. J.; Hicks, K. B. J. Agric. Food Chem. 1996, 44, 2149-2154. (5) Wei, Y.; Ding, M. Y. J. Chromatogr., A 2000, 904, 113-117. (6) Asmus, P. A.; Landis, J. B. J. Chromatogr. 1984, 316, 461-472. (7) Strege, M. A.; Stevenson, S.; Lawrence, S. M. Anal. Chem. 2000, 72, 46294633. (8) Kohler, M.; Haerdi, W.; Christen, P.; Veuthey, J. L. TRAC, Trends Anal. Chem. 1997, 16, 475-484. (9) Fe´rey, G.; Cheetham, A. K. Science 1999, 283, 1125-1126. (10) Chae, H. K.; Siberio-Pe’rez, D. Y.; Kim, J.; Go, Y. B.; Eddaoudi, M. Nature 2004, 427, 523-527. (11) Sen, T.; Tiddy, G. J. T.; Casci, J. L.; Anderson, M. W. Chem. Mater. 2004, 16(11), 2044-2054. (12) Jiang, Z. T.; Zuo, Y. M. Anal. Chem. 2001, 73, 686-688. 10.1021/ac048816w CCC: $30.25
© 2005 American Chemical Society Published on Web 01/25/2005
Figure 1. Schematic diagram of the aerosol cataluminescence detection system.
gas storage,13,14 and molecular recognition15,16 due to their high surface area, stable structure, good adsorption, and high activity. CL generated on the surface of solid porous materials is a very interesting phenomenon, which was observed during the mixing of porous silicon with nitric acid or persulfate by McCord and co-workers in 1992.17 On the other hand, Breysse et al.18 reported that the catalytic oxidation of carbon monoxide on the surface of thoria, a nonporous material, could produce a weak CL emission and established a concept of “cataluminescence”. The phenomenon of CL emission based on the catalytic oxidation of organic vapors could be used for the design of gas sensor. In previous work,19-22 we have investigated CL emission on the surface of several nanosized materials such as titanium dioxide, strontium carbonate, and zirconium dioxide and constructed a series of gas sensors for measuring concentrations of organic vapors, such as ethanol, acetaldehyde, and H2S. In the present work, we developed a novel aerosol chemiluminescence detector, which can be coupled to HPLC for the detection of the compounds without or with only weak UV-visible absorption. In comparison with an ELSD detector, the most distinctive characteristic of the present detector is that no radiation source was required for the detection of light scattering from (13) Dybtsev, D. N.; Chun, H.; Yoon, S. H.; Kim, D.; Kim, K. J. Am. Chem. Soc. 2004, 126(1), 32-33. (14) Sharma, A. C.; Borovik, A. S. J. Am. Chem. Soc. 2000, 122(37), 89468955. (15) Gross, E.; Kovalev, D.; Kunzner, N.; Timoshenko, V. Y.; Diener, J.; Koch, F. J. Appl. Phys. 2001, 90, 3529-3532. (16) Ito, T.; Hioki, T.; Yamaguchi, T.; Shinbo, T.; Nakao, S.; Kimura, S. J. Am. Chem. Soc. 2002, 124, 7840-7846. (17) McCord, P.; Yau, S. L.; Bard, A. J. Science 1992, 257, 68-69. (18) Breysse, M.; Claudel, B.; Faure, L.; Guenin M.; Williams, R. J. J. J. Catal. 1976, 45, 137-144. (19) Zhu, Y.; Shi, J.; Zhang, Z.; Zhang, C.; Zhang, X.; Anal. Chem. 2002, 74, 120-124. (20) Cao, X. O.; Zhang, Z. Y.; Zhang, X. R. Sens. Actuators, B 2004, 99 (1), 30-35. (21) Shi, J. J, Yan, R. X.; Zhu, Y. F.; Zhang, X. R. Talanta 2003, 61 (2), 157164. (22) Zhang, Z. Y.; Zhang, C.; Zhang, X. R. Analyst 2002, 127 (6), 792-796.
particles, leading to a simplified setup. The detector was fabricated by immobilizing the porous alumina onto the ceramic tube; the HPLC effluent was first introduced into a nebulizer in order to be converted into the aerosol, which then aroused a strong CL emission on the surface of porous alumina at optimal temperature. Our preliminary investigation showed that many organic compounds, without or with only weak UV-visible absorption, such as saccharides, poly(ethylene glycol)s, amino acids, and steroid pharmaceuticals in solution could be detected by the present aerosol chemiluminescence detector. The second advantage of the present detector is that it has a good quality of response to the volatile organic compounds such as acetone and ethanol with water as mobile phase, which is considered difficult by using the ELSD detector. The third advantage is that the present detector is free from the interference of inorganic modifiers such as phosphate in mobile phase. All the advantages mentioned indicated that this detector would be used as a supplement for ELSD to a certain extent. EXPERIMENTAL SECTION Aerosol Chemiluminescence Detector Coupled to Liquid Chromatography. A schematic diagram illustrating the main features of aerosol chemiluminescence detector as used in this study is shown in Figure 1. The aerosol chemiluminescence detector is composed of three main components that individually govern three successive processes: nebulization of HPLC effluent, CL reaction on the surface of porous alumina, and optical detection by PMT. A stainless steel capillary tube (0.35-mm i.d., 0.80-mm o.d.) is used for the continuous sampler, through which the effluent from the liquid chromatography is introduced into the aerosol chemiluminescence detector. The capillary tube is surrounded by a larger tube (1.29-mm i.d., 3.6-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 Analytical Chemistry, Vol. 77, No. 5, March 1, 2005
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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 tee quartz tube of 15-mm inner diameter to avoid the eluent without complete nebulization pouring directly on the surface of porous alumina, which would cause the temperature to vary sharply. The experiment was performed with water as the mobile phase at a liquid flow rate of 1.0 mL/min, because this is a typical flow rate for HPLC; an Alltech 526 HPLC pump was used to deliver the eluent; no chromatographic columns were used in these studies except for a special requirement. Samples dissolved in the eluent were introduced into the aerosol chemiluminescence detection system by means of a six-port injection valve equipped with a 40-µL capacity sample loop. The effluent from the liquid chromatography is converted into aerosol by the nebulizer; the formed aerosol is carried through the outside of ceramic heater. 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 and Synthesis of Chemicals. All the chemicals were of reagent grade or better and used as purchased. Carbon tetrachloride was used for the preparation of steroid solutions, and doubly distilled water was used for others. L-Leucine, Lthreonine, L-histidine, and L-phenylalanine were obtained from Sigma. Saccharides including raffinose, sucrose, glucose, maltose, and R-lactose were purchased from Tianjin Chemical Co. Ltd. (Tianjin, China). Steroid standards containing cholesterol, methyltestosterone, stanozolol, testosterone propionate, and nandrolone phenylpropionate were obtained from the Institute of Pharmaceutical and Biomaterial Authentication (Beijing, 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). 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) 10000 (10 g) were added into 500-mL aqueous solutions of Al(NO3)3 (0.1 mol‚L-1) 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. The morphology of synthesized alumina was examined on a Hitachi H-800 transmission electron microscope (TEM) at an accelerating voltage of 200 kV. Figure 2 showed that the synthesized alumina have a porous appearance. An X-ray power diffraction (XRD) 1520 Analytical Chemistry, Vol. 77, No. 5, March 1, 2005
Figure 2. TEM image of the synthesized porous alumina. The image was obtained from a Hitachi H-800 TEM operated at an accelerating voltage of 200 kV.
Figure 3. X-ray diffraction pattern of porous alumina. The diffraction data were collected on a Rigaku DMAX-2400 diffractometer using Cu KR radiation. The sample was scanned from 10° to 70° (2θ) with a step size of 0.02°.
experiment was carried out in a Rigaku DMAX-2400 diffractometer using Cu KR radiation. From Figure 3, we can found that these peaks are attributed to the γ-phase according to the XRD standard spectrum of alumina. A typical nitrogen adsorption-desorption isotherm of the synthesized alumina was further measured at 77 K using an Autosorb-1 instrument (Quantachrome Co.). The surface area and pore size distribution were calculated by the BET method and the BJH model from the desorption branch, respectively. The results showed that the BET surface area and BJH pore volume of this alumina were equal to 237.1 m2/g and 1.5 cm3/g, respectively. The pore size distribution is approximately, mainly broad from 5 to 40 nm with a BJH desorption average pore diameter (4 V/A) of 17.3 nm. RESULTS AND DISCUSSION 1. Examination of CL Behaviors of the Present Detector. The preliminary experiments were carried out to examine the CL behaviors of different compounds that might not be detected easily by the UV-visible detector currently used in HPLC, since these
compounds have no or only have weak UV-visible absorbance. Another purpose of these experiments was to make differences between the analytes eluted from HPLC column and the inorganic salts used in buffer composition in the mobile phase, because we expected that the latter species would cause serious background interference during detection with the ELSD detector but may not interfere with the present detector. The analytes that would be easily evaporated in comparison with the mobile phase were also examined because these compounds were considered to be difficult for detection by ELSD as reported by numerous publications. Detection of the Analytes with Weak Optical Absorption in the UV-Visible Region. The most distinguished characteristic of ELSD is its application to the measurements of the nonvolatile analytes without UV-visible absorbance, such as carbohydrates, fatty acid esters, and poly(ethylene glycol)s. Furthermore, it is also an efficient detection technique for some nonvolatile pharmaceuticals with weak optical absorption in the UV-visible region, e.g., steroid pharmaceuticals. Several families of compounds including poly(ethylene glycol)s (molecular weights are 400, 2000, 10 000, respectively), saccharides (including raffinose, maltose, glucose, sucrose, and R-lactose), and steroids (including cholesterol, methyltestosterone, stanozolol, testosterone propionate, and nandrolone phenylpropionate) were introduced into the present aerosol chemiluminescence detection system in sequence. Each of the analytes injected into the carrier is 40 µL with a concentration of 100 µg‚mL-1. The results shown in Figure 4a-c, respectively. It is very interesting to note that CL intensities of these PEGs were approximately equal; signals of CL emission for PEG 400, 2000, and 10 000 were about 5150, 4790, and 4650 counts (Figure 4a), but glucose, with the most small molecular weight in these saccharides, has the most strong CL response to about 5250 counts (Figure 4b). This study indicated that the abovementioned analytes with the characteristic of weak optical absorption in the UV-visible region could produce strong CL emission. Amino acids including L-leucine, L-threonine, L-histidine, and L-phenylalanine have also been examined and strong CL emission was also obtained. The determination of amino acids will be discussed in a later section. Detection of the Analytes with Volatile Properties. It is well known that the detection signal of the ELSD is based on the scattering light producded from the particle of analyte via evaporating the solvent in the eluent; as a consequence, the obvious disadvantage of the ELSD is that it can only be suitable for the molecules with nonvolatile properties compared with that of the mobile phase, and compounds with high volatile properties cannot be detected. Our preliminary experience indicated that the present detector could also be suitable for the compounds with high volatile properties. Figure 5 shows the CL responses from 500 µg‚mL-1 ethanol, 200 µg‚mL-1 propyl alcohol, and 100 µg‚mL-1 acetone with an injection volume of 40 µL for each sample solution. They are all volatile in comparison with water as mobile phase. These results indicated that the present detector has more potential for the application than the ELSD when using water as mobile phase. The methanol and acetonitrile, which were widely used as the mobile phase in liquid chromatography, were also tested by the present detector. The result showed that aqueous methanol at the concentration of above 10% could arouse a CL;
Figure 4. Typical recording of CL response for chemicals with weak UV-visible absorbance. Aerosol CL detector condition: carrier flow rate, 1.0 mL‚min-1; nebulizer air flow, 12 dm3‚min-1; temperature, 400 °C; wavelengths 460, 460, and 420 nm for a-c, respectively. (a) Poly(ethylene glycol)s (100 µg‚mL-1); (b) saccharides (100 µg‚mL-1); (c) steroids (100 µg‚mL-1).
however, acetontrile at any concentration could not do that. This result indicated that acetontrile had a potential use as the mobile phase, when the present detector was coupled to reversed-phase liquid chromatography. Detection of Amino Acid in a Different Inorganic Medium. Although the ELSD is an ideal detector that could be especially applied to the molecules without UV absorption, detection could only be carried out in the mobile phase without high inorganic salt as buffer (e.g., phosphate buffer). The presence of nonvolatile and sparingly eluent modifiers would cause an elevated backAnalytical Chemistry, Vol. 77, No. 5, March 1, 2005
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Figure 5. Typical recording of CL response for volatile compounds. Aerosol CL detector condition: carrier flow rate, 1.0 mL‚min-1; nebulizer air flow, 12 dm3‚min-1; temperature, 400 °C; wavelength, 460 nm. (A) Acetone (100 µg‚mL-1); (E) ethanol (500 µg‚mL-1); (P) isopropyl alcohol (200 µg‚mL-1).
Figure 7. CL spectra of saccharides on porous alumina surface at temperatures of 375, 400, and 425 °C. Aerosol CL detector condition: carrier flow rate, 1.0 mL‚min-1; nebulizer air flow, 12 dm3‚min-1; temperature, 400 °C; wavelength, 460 nm.
Figure 6. Typical recording of the 200 µg‚mL-1 L-histidine and L-phenylalanine in different concentrations of phosphate medium (pH7.4, NaH2PO4/Na2HPO4); 0 (1), 0.01 (2), and 0.1(3) mol‚L-1. RSDs (CL peak areas) for L-histidine and L-phenylalanine in different phosphate buffers were 2.1 and 1.9%.
ground, decreasing the sensitivity of the detector and inducing a rapid degradation of the performance of the instruments, due to their deposit in the optical cell. Unfortunately, the inorganic chemicals, such as phosphate, are widely, sometimes inevitably used as pH modifiers of mobile phases and sample solutions in liquid chromatorgraphy. To demonstrate the ability of the present detector to tolerate high inorganic salt medium, an experiment was designed for the detection of amino acids in different concentrations of phosphate buffer. Figure 6 showed the chemilumiescence response profile of 200 µg‚mL-1 L-histidine and 200 µg‚mL-1 L-phenylalanine in different phosphate buffers (NaH2PO4/ Na2HPO4, pH 7.4); the phosphate concetractions corresponding to curves 1-3 were 0, 0.01, and 0.1 mol/L, respectively. The results indicated that there was no significant difference among the peak areas for the same compounds at different concentrations 1522 Analytical Chemistry, Vol. 77, No. 5, March 1, 2005
of phosphate medium. The RSD for the three peaks by measuring the peak areas were 2.1 and 1.9% for L-histidine and L-phenylalanine, respectively. These results indicated that different concentrations of phosphate medium would not affect the CL detection of the L-histidine and L-phenylalanine. 2. Application for the Determination of Saccharides. To estimate the reliability of the aerosol chemiluminescence detector, the detection of saccharides by the present aerosol chemiluminescence detector was conducted in detail. CL Spectra and Analytical Charactersitics. The CL spectra of the saccharides inluding sucrose, glucose, maltose, R-lactose, and raffinose on the surface of porous alumina at different temperature (375, 400, 425 °C) were investigated through a series of optical filters. The results were shown in Figure 7. It can be seen that the profile of curves for each saccharide at different temperatures are similar and each curve has the same peak at around 425 and 460 nm; furthermore, all of the curves have a same maximum of CL emission at 460 nm. The experiment for CL spectra implies that the same luminescent intermediate would be produced during catalytic oxidation of the analytes mentioned. It is very interesting to note that the CL spectra profile of these analytes is very similar to the one during the oxidation of ethylene (C2H2) described in our previous work.23 Alumina is well known for its wide application in catalytic cracking of aliphatic hydrocarbons (such as alkene and alkane)24 and catalytic decomposition of polymers (such as polyethylene, polypropylene, and poly(ethylene glycol)).25,26 This implies that ethylene produced during (23) Shi, J.; Li, J.; Zhu, Y.; Wei, F.; Zhang, X. Anal. Chim. Acta 2002, 466, 6978. (24) Kissin Y. V. Catal. Rev. Sci. Eng. 2001, 43 (1-2), 85-146.
Figure 9. Effects of nebulizer gasflow rate and temperature on detector response for sucrose at injected concentration of 200 µg.mL-1. b, 375 °C; 9, 400 °C; 2, 425 °C). Figure 8. Typical recording of CL response (sucrose). Aerosol CL detector condition: carrier flow rate, 1.0 mL‚min-1; nebulizer air flow, 14 dm3‚min-1; temperature, 400 °C; wavelength, 460 nm. Table 1. Analytical Characteristics of Saccharide Detection
saccharide
regression equationa
linear range (C. µg/mL)
rel coeff
limit of detection (µg/mL)
sucrose glucose maltose R-lactose raffinose
I ) 15.95C + 137.6 I ) 21.03C + 191.5 I ) 14.31C + 173.5 I ) 15.23C + 181.2 I ) 16.15C + 157.9
50-1000 10-1000 50-1000 50-1000 40-800
0.999 0.999 0.997 0.998 0.998
19 3.1 23 35 17
Figure 10. Effect of temperature on the CL intensity of 100 µg‚mL-1 sucrose at 460 nm. (b, relative CL intensity; 2, signal/noise).
a Where I is the CL intensity and C is the concentration of saccharide solution.
C-C-bond fission in a cracking and decomposition reaction may be responsible for the CL emission. Further work is required to identify the radical intermediates and explain the CL mechanism. This exploration of CL spectra also indicated that the detection of these analytes in liquid chromatography with the present detector could be conducted at the same wavelength band. As a consequence, the measurements of saccharides were conducted at a selected wavelength of 460 nm. In the calibration study, sucrose standards with concentrations ranging from 50 to 1000 µg‚mL-1 were injected multiple times inside an hour. Figure 8 showed typical CL response traces for 50, 100, 250, 500, and 1000 µg‚mL-1 sucrose standards injected in triplicate. With the peak height as a quantitative parameter, the calibration curve of CL intensity versus sucrose concentration was linear over the range of 50-1000 µg‚mL-1 with a relative coefficient of 0.998. Reproducibility based on the variability of peak height of the standard was estimated; the result showed that the relative standard deviation is 3.1% (n ) 7) for 100 µg‚mL-1 sucrose standard. The linear range and limit of detection of the method for saccharides are shown in Table 1. However, it is worth mentioning here that the data obtained by the direct injection procedures do not represent true peak shapes obtained with chromatographic columns. The practical sensitivity of the aerosol CL detector would actually be worse than stated by the direct injection procedures because of the (25) Voorhees, K. J.; Baugh, S.; Stevenson, D. N. Thermochim. Acta 1996, 274, 187-207. (26) Lin, Y. H.; Hwu, W. H.; Ger, M. D.; Yeh, T. F.; Dwyer, J. J. Mol. Catal., A 2001, 171, 143-151.
broadened chromatographic peaks. This phenomenon in fact has been observed in our complementary experiment. A peak broadening has occurred, especially when an unsuitable ratio of acetonitrile and water was used as the mobile phase. Effect of Nebulizer Gas Flow Rate and Temperature. Nebulizer gas flow rate and temperature are the major factors that have a strong and definitive effect on the performance of an aerosol chemiluminescence detector. Figure 9 showed a typical example of the sucrose in response over the ranges of 4-18 dm3‚min-1 at temperatures of 375, 400, and 425 °C. The results showed that the response of chemiluminescence increases with increasing of gas flow rate up to 14 dm3‚min-1 at temperatures of 375, 400, and 425 °C, respectively, above which it decreases. The mean diameter of the drop produced by a Venturi-type nebulizer, in micrometers, as a function of liquid and gas flow parameters, has been approximated by a typical equation developed by Nukiyama and Tanasawa, which indicates that the diameter of drop decreases with increasing nebulizer gas flow rate.3 In the present detector, only the drop with a small diameter can be carried through the chemilumiescence system; therefore, strong CL intensity can be obtained at a high gas flow rate. However, the CL intensity produced by the oxidation of analytes on the surface of porous alumina, and a too high gas flow rate, would bring a decrease of adsorbing analytes on the surface of alumina, which also leads to the decrease of CL intensity. The effect of temperature on the chemilumiescence intensity of 100 µg‚mL-1 sucrose was also investigated under the bandpass of 460 nm and at the air flow rate of 14 dm3‚min-1. Figure 10 denotes that the CL signal increases with the increase in temperature from 350 to 450 °C. However, according to the value Analytical Chemistry, Vol. 77, No. 5, March 1, 2005
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Figure 11. Separation of saccharides (glucose and sucrose) by HPLC with the aerosol CL detector. Aerosol CL detector condition: nebulizer gas flow, 14 dm3‚min-1; temperature, 400 °C; wavelength, 460 nm. Separation condition: flow rate, 1.0 mL‚min-1; sample, mixture of glucose (100 µg‚mL-1) and sucrose (100 µg‚mL-1); sample volume, 50 µL; column, Altima Amino (Alltech Associates, Inc., 250 mm × 4.6 mm, i.d. 5 µm); eluent, acetonitrile 100 (a), 85 (b), 75 (c), 50. (d), and 25% (e).
of S/N versus temperature, the maximum of the value of S/N can be obtained when the working temperature is 400 °C; under or above the temperature causes a decrease in the value of S/N. The reason would probably be that the noise signal also increases with increasing temperature and even increases faster than the CL intensity at higher temperature. Therefore, the temperature of 400 °C was an optimum condition for sucrose detection. In addition, other saccharides including glucose, maltose, and R-lactose were also investigated by the same procedure. The results showed that all the above-mentioned saccharides have the same optimum temperature, which indicated that the measurement of these saccharides in liquid chromatography with the present detector could be conducted at the same temperature. 1524 Analytical Chemistry, Vol. 77, No. 5, March 1, 2005
Lifetime of the Aerosol Chemiluminescence Detector. To examine the stability and durability of this aerosol CL detector, one experiment was carried out by sampling 40 µL of sucrose (100 µg‚mL-1) in the detection system every 30 min at 400 °C with the nebulizer gas flow rate of 14 dm3‚min-1. A 30 mmol‚L-1 phosphate buffer was used as the mobile phase. The CL intensity was measured at the wavelength band-pass of 460 nm. The results showed that during 200 times sampling, no obvious change could be found. The RSD (n ) 19) by a random data acquisition during the 100-h detection is ∼1.2%. Study of the HPLC Coupled to the Aerosol CL Detector for the Separation of Glucose and Sucrose. The experiments were carried out by using an HPLC column with acetonitrile-
water as mobile phase (different volume ratio from 100 to 25% acetonitrile) for the separation of glucose and sucrose. The detail procedures are shown as follows: the experiments were conducted at ambient temperature (∼20 °C). An Alltech 526 HPLC pump with a six-port injection valve was employed in the complementary experiments. The homemade aerosol CL detector presented in the paper was used for the detection (detection condition: nebulizer gas flow, 14 dm3‚min-1; temperature, 400 °C; wavelength, 460 nm). The chromatographic column used for separation of saccharides is the Altima Amino (Alltech Associates, Inc., 250 mm × 4.6 mm, i.d. 5 µm). The column was conditioned by passing the mobile phase for 40 min; the on-column pressure limit was set to 0-3000 psi. The sample solution was prepared by dissolving 0.010 g of glucose and sucrose in 100 mL of water, a 5-mL aliquot of this mixture was passed through a 0.45-mm poly(vinylidene difluoride) (PVDF) disk filter, and the filtrate was collected for HPLC analysis. Acetonitrile (Concord Tech Reagent, Tianjin, China) (HPLC grade) was filtered through a 0.45-µm PVDF membrane filter and degassed ultrasonically. The mobile phase was acetonitrile-water at a flow rate of 1.0 mL‚min-1. The volume ratios of acetonitrile and water were 100:0; 85:15; 75:25; 50:50, and 25:75, respectively. The corresponding results were shown in Figure 11. When the mobile phase was the 100% acetonitrile, there was no signal in 1 h (Figure 11a), indicating that the pure acetonitrile is not strong enough to elute the glucose and sucrose. Both of these saccharides were retained on the column. When the concentration of acetonitrile in the mobile phase was decreased down to 85% both the glucose and sucrose were well separated but the second compound (sucrose) shows serious peak broadening (Figure 11b), indicating the the separation of glucose and sucrose at this acetonitrile-water ratio was not acceptable. The optimal acetonitrile-water ratio was 75:25, both glucose and sucrose were well separated with baseline resolution; therefore, a 75% acetonitrile could be used for the separation of glucose and sucrose (Figure 11c). When the acetonitrile-water ratio was decreased to 50% (Figure 11d), both the glucose and sucrose were eluted at the same retention time, indicating a poor separation at that condition. The same situation was also observed at the 25% acetonitrile (Figure 11e). It is also worth mentioning here that, the concentration of acetonitrile in the mobile phase has no significant influence on the baseline, indicating that the present CL detector could be used with different concentrations of acetonitrile as mobile phase. CONCLUSIONS A novel aerosol CL detector for HPLC based on porous alumina was developed according to the generated CL emission from catalytic oxidation of the analytes. Studies have shown that the detection of compounds with weak optical absorption in the UVvisible region such as saccharides, poly(ethylene glycol)s, and
steroid pharmaceuticals can be completed successfully. Compared with the ELSD method, the present aerosol CL detector offers advantages for the measurement of volatile compounds and with less interference from inorganic modifiers in mobile phase or preparation of sample solutions. Studies have also demonstrated that the present aerosol CL detector has advantages of wide linear response, satisfied stability, simplicity, low cost, minimal size, and easiness for fabrication. Before this detector is suitable for routine applications, several challenges need to be addressed. First, one drawback is the relatively poor sensitivity, which probably and mainly depends on the catalytic activity and ability of materials for the analytes. To overcome this problem, the bulk of further work for the investigation of suitable materials is necessary. The first attempt would be made by searching for materials with relatively higher catalytic activity as a substitute for alumina. In the previous work, we found that the catalytic activity of some materials could be improved by noble metal atoms, so doping the noble metal atoms into the materials may be used. A preliminary experiment has also been carried out by doping the rare earth ions such as Eu3+, Tb3+, and Er3+ into the catalysts; the results showed that an energy-transfer CL reaction happened, and the sensitivity of the detector was increased to a certain extent. Therefore, a further investigation on doping rare earth ions would also be valuable. Second, this detector is an ideal detector for HPLC with waterbased eluents. Although acetonitrile has no CL emission on the porous alumina, the CL response of methanol appears to be an obvious barrier to the application of methanol in mobile phase. Acetonitrile and methanol could not produce strong CL emissions, but exploration of material on which the analytes could do that is necessary. Third, all experiments described in this paper only represent a proof of principle for the process of converting the sample solution into aerosol and taking it to produce CL emission. Therefore, two problems in fabrication of the detector including the rationality of the structure and the efficiency of the nebulization should be considered. In other words, application of the aerosol CL detector in HPLC is anticipated to be promising. ACKNOWLEDGMENT We gratefully acknowledged financial support of the work by the National Natural Science Foundation of China (20375022, 20345005). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
Received for review August 10, 2004. Accepted December 9, 2004. AC048816W
Analytical Chemistry, Vol. 77, No. 5, March 1, 2005
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