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Chemiluminescence of bis(2,4,6-trichlorophenyl) oxalate in aqueous micellar systems
Analytica Chimica Acta, 217 (1989) 229-237 Elsevier Science Publishers B:V., Amsterdam -
229
Printed in The Netherlands
CHEMILUMINESCENCE OF BIS(2,4,6-TRICHLOROPHENYL) OXALATE IN AQUEOUS MICELLAR SYSTEMS
O.M. STEIJGER*, H.M. VAN MASTBERGEN and J.J.M. HOLTHUIS Department of Pharmaceutical Analysis, University of Utrecht, Catharijnesingel60,3511 Utrecht (The Netherlands)
GH
(Received 1lth May 1988)
SUMMARY The chemiluminescent reaction of bis(2,4,6-trichlorophenyl) oxalate (TCPO) in aqueous micellar systems is compared with the reaction in a mixture of acetonitrile and aqueous phosphate buffer. The chemiluminescence was studied in batch experiments with perylene as the fluorophore. The oxidation of TCPO produced the same intensity of chemiluminescence in the buffered acetonitrile as in Arkopal N-300 micelles, the best micellar system. The solubility of TCPO in an aqueous micellar system is greater than that in the acetonitrile/aqueous buffer (80:20, v/v), but TCPO is less stable in the former system.
Bis (2,4,6+ichlorophenyl) oxalate (TCPO ) is frequently used in chemiluminescence reactions. In the presence of hydrogen peroxide or another oxidant and a suitable fluorophore, TCPO chemiluminescence is one of the most efficient reactions [ 11. Figure 1 shows the generally proposed mechanism of the reaction. Chemiluminescence detection in reversed-phase liquid chromatography is
-
F’
-
F*+
2C02
F+LffiHT
Fig. 1. Generally proposed mechanism for TCPO chemiluminescence (F, fluorophore).
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0 1989 Elsevier Science Publishers B.V.
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often based on TCPO. Dansylated amino acids [ 2,3], dansylated steroids [ 41, fluorescamine-labelled catecholamines [ 51, o-phthalaldehyde- and nitrobenzofurazan-labelled amines [ 61 and polynuclear aromatic hydrocarbons [ 7,8 ] can be determined at lower concentrations with chemiluminescence detection than with fluorescence detection. A disadvantage of chemiluminescence detection involving TCPO is the poor solubility and stability of TCPO in aqueous systems. Because of its poor stability, the TCPO solution must be delivered to the eluent stream via a separate flow line. The poor solubility of TCPO means that a large amount of organic solvent is required to dissolve sufficient TCPO. A mixing problem arises because the chemiluminescence reagents in organic solvents must be mixed postcolumn with the partly aqueous column effluent. Therefore, it is important to choose the right type of buffer because it must be soluble in mixtures of water and organic solvents [ 2-71. The study described here was designed to investigate the usefulness and influence of micellar systems in the TCPO chemiluminescence reaction. Micelles can affect the solubility and the physical and chemical properties of various compounds [ 91, and have been used successfully in several analytical techniques [lo]. However, there have been only a few investigations of chemiluminescence reactions in micellar media [ 11-151. Chemiluminescence assays in aqueous solutions and based on TCPO and the use of micelles have not been described before. Here, the intensity of the chemiluminescence and the stability of TCPO in several micellar systems are compared with these parameters in an acetonitrile/water buffer solution (80:20, v/v) with perylene as the fluorophore. EXPERIMENTAL
Chemicals and solutions Bis (2,4,6-trichlorophenyl ) oxalate was prepared by the method developed by Mohan and Turro [ 161, and was stored under nitrogen at - 18” C. Solutions of TCPO in acetonitrile (Merck; analytical-reagent grade) were freshly prepared every day, as were the solutions of perylene (Janssen Chimica, Beerse, Belgium) in acetone (Baker, analyzed grade). Hydrogen peroxide (30%, w/v) was obtained from O.P.G. (Utrecht, The Netherlands). The buffers used were 0.01 M sodium phosphate buffer, pH 7.0 (NaH,PO,*2H,O) (Brocacef, Maarssen, The Netherlands) and 0.01 M Tris buffer, pH 7.0 [ tris (hydroxymethyl) aminomethane; Merck; analytical-reagent grade]. The pH of the buffers was adjusted with sodium hydroxide. The following micellar solutions were prepared in phosphate buffer: Triton X-100 (a condensate of polyoxyethylene with a mean chain length of 9.5 and p-1,1,3,3-tetramethylbutylphenol), sodium dodecyl sulphate (SDS; Merck), Genapol C-100 (a condensate of polyoxyethylene with a mean chain length of
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10 and the alcohol of coconut oil), Arkopal N-90, N-130, N-150 and N-300 (condensates of polyoxyethylene, n = 9,13,15 and 30, respectively, and nonylphenol; Hoechst ) and cetyltrimethylammonium bromide (CTAB; BDH ) . The water used was purified by a Millipore Milli-Q system. All the chemicals were used as received. Apparatus Measurements were made in a quartz cuvette (lo-mm path length) placed in the sample holder of a Perkin-Elmer 204 fluorescence spectrophotometer. The lamp of the spectrophotometer was disconnected from the power supply. The solution in the cuvette was mixed with a magnetic stirrer. Hydrogen peroxide solution was added to the sample in the cuvette by a Multi-Burette E485 (Metrohm) through a PTFE capillary equipped with a micro-valve. The capillary was shielded from ambient light. The chemiluminescence was measured over the wavelength interval 220-780 nm. The output was recorded on a BD 8 flat-bed recorder (Kipp and Zonen) . Procedure A solution of TCPO in acetonitrile (100 ~1) was pipetted into the cuvette and 30 ~1 of a solution of perylene in acetone were added, followed by 3 ml of a solution of surfactant in phosphate buffer or 3 ml of acetonitrile/buffer (80:20, v/v). The cuvette was placed in the fluorescence spectrophotometer and the cuvette compartment was closed. After the sample had been mixed by stirring for 15 s, hydrogen peroxide solution (20-150 ~1) was added to initiate the chemiluminescent reaction. The emitted light was detected directly and the output was recorded until the intensity had fallen to zero. The chemiluminescence intensity was measured as the peak-height signal in millimetres. RESULTS AND DISCUSSION
The TCPO chemiluminescent reaction was studied in different micellar systems and compared with the reaction in acetonitrile/buffer (80:20, v/v). This ratio was chosen because it gave the best compromise between TCPO solubility and reproducibility [ 171. Perylene, one of the most efficient sensitizers [ 181, was used in all the systems. The reaction between TCPO and hydrogen peroxide is very fast (the maximum signal is reached within 1 s). Therefore, it is important to add the hydrogen peroxide rapidly to the solution in the cuvette while the cuvette holder is closed. In order to optimize the conditions for the chemiluminescent reaction, the optimum sequence for the addition of the solutions to the cuvette in acetonitrile/buffer was first studied. The sequence influences the emission intensity because TCPO is hydrolysed in the aqueous solution to trichlorophenol
232
(TCP ), which can quench the chemiluminescence emission [ 191. Hence there are two possibilities: either TCPO or the aqueous solution should be added just before the addition of hydrogen peroxide. Addition of the aqueous solution just before hydrogen peroxide was chosen because this makes it possible at a later stage to add the hydrogen peroxide combined with the aqueous solution. Therefore, the sequence chosen was addition of TCPO in acetonitrile and the solution of perylene, followed by the acetonitrile/buffer mixture or micellar solution and finally the hydrogen peroxide solution to initiate the chemiluminescent reaction. The interval between the addition of the acetonitrile/buffer and the hydrogen peroxide must be kept as short as possible. An increase in the time interval from 15 to 60 s led to a 35% decrease in intensity. For the TCPO reaction in a micellar medium, the influence of micelles was investigated in relation to both the chemiluminescence intensity and the stability of TCPO. Chemiluminescent reaction in micellar systems The chemiluminescence intensity was measured in the following types of micelles: six non-ionic micelles (Genapol C-100, Triton X-100 and Arkopal N90, N-130, N-150 and N-300), one anionic micelle (SDS) and one cationic micelle (CTAB ) . Some of the surfactant solutions were used at two concentrations (50 and 5 mM) and others only at 50 mM. The concentration of the hydrogen peroxide solution added was 12 or 30%. It was found that the TCPO chemiluminescence did not occur in CTAB micelles, and that the intensity in SDS micelles was appreciably less than that in neutral micellar solutions (Fig. 2 ). The ionized head groups of the micelles of SDS and CTAB probably depress the formation of the intermediate 1,2dioxetanedione or the energy transfer from the intermediate to the fluorophore. As a result, the concentration of excited fluorophore is diminished and the chemiluminescence intensity is decreased. The intensity may also be quenched by the bromide ions of CTAB [ 201. The chemiluminescence intensity in Genapol C-100 is about 25% less than that in Arkopal N-150 and Triton X-100 (Fig. 2). A marked difference between Genapol C-100 and both Arkopal N-150 and Triton X-100 is the lack of a phenol group in Genapol C-100; this might explain the difference in chemiluminescence intensity, but the effect was not investigated further. As is shown in Fig. 2, the concentration of the surfactant does not have any influence on. the chemiluminescence intensity when the concentration of the surfactant is above the critical micellar concentration (c.m.c. ) . The 5 mM solutions of all the surfactants, except SDS, are above the c.m.c. of the surfactants [ 21,221. In the 5 mM solution of SDS, there is a perceptible decrease in intensity compared with the intensity in 50 mM SDS. For the other surfactants, the intensity is the same in the 5 and 50 mM surfactant solutions. Figure 2 also shows the chemiluminescence intensity in the presence of Ar-
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Fig. 2. TCPO chemiluminescence intensity in several micellar solutions and in the acetonitrile/ buffer (80:20, v/v): (solid boxes) 50 mM surfactant, 100 ~1 H,Oz (12%); (single hatched boxes) 5 mM surfactant, 100 ~1 H202 (12%); (open boxes) 50 mM surfactant, 50 ,nlH,O, (30%); (crosshatched box) acetonitrile/buffer (80:20), 50 ,nl of H202 (30% ). General conditions: 100 ~1 of TCPO in acetonitrile, 30 ,nl of perylene in acetone and 3 ml of surfactant in buffer or 3 ml of acetonitrile/buffer.
kopal micelles having polyoxyethylene chains of different length. It is evident that the longer the polyoxyethylene chain, the higher is the chemiluminescence intensity. The highest intensity in micelles is attained in Arkopal N-300, and is similar to the intensity in the acetonitrile/buffer. The structure of the hydrophobic centre of the micelles apparently has no effect on the intensity; in Triton X-100 and in Arkopal N-90 the intensities are similar, although Triton X-100 has a branched and Arkopal N-90 an unbranched hydrocarbon chain. A possible explanation of the increase in intensity with the longer polyoxyethylene chains could lie in the aggregation number of the micelles. The aggregation number decreases with increase in the number of oxyethylene groups (the aggregation number of Arkopal N-90 is 265 and that of Arkopal N-300 is 55) [ 211.The decrease in the aggregation number causes a decrease in the size of the micelle core and an increase in the number of micelles at a given surfactant concentration. Perylene, an aromatic, hydrophobic compound, is expected to be found in the hydrophobic core of the micelle. Polyoxyethylene glycol and aromatic hydrocarbons, however, are miscible. Thus perylene may dissolve to some extent
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in the hydrated polyethylene core and also in the inner hydrocarbon core [ 231. Perylene, in a micelle with a low aggregation number, may be present more in the polyethylene core than in the micelle core, because the micelle core is small. Thus the reaction of the product from TCPO with hydrogen peroxide (1,2dioxetanedione) and perylene will be more effective, because 1,2-dioxetanedione, a polar compound, will also be present in the polyethylene core. Therefore, the Arkopal N-300 micelles, which have a small core and long polyoxyethylene chains, induce the highest chemilummescence intensity. The influence of TCPO and hydrogen peroxide concentrations in this micellar solution was investigated. An increase in either concentration results in an increase in intensity (Figs. 3 and 4). The TlCPO concentration in the stock solution is in the range 0.33-1.67 mg ml-‘, resulting in final concentrations of 23-117 ,ug 1-l. A concentration exceeding 1.67 mg ml-’ is not possible in acetonitrile. The influence of hydrogen peroxide concentration was investigated by adding 20-150 ,~l of 30% hydrogen peroxide solution to the cuvette. Addi-
D
.
I+
0
1
cont. TWO
(mg/ml)
c,
1 min
Fig. 3. Effect of TCPO concentration (in the stock solution) on the chemiluminescence intensity. General conditions: 100 ,nl of TCPO in acetonitrile, 30 ~1 of perylene solution in acetone, 3 ml of 50 mM Arkopal N-300 solution in buffer and 50 ~1 of HzOz (30% ) . Fig. 4. Effect of amount of H,O, (30%) on the peak response for the chemiluminescence reaction. H,O, added (~1): (A) 20, (B) 70, (C) 100, (D) 150. Recorder range (mVf.s.d.): (A) 10, (B-D) 20. General conditions: 100 ,~l of TCPO in acetonitrile, 30 pl of perylene in acetone and 3 ml of 50 mM Arkopal N-300 solution in buffer.
235
tion of more than 70 ,~l of hydrogen peroxide does not increase the intensity further. An increasing concentration of hydrogen peroxide causes an increase in peak height and a decrease in peak width (Fig. 4). The surfactant concentration above the c.m.c., as mentioned above, had no influence on the peak height, but an increase in the surfactant concentration decreased the peak width, as did increases in the hydrogen peroxide concentration. This narrowing did not occur when the TCPO concentration was increased. The optimum parameters found, therefore, are a TCPO concentration of 1.67 mg ml-’ in the stock solution in acetonitrile, a surfactant with a long polyoxyethylene chain (Arkopal N-300) at a concentration of 50 mM in phosphate buffer (0.01 M, pH 7.0) and 100 ~1 of 30% hydrogen peroxide, in a total solution volume of 3230 ~1. When these optimum parameters had been established, the concentration of perylene was varied from 0.05 to 1.5 ,uM in both Arkopal N-300 and the mixed acetonitrile/phosphate buffer. The chemiluminescence reaction in Arkopal N-300 gave a linear intensity/concentration plot over three orders of magnitude up to ca. 1.0 ,uM perylene (rz0.994, n=9), whereas in acetonitrile/buffer the plot was linear only up to ca. 0.5 PM (r=O.999, n=5). The limit of detection calculated for a signal-to-noise ratio of 2 was in the low nM range. The most important finding, however, is that the intensity in Arkopal N-300 is similar to that in the acetonitrile/phosphate buffer. When 0.01 M Tris buffer (pH 7.0) was used instead of the 0.01 M phosphate buffer (pH 7.0) the same results were obtained. Stability of TCPO in micellar systems To establish the stability of TCPO in micelles, the TCPO solution was added at time zero to a surfactant solution, and the perylene solution and the hydrogen peroxide solution were added at set intervals. The stability of TCPO is expressed as the half-life of the hydrolysis of TCPO. The hydrolysis was monitored by measurement of the chemiluminescence intensity. In Arkopal N-300,
Fig. 5. Stability of TCPO: (0 ) in acetonitrile/buffer (80:20, v/v); ( A ) in Arkopal N-300 in buffer (50 mM); (0 ) in Arkopal N-90 in buffer (50 mM).
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TCPO hydrolysis was very rapid, with a very short half-life (2 min, Fig. 5). The stability of TCPO in micelles with a shorter polyoxyethylene chain (Arkopal N-90) was greater, with a half-life of 3 min (Fig. 5). The lower stability in Arkopal N-300 depends on the hydration number (i.e., the number of water molecules per polyoxyethylene chain) of the micelle. The hydration number increases strongly with increasing number of oxyethylene groups [ 241. The higher the hydration number, the faster is the hydrolysis of TCPO. However, the half-life of the reaction in Arkopal is of a similar order of magnitude to that in acetonitrile/buffer, which is about 5 min (Fig. 5). Conclusions The intensity of the TCPO chemiluminescence reaction in micelles is similar to the intensity of the reaction in the acetonitrile/phosphate buffer. The problem of TCPO solubility can be solved by using a micellar system. Therefore, it might be possible to use micellar chromatography with chemiluminescence detection based on this system. This is under investigation. The stability of TCPO is not increased in the micellar systems compared with the acetonitrile/buffer. Because of the poor stability of TCPO, it would not be possible to add TCPO to the mobile phase pre-column, but TCPO dissolved in acetonitrile can be added post-column to the micellar effluent. The authors are grateful to Hoechst for supplying the surfactants. REFERENCES
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M.M. Rauhut, Act. Chem. Res., 2 (1969) 80. K. Kobayashi and K. Imai, Anal. Chem., 52 (1980) 424. G. Mellbin, J. Liq. Chromatogr., 6 (1983) 1603. G.J. de Jong, N. Lammers, F.J. Spruit, U.A.Th. Brinkman and R.W. Frei, Chromatographia, 18 (1984) 129. S. Kobayashi, J. Sekino, K. Honda and K. Imai, Anal. Biochem., 112 (1981) 99. G. Mellbin and B.E.F. Smith, J. Chromatogr., 312 (1984) 203. K.W. Sigvardson and J.W. Birks, Anal. Chem., 55 (1983) 432. M.L. Grayeski and A.J. Weber, Anal. Lett., 17 (1984) 1539. K.L. Mittal, Micellization, Solubilization, and Microemulsions, Plenum, New York, 1977. W.L. Hinze, in K.L. Mittal (Ed.), Solution Chemistry of Surfactants, Vol. 1, Plenum, New York, 1979, p. 79. W.L. Hinze, T.E. Riehl, H.N. Singh and Y. Baba, Anal. Chem., 56 (1984) 2180. J. Lasovsky and F. Grambal, Bioelectron. Bioenerg., 15 (1986) 95. M. Yamada and S. Suzuki, Anal. Lett., 17 (1984) 251. M. Yamada and S. Suzuki, Anal. Chim. Acta, 193 (1987) 337. C.M. Paleos, G. Vassilopoulos and J. Nikokavouras, J. Photochem., 18 (1982) 327. A.G. Mohan and M.J. Turro, J. Chem. Educ., 51 (1974) 528. P. van Zoonen, D.A. Kamminga, C. Gooijer, N.H. Velthorst and R.W. Frei, Anal. Chim. Acta, 167 (1985) 249. K. Honda, K. Miyaguchi and K. Imai, Anal. Chim. Acta, 177 (1985) 111. K. Honda, J. Sekino and K. Imai, Anal. Chem., 55 (1983) 940.
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P. van Zoonen, H. Bock, C. Gooijer, N.H. Velthorst and R.W. Frei, Anal. Chim. Acta, 200 (1987) 131. P. Becher, in M.J. Schick and F.M. Fowkes (Eds.), Surfactant Science Series, Vol. 1, Nonionic Surfactants, M. Dekker, New York, 1967, p. 478. E.J. Fendler and J.H. Fendler, Adv. Phys. Org. Chem., 8 (1970) 271. K. Shinoda, in M.J. Schick and F.M. Fowkes (Eds.), Surfactant Science Series, Vol. 2, Solvent Properties of Surfactant Solutions, M. Dekker, New York, 1967, p. 27. B. Lindman and H. WennerstrSm, in F.L. Boschke (Ed.), Topics in Current Chemistry, Vol. 87, Springer, Berlin, 1980, p. 54.
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Printed in The Netherlands
CHEMILUMINESCENCE OF BIS(2,4,6-TRICHLOROPHENYL) OXALATE IN AQUEOUS MICELLAR SYSTEMS
O.M. STEIJGER*, H.M. VAN MASTBERGEN and J.J.M. HOLTHUIS Department of Pharmaceutical Analysis, University of Utrecht, Catharijnesingel60,3511 Utrecht (The Netherlands)
GH
(Received 1lth May 1988)
SUMMARY The chemiluminescent reaction of bis(2,4,6-trichlorophenyl) oxalate (TCPO) in aqueous micellar systems is compared with the reaction in a mixture of acetonitrile and aqueous phosphate buffer. The chemiluminescence was studied in batch experiments with perylene as the fluorophore. The oxidation of TCPO produced the same intensity of chemiluminescence in the buffered acetonitrile as in Arkopal N-300 micelles, the best micellar system. The solubility of TCPO in an aqueous micellar system is greater than that in the acetonitrile/aqueous buffer (80:20, v/v), but TCPO is less stable in the former system.
Bis (2,4,6+ichlorophenyl) oxalate (TCPO ) is frequently used in chemiluminescence reactions. In the presence of hydrogen peroxide or another oxidant and a suitable fluorophore, TCPO chemiluminescence is one of the most efficient reactions [ 11. Figure 1 shows the generally proposed mechanism of the reaction. Chemiluminescence detection in reversed-phase liquid chromatography is
-
F’
-
F*+
2C02
F+LffiHT
Fig. 1. Generally proposed mechanism for TCPO chemiluminescence (F, fluorophore).
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0 1989 Elsevier Science Publishers B.V.
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often based on TCPO. Dansylated amino acids [ 2,3], dansylated steroids [ 41, fluorescamine-labelled catecholamines [ 51, o-phthalaldehyde- and nitrobenzofurazan-labelled amines [ 61 and polynuclear aromatic hydrocarbons [ 7,8 ] can be determined at lower concentrations with chemiluminescence detection than with fluorescence detection. A disadvantage of chemiluminescence detection involving TCPO is the poor solubility and stability of TCPO in aqueous systems. Because of its poor stability, the TCPO solution must be delivered to the eluent stream via a separate flow line. The poor solubility of TCPO means that a large amount of organic solvent is required to dissolve sufficient TCPO. A mixing problem arises because the chemiluminescence reagents in organic solvents must be mixed postcolumn with the partly aqueous column effluent. Therefore, it is important to choose the right type of buffer because it must be soluble in mixtures of water and organic solvents [ 2-71. The study described here was designed to investigate the usefulness and influence of micellar systems in the TCPO chemiluminescence reaction. Micelles can affect the solubility and the physical and chemical properties of various compounds [ 91, and have been used successfully in several analytical techniques [lo]. However, there have been only a few investigations of chemiluminescence reactions in micellar media [ 11-151. Chemiluminescence assays in aqueous solutions and based on TCPO and the use of micelles have not been described before. Here, the intensity of the chemiluminescence and the stability of TCPO in several micellar systems are compared with these parameters in an acetonitrile/water buffer solution (80:20, v/v) with perylene as the fluorophore. EXPERIMENTAL
Chemicals and solutions Bis (2,4,6-trichlorophenyl ) oxalate was prepared by the method developed by Mohan and Turro [ 161, and was stored under nitrogen at - 18” C. Solutions of TCPO in acetonitrile (Merck; analytical-reagent grade) were freshly prepared every day, as were the solutions of perylene (Janssen Chimica, Beerse, Belgium) in acetone (Baker, analyzed grade). Hydrogen peroxide (30%, w/v) was obtained from O.P.G. (Utrecht, The Netherlands). The buffers used were 0.01 M sodium phosphate buffer, pH 7.0 (NaH,PO,*2H,O) (Brocacef, Maarssen, The Netherlands) and 0.01 M Tris buffer, pH 7.0 [ tris (hydroxymethyl) aminomethane; Merck; analytical-reagent grade]. The pH of the buffers was adjusted with sodium hydroxide. The following micellar solutions were prepared in phosphate buffer: Triton X-100 (a condensate of polyoxyethylene with a mean chain length of 9.5 and p-1,1,3,3-tetramethylbutylphenol), sodium dodecyl sulphate (SDS; Merck), Genapol C-100 (a condensate of polyoxyethylene with a mean chain length of
231
10 and the alcohol of coconut oil), Arkopal N-90, N-130, N-150 and N-300 (condensates of polyoxyethylene, n = 9,13,15 and 30, respectively, and nonylphenol; Hoechst ) and cetyltrimethylammonium bromide (CTAB; BDH ) . The water used was purified by a Millipore Milli-Q system. All the chemicals were used as received. Apparatus Measurements were made in a quartz cuvette (lo-mm path length) placed in the sample holder of a Perkin-Elmer 204 fluorescence spectrophotometer. The lamp of the spectrophotometer was disconnected from the power supply. The solution in the cuvette was mixed with a magnetic stirrer. Hydrogen peroxide solution was added to the sample in the cuvette by a Multi-Burette E485 (Metrohm) through a PTFE capillary equipped with a micro-valve. The capillary was shielded from ambient light. The chemiluminescence was measured over the wavelength interval 220-780 nm. The output was recorded on a BD 8 flat-bed recorder (Kipp and Zonen) . Procedure A solution of TCPO in acetonitrile (100 ~1) was pipetted into the cuvette and 30 ~1 of a solution of perylene in acetone were added, followed by 3 ml of a solution of surfactant in phosphate buffer or 3 ml of acetonitrile/buffer (80:20, v/v). The cuvette was placed in the fluorescence spectrophotometer and the cuvette compartment was closed. After the sample had been mixed by stirring for 15 s, hydrogen peroxide solution (20-150 ~1) was added to initiate the chemiluminescent reaction. The emitted light was detected directly and the output was recorded until the intensity had fallen to zero. The chemiluminescence intensity was measured as the peak-height signal in millimetres. RESULTS AND DISCUSSION
The TCPO chemiluminescent reaction was studied in different micellar systems and compared with the reaction in acetonitrile/buffer (80:20, v/v). This ratio was chosen because it gave the best compromise between TCPO solubility and reproducibility [ 171. Perylene, one of the most efficient sensitizers [ 181, was used in all the systems. The reaction between TCPO and hydrogen peroxide is very fast (the maximum signal is reached within 1 s). Therefore, it is important to add the hydrogen peroxide rapidly to the solution in the cuvette while the cuvette holder is closed. In order to optimize the conditions for the chemiluminescent reaction, the optimum sequence for the addition of the solutions to the cuvette in acetonitrile/buffer was first studied. The sequence influences the emission intensity because TCPO is hydrolysed in the aqueous solution to trichlorophenol
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(TCP ), which can quench the chemiluminescence emission [ 191. Hence there are two possibilities: either TCPO or the aqueous solution should be added just before the addition of hydrogen peroxide. Addition of the aqueous solution just before hydrogen peroxide was chosen because this makes it possible at a later stage to add the hydrogen peroxide combined with the aqueous solution. Therefore, the sequence chosen was addition of TCPO in acetonitrile and the solution of perylene, followed by the acetonitrile/buffer mixture or micellar solution and finally the hydrogen peroxide solution to initiate the chemiluminescent reaction. The interval between the addition of the acetonitrile/buffer and the hydrogen peroxide must be kept as short as possible. An increase in the time interval from 15 to 60 s led to a 35% decrease in intensity. For the TCPO reaction in a micellar medium, the influence of micelles was investigated in relation to both the chemiluminescence intensity and the stability of TCPO. Chemiluminescent reaction in micellar systems The chemiluminescence intensity was measured in the following types of micelles: six non-ionic micelles (Genapol C-100, Triton X-100 and Arkopal N90, N-130, N-150 and N-300), one anionic micelle (SDS) and one cationic micelle (CTAB ) . Some of the surfactant solutions were used at two concentrations (50 and 5 mM) and others only at 50 mM. The concentration of the hydrogen peroxide solution added was 12 or 30%. It was found that the TCPO chemiluminescence did not occur in CTAB micelles, and that the intensity in SDS micelles was appreciably less than that in neutral micellar solutions (Fig. 2 ). The ionized head groups of the micelles of SDS and CTAB probably depress the formation of the intermediate 1,2dioxetanedione or the energy transfer from the intermediate to the fluorophore. As a result, the concentration of excited fluorophore is diminished and the chemiluminescence intensity is decreased. The intensity may also be quenched by the bromide ions of CTAB [ 201. The chemiluminescence intensity in Genapol C-100 is about 25% less than that in Arkopal N-150 and Triton X-100 (Fig. 2). A marked difference between Genapol C-100 and both Arkopal N-150 and Triton X-100 is the lack of a phenol group in Genapol C-100; this might explain the difference in chemiluminescence intensity, but the effect was not investigated further. As is shown in Fig. 2, the concentration of the surfactant does not have any influence on. the chemiluminescence intensity when the concentration of the surfactant is above the critical micellar concentration (c.m.c. ) . The 5 mM solutions of all the surfactants, except SDS, are above the c.m.c. of the surfactants [ 21,221. In the 5 mM solution of SDS, there is a perceptible decrease in intensity compared with the intensity in 50 mM SDS. For the other surfactants, the intensity is the same in the 5 and 50 mM surfactant solutions. Figure 2 also shows the chemiluminescence intensity in the presence of Ar-
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Fig. 2. TCPO chemiluminescence intensity in several micellar solutions and in the acetonitrile/ buffer (80:20, v/v): (solid boxes) 50 mM surfactant, 100 ~1 H,Oz (12%); (single hatched boxes) 5 mM surfactant, 100 ~1 H202 (12%); (open boxes) 50 mM surfactant, 50 ,nlH,O, (30%); (crosshatched box) acetonitrile/buffer (80:20), 50 ,nl of H202 (30% ). General conditions: 100 ~1 of TCPO in acetonitrile, 30 ,nl of perylene in acetone and 3 ml of surfactant in buffer or 3 ml of acetonitrile/buffer.
kopal micelles having polyoxyethylene chains of different length. It is evident that the longer the polyoxyethylene chain, the higher is the chemiluminescence intensity. The highest intensity in micelles is attained in Arkopal N-300, and is similar to the intensity in the acetonitrile/buffer. The structure of the hydrophobic centre of the micelles apparently has no effect on the intensity; in Triton X-100 and in Arkopal N-90 the intensities are similar, although Triton X-100 has a branched and Arkopal N-90 an unbranched hydrocarbon chain. A possible explanation of the increase in intensity with the longer polyoxyethylene chains could lie in the aggregation number of the micelles. The aggregation number decreases with increase in the number of oxyethylene groups (the aggregation number of Arkopal N-90 is 265 and that of Arkopal N-300 is 55) [ 211.The decrease in the aggregation number causes a decrease in the size of the micelle core and an increase in the number of micelles at a given surfactant concentration. Perylene, an aromatic, hydrophobic compound, is expected to be found in the hydrophobic core of the micelle. Polyoxyethylene glycol and aromatic hydrocarbons, however, are miscible. Thus perylene may dissolve to some extent
234
in the hydrated polyethylene core and also in the inner hydrocarbon core [ 231. Perylene, in a micelle with a low aggregation number, may be present more in the polyethylene core than in the micelle core, because the micelle core is small. Thus the reaction of the product from TCPO with hydrogen peroxide (1,2dioxetanedione) and perylene will be more effective, because 1,2-dioxetanedione, a polar compound, will also be present in the polyethylene core. Therefore, the Arkopal N-300 micelles, which have a small core and long polyoxyethylene chains, induce the highest chemilummescence intensity. The influence of TCPO and hydrogen peroxide concentrations in this micellar solution was investigated. An increase in either concentration results in an increase in intensity (Figs. 3 and 4). The TlCPO concentration in the stock solution is in the range 0.33-1.67 mg ml-‘, resulting in final concentrations of 23-117 ,ug 1-l. A concentration exceeding 1.67 mg ml-’ is not possible in acetonitrile. The influence of hydrogen peroxide concentration was investigated by adding 20-150 ,~l of 30% hydrogen peroxide solution to the cuvette. Addi-
D
.
I+
0
1
cont. TWO
(mg/ml)
c,
1 min
Fig. 3. Effect of TCPO concentration (in the stock solution) on the chemiluminescence intensity. General conditions: 100 ,nl of TCPO in acetonitrile, 30 ~1 of perylene solution in acetone, 3 ml of 50 mM Arkopal N-300 solution in buffer and 50 ~1 of HzOz (30% ) . Fig. 4. Effect of amount of H,O, (30%) on the peak response for the chemiluminescence reaction. H,O, added (~1): (A) 20, (B) 70, (C) 100, (D) 150. Recorder range (mVf.s.d.): (A) 10, (B-D) 20. General conditions: 100 ,~l of TCPO in acetonitrile, 30 pl of perylene in acetone and 3 ml of 50 mM Arkopal N-300 solution in buffer.
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tion of more than 70 ,~l of hydrogen peroxide does not increase the intensity further. An increasing concentration of hydrogen peroxide causes an increase in peak height and a decrease in peak width (Fig. 4). The surfactant concentration above the c.m.c., as mentioned above, had no influence on the peak height, but an increase in the surfactant concentration decreased the peak width, as did increases in the hydrogen peroxide concentration. This narrowing did not occur when the TCPO concentration was increased. The optimum parameters found, therefore, are a TCPO concentration of 1.67 mg ml-’ in the stock solution in acetonitrile, a surfactant with a long polyoxyethylene chain (Arkopal N-300) at a concentration of 50 mM in phosphate buffer (0.01 M, pH 7.0) and 100 ~1 of 30% hydrogen peroxide, in a total solution volume of 3230 ~1. When these optimum parameters had been established, the concentration of perylene was varied from 0.05 to 1.5 ,uM in both Arkopal N-300 and the mixed acetonitrile/phosphate buffer. The chemiluminescence reaction in Arkopal N-300 gave a linear intensity/concentration plot over three orders of magnitude up to ca. 1.0 ,uM perylene (rz0.994, n=9), whereas in acetonitrile/buffer the plot was linear only up to ca. 0.5 PM (r=O.999, n=5). The limit of detection calculated for a signal-to-noise ratio of 2 was in the low nM range. The most important finding, however, is that the intensity in Arkopal N-300 is similar to that in the acetonitrile/phosphate buffer. When 0.01 M Tris buffer (pH 7.0) was used instead of the 0.01 M phosphate buffer (pH 7.0) the same results were obtained. Stability of TCPO in micellar systems To establish the stability of TCPO in micelles, the TCPO solution was added at time zero to a surfactant solution, and the perylene solution and the hydrogen peroxide solution were added at set intervals. The stability of TCPO is expressed as the half-life of the hydrolysis of TCPO. The hydrolysis was monitored by measurement of the chemiluminescence intensity. In Arkopal N-300,
Fig. 5. Stability of TCPO: (0 ) in acetonitrile/buffer (80:20, v/v); ( A ) in Arkopal N-300 in buffer (50 mM); (0 ) in Arkopal N-90 in buffer (50 mM).
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TCPO hydrolysis was very rapid, with a very short half-life (2 min, Fig. 5). The stability of TCPO in micelles with a shorter polyoxyethylene chain (Arkopal N-90) was greater, with a half-life of 3 min (Fig. 5). The lower stability in Arkopal N-300 depends on the hydration number (i.e., the number of water molecules per polyoxyethylene chain) of the micelle. The hydration number increases strongly with increasing number of oxyethylene groups [ 241. The higher the hydration number, the faster is the hydrolysis of TCPO. However, the half-life of the reaction in Arkopal is of a similar order of magnitude to that in acetonitrile/buffer, which is about 5 min (Fig. 5). Conclusions The intensity of the TCPO chemiluminescence reaction in micelles is similar to the intensity of the reaction in the acetonitrile/phosphate buffer. The problem of TCPO solubility can be solved by using a micellar system. Therefore, it might be possible to use micellar chromatography with chemiluminescence detection based on this system. This is under investigation. The stability of TCPO is not increased in the micellar systems compared with the acetonitrile/buffer. Because of the poor stability of TCPO, it would not be possible to add TCPO to the mobile phase pre-column, but TCPO dissolved in acetonitrile can be added post-column to the micellar effluent. The authors are grateful to Hoechst for supplying the surfactants. REFERENCES
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