Langmuir 1994,10, 441-453
447
Effects of Reversed Micelles on Peroxyoxalate Chemiluminescence and Analytical Implications for Determination of Fluorophors Niya Dan and Mary Lynn Grayeski* Department of Chemistry, Seton Hall University, South Orange, New Jersey 07079 Received August 9,1993. In Final Form: November 1 , 1 9 9 P The effects of reversed micelles on the bis(2,4,6-trichlorophenyl)oxalate (TCP0)-hydrogen peroxide chemiluminescence reaction was evaluated in two micellar systems: (1)hexadecyltrimethylammonium chloride (CTAC)and (2) sodium bis(2-ethylhexyl)sulfosuccinate (AOT),each formed in 1:2 (v/v) 1-butanol/ cyclohexane and 1:l (v/v) chloroform/cyclohexane solvent mixtures with varying amounts of water. Chemiluminescence intensity was increased 6 to 11times in CTAC and 4.4 to 55 times in AOT reversed micelles. The chemiluminescenceefficiency enhancement was caused mainly by the increase of chemical excitation efficiency. The experimental variables that influence the magnitude of the chemiluminescence enhancements were studied. The analytical figures of merit for this chemiluminescencereaction system have been evaluated for the determination of fluorophors. Using a flow injection system, the detection limits for 8-anilino-1-naphthalenesulfonicacid (ANS) were improved 20 and 7 times in CTAC and AOT reversed micellar medium, respectively. The detection limits for Rhodamine B (Rh B) were improved 5 and 50 times in the same reversed micellar medium. The detection linear range and precision were also improved.
Introduction Peroxyoxalate chemiluminescence detection has been demonstrated to be a sensitive technique for the determination of a variety of analytes including hydrogen peroxide, glucose, fluorophors, and fluorophor-labeled compounds.14 The possible mechanism for peroxyoxalate CL (Scheme 1)involves the oxidation of oxalic esters with hydrogen peroxide and energy transfer to the fluorophor by the formation of one or more intermediate-fluorophor complexes has been studied. Dioxetanedione was first suggested by Rauhut as a reaction intermediate but was never directly observed.9 Other reaction intermediate(s1 such as a substituted dioxetanone and peroxyoxalate were also proposed.'('-16 However, the development of assays using this reaction is restricted because most of the efficient oxalic derivatives have limited solubility in aqueous solvents typically used for analytical applications and even
* Current address: Research Corp., 101 N. W h e t Rd., Suit 250, Tucson, A2 85711-3332. 0 Abstract published in Advance ACS Abstracts, January 15,1994. (1) Tseng, S. S.; Mohan, A. G.; Haines, L. G.; Vizcarra, L. S.;Rauhut, M. M. J. Org. Chem. 1979,44,4113. (2) Seitx, W. R.Meth& in Enzymology;Deluca, M. A,,Eds.;Academic Press: New York, 1978; Vol. 57, p 445. (3) Scott, G.; Seitz, W. R.; Ambrose, J. Anal. Chim. Acta 1980,115, 221. (4) Miyaguchi,K.; Honda, K.; Imai, K. J. Chromatogr. 1984,316,501. (5) Kobayashi, K.;Sekino, J.; Honda, K. Anal. Biochem. 1981,112,99. (6) Sigvardson, K. W.; Birks, J. W. Anal. Chem. 1983,55,432. (7) Sigvardson, K. W.; Kennish, J. M.; Birks, J. W. Anal. Chem. 1984, 56, 1096. (8) Grayeski, M. L.; Seitz, W. R. Anal. Biochem. 1984, 136, 277. (9) Rauhut, M. M.; Bollyky, L. J.; Roberta,B. G.; Loy, M.; Whitman, R. H.; Iannotta, A. V.; Semsel, A. M.; Clarke, R. A. J. Am. Chem. SOC. 1967,89,6515. (10) McCapre, F.; Perring, K.; Hard, R. J.; Hann, R. A. Tetrahedron Lett. 1981, 5087. (11) Givens, R. S.; Schowen, R. L. Chemiluminescence and Photochemical Reaction Detection in Chromatography, Birks, J. W., Eds.; VCH: New York, 1989; Chapter 5, p 125. (12) Schuater, G. B.; Schmidt, S.P. Adu. Phys. Org. Chem. 1982,18, __
187. ..
(13) Orlovic,M.;Schowen,R. L.;Givens,R. S.;Alvarez,F.;Matuszewski, B.; Parekh, N. J. Org. Chem. 1989,54,3606. (14) Catherall, C. L. R.; Palmer, T. F.; Cundall, R. B. J. Chem. SOC., Faraday Trona. 2 1984,80,823. (15) Alvarez, F.; Parekh, N.; Matuszewski, B.; Givens, R. S.;Higuchi, T.; Schowen, R. L. J. Am. Chem. SOC.1986,108,6435.
Scheme 1. Proposed Mechanism for Peroxyoxalate Chemiluminescencea H24 + A Q C C O ~ A I
I
+ FL
B'
n' a
I intermediate
[ I"
='+I
FL. + hv
FL is the fluorescent acceptor and B- is a base catalyst.
the most efficient esters have much lower efficiencies than bioluminescent systems. Some studies have been done to overcome the difficulties associated with the analytical applications of peroxyoxalate chemiluminescence. In our laboratory, cyclodextrins and normal micelles have been used to enhance the aqueous peroxyoxalate c h e m i l u m i n e s ~ e n c e . ~ ~ChemiluminJ~ escence intensity was increased by as much as 130-fold and analytical figures of merit were also improved. Microemulsion systems were also evaluated as a medium for the nonaqueous peroxyoxalate chemiluminescence.'8 Here we report the study of the effects of reversed micelles on the bis(2,4,6-trichlorophenyl)oxalate (TCP0)-hydrogen peroxide chemiluminescence reaction. Reversed micelles are formed by dissolving surfactants in apolar solvents. Because of the unusual properties of the three phases, the water pool, the interface of amphiphilic molecules, and the organic phase, these systems have been used as a medium for chemiluminescence assays. (16) Woolf, E. J.; Grayeski, M. L. J. Lumin. 1987,39, 19. (17) Grayeski, M. L.; Liu, M.; Dan, N. Anal. Chem. 1991, 63, 1766. (18) Thompson, R. B.; McBee, S. E. S.Langmuir 1988,4,106.
0143-7463/94/2410-0447$04.50/00 1994 American Chemical Society
448 Langmuir, Vol. 10,No. 2, 1994
Dan and Grayeski
cyclohexaneto dissolve hydrogen peroxide. TCPO solution was For example, cationic hexadecyltrimethylammonium chlorprepared by dissolving 18 mg of dried TCPO in 10mL of solvent ide (CTAC) reversed micelleshave been used as a medium mixture. Triethylamine was diluted to 2.0 mM in a solvent to improve the luminol chemiluminescent assays for ~ hydrogen peroxide, glucose, or glucose oxidasea ~ t i v i t y . ~ ~ *mixture. In the measurementof chemiluminescencespectra,fluorophors Anionic sodium bis( 2-ethylhexyl) sulfoguecinate (AOT) were dissohed in methanol (1.0 M) and then diluted to 9.0 mM reversed micelles have been studied in detail by the changes in solvent mixtures. CTAC and AOT surfactants were dissolved of fluorescence quantum yield of 8-anilino-1-naphthalene- in the mixture of 1:2 (v/v) 1-butanol/cyclohexane, with concensulfonic acid (ANS).21*22In our study, both cationic CTAC trations of 4.0 mM and 0.06 M, respectively. The R ratio was 10 and anionic AOT reversed micelles were evaluated. The for both reversed micelles. TCPO solution was prepared by dissolving 95 mg of dried TCPO in 15 mL of ethyl acetate (to effects of experimental variables (i.e., Surfactant concenincrease the solubility of TCPO). Hydrogen peroxide was tration, ratio of water to surfactant, viscosity, catalyst dissolved to 0.8 M in the solvent mixture of 1:2 (v/v) 1-butanol/ concentration, hydrogen peroxide concentration, surfaccyclohexane by adding 30% of 2-propanol. Triethylamine was tant chain length, and surfactant counterions) upon the diluted to 2.5 mM in a solvent mixture. chemiluminescence intensity were studied. In the measurement of fluorescence spectra, fluorophors were dissolvedin distilled water (0.01 M) and then diluted to 6 X 1od Experimental Section M in reversed micellar solutions and solvent mixtures. The concentrations of surfactants were the same as in the study of Apparatus. All CL measurements were made on a Turner chemiluminescence spectra Designs (Mountain View, CA) Model TD20-e photometer In the measurement of UV spectra, fluorophors were dissolved equipped with a manual injector. An IBM-XT computer or an in methanol (1.0 M) and then diluted to 9.0 mM in reversed AT&TPC6300computer with ASYSTANT+ software equipped micellar solutions and solvent mixtures. The concentrations of with a Data Translation DT2808 acquisition board was used to surfactants were the same as in the study of fluorescencespectra. collect all CL intensity-time profiles. A Vortex-Genie K-550-G In the study of determination of fluorophors using a flow mixer was used for agitation of the reversed micellar solutions. injection analysis system, reversed micellar solutions were The absorption spectra were carried out with a Varian 2200 prepared by dissolving CTACand AOT in the 1:2 (v/v) 1-butanol/ spectrophotometeror a Perkin-Elmer UV-Vis spectrophotometer, cyclohexane bulk solvent mixture. The concentrations of CTAC Model lamda-4c. The fluorescence and chemiluminescence and AOT were 4.0 mM and 0.06 M; R ratios were 10 and 5, spectra were recorded on a Perkin-Elmer Model MPF-66 respectively. The concentration of TCPO was 0.8 mM. The spectrofluorometer. solution mixture of 9.7 mM hydrogen peroxide and 0.3 mM The water content was determined by the Karl Fisher titration triethylamine was prepared by mixing 307% hydrogen peroxide using a Brinkmann 658 KF Processor equipped with a Metrohm with 1M triethylamine in the same bulk solvent as in the above 655 Dosimat. reversed micelles. ANSand Rh B sample solutionswere prepared The viscosity was measured by a Brookfield DigitalViscometer by diluting 0.01 M aqueous solution in the above bulk solvent. Model DV-11. In the study of condition optimizations, concentrations of ANS In the flow injection analysis (FIA) system two ISCO Model and Rh B were 3 X 10-6 and 6 X lod M, respectively. LC-2600 syringe pumps and an Applied Biosystems Spectroflow Procedures. ChemiluminescenceExperiments. The general Model 400 pump were used to deliver solutions. A Rheodyne CL analysis procedure consists of using Gilson microliter pipets injector with a 10-pLloop was used for introduction of samples. to add 25 pL of hydrogen peroxide solution, 25 p L of fluorophor An Applied Biosystems Spectroflow 980 programmable fluorsolution, 150 pL of triethylamine solution, and 500 pL of bulk escence detector with the lamp turned off and a 25-pL flow cell solvent or surfactant solution into a polypropylene (8 X 40 mm, was used for detection. A Spectra-Physics integrator Model 1.6mL, Evergreen Scientific)disposable culture tube, which was SP4290 was used to record data. subsequently agitated by a Vortex mixer for 10sand then placed &agents and Solutions. Hexadecyltrimethylammonium into the cuvette holder of the luminometer. Then 50pL of TCPO chloride (CTAC)(Eastman Kodak Co.), sodiumbis(2-ethylhexyl) solution was injected manually and output was read from the sulfosuccinate(AOT)(Fisher),stearyldimethylbenzylammonium Turner and collected by a computer. A delay time of 0 s and run chloride (SBAC) (K&K Laboratories, Inc.), dodecyltrimethyltime of 1.5 or 2 min was used. The CL signal was collectad as ammonium bromide (DTAB) (Sigma), dodecyltrimethylthe integrated CL intensity and the peak CL intensity. ammonium chloride (DTAC) (Eastman Kodak), cetyltrimethRelative Fluorescence Efficiency. Fluorescence spectra were ylammonium bromide (CTAB) (Aldrich), tetradecylbenzylobtained by excitinga mixture of 13pL of ANS or Rh B solution dimethylammonium chloride (TBAC) (Chemical Dynamics and 20 mL of surfactant solution or bulk solvent at 365 or 320 Corp.), triethylamine (Aldrich),8-anilino-1-naphthalenesulfonic nm. The emission spectra were scanned from 380 to 600 nm for acid (ANS) (Sigma), Rhodamine B (Rh B) (Aldrich), 30 wt % ANS and 530 to 680 nm for Rh B. The slits and scan rates of hydrogen peroxide (Aldrich),polyethylene glycol (M. W. 35 OOO) the fluorometer were 5 nm and 120 nm/min. (Fluka), and bis(2,4,6-trichlorophenyl)oxalate (TCPO) (given Absorbances of ANS or Rh B in surfactant solution and bulk by Dr. Mohan) were obtained. HPLC grade cyclohexane solvent were measured at 365 or 320 nm. The UV absorption (Aldrich),chloroform (Aldrich),1-butanol (Aldrich),and 2-prospectra were scanned from 220 to 380 nm for ANS and 250 to 600 pan01 (Aldrich) were used. All reagents were used as received. nm for Rh B. Reversed micellar solutions were prepared by dissolving an Chemiluminescence Spectra. Chemiluminescence spectra appropriate amount of surfactant in the solvent mixtures (1:l were measured on a fluorometer with the lamp off. The slits and (v/v) chloroform/cyclohexaneand 1:2 (v/v) 1-butanol/cyclohexscan rates of the fluorometer were 20 nm and 240 nm/min. The ane). Then a small amount of distilled water was added to the procedure involved adding 500 pL of hydrogen peroxide solution, solution to obtain a constant R ratio ([HzO]/[surfactant]). The 60 pL of fluorophor solution, 50 pL of triethylamine solution, final water content was determined by the Karl Fisher titration. and 1500 pL of bulk solvent or surfactant solution, then the In the study of the effects of surfactant concentration, reaction was initiated by pipetting 500 pL of TCPOsolution into fluorophorswere dissolvedin distilledwater (0.01M),then diluted a quartz fluorescence cell, and the spectrum was measured. to 6 X 10-6M in solventmixtures. Hydrogenperoxide was diluted Analytical Application. The flow injection analysis system to 0.08 M in the solvent mixture of 1:2 (v/v) 1-butanol/ is shown schematically in Figure 1. A syringepump waa used for cyclohexane. 2-Propanol (5% )wasaddedto 1:l(v/v)chloroform/ deliveringa solution mixture of hydrogen peroxide and triethylamine. TCPO solution was delivered by another syringe pump. (19) Hoshino, H.; Hinze, W. L. Anal. Chem. 1987,59,496. A peristaltic pump was used to deliver reversed micellarsolution (20) Igarash, S.; Hinze, W. L. Anal. Chem. 1988, 60, 446. or bulk solvent. Fluorophor sample solutions were injected by (21) Wong, M.; Gratzel, M.; Thomas, J. K. J. Am. Chem. SOC.1976, a Rheodyne injector into the reversed micellar solution or bulk 98,2391. solvent. These three solutions were mixed through two miring (22) Wong, M.;Thomas, J. K.; Now& T. J.Am. Chem. SOC.1977,99, tees. The distances between mixing tee M1 and M2 was kept as 4730.
Langmuir, Vol. 10, No. 2, 1994 449
Effects of Reversed Micelles on Chemiluminescence
I
Waste
H202 +TEA
TCX"J
MisoCtfsn wbuksoUm
Figure 1. Chemiluminescence FIA system.
3
-* IC n
C
smallas possible (lessthan 2 cm). The distance between M2 and inlet of detector was adjusted to optimize response. The flow rates of pumps were also optimized versus response.
Results and Discussion The effects of micelles on chemiluminescenceefficiency and rate are studied by monitoring the intensity-time curve and the observed pseudo-first-order reaction rate constant, k. Comparison of integrated area under the intensitytime curves is indicative of relative chemiluminescence efficiency, &I, which is the product of the excitation efficiency of the reaction, #ex, and fluorescence efficiency of the emitting species, &. Intensity-Time Profiles. Typical CL intensity-time profiles for the reaction of TCPO-hydrogen peroxide CL reaction in the presence of the fluorophor ANS in three different media, i.e., 1:2 (v/v) 1-butanol/cyclohexane(bulk solvent), 2.0 X 103M CTAC in the bulk solvent, and 0.08 M AOT in the bulk solvent, are shown in Figure 2a. The highest intensity is achieved with CTAC reversed micelles. An integration time of 90 s or 120 s was used throughout the entire study; although the reaction is not complete in this time, relative intensities can still be compared because the shapes of these curves are similar. Similar profiles were also observed with Rh B as the fluorophor except that the highest intensity was obtained with AOT reversed micelles (Figure 2b). TCPO-Hydrogen Peroxide CL Reaction in CTAC and AOT Reversed Micelles (Effects of Surfactant and R Ratio). The typical net integrated CL intensitysurfactant concentration profiles for the TCPO-hydrogen peroxide reaction in CTAC and AOT reversed micelles are shown in Figure 3. Both ANS and Rh B were used as fluorophors. As can be seen, the net CL intensity depends upon the charge type of the micelles and the fluorophor and the surfactant concentrations. In CTAC reversed micelles, the largest increase of CL intensity was obtained at concentrations of about 2 mM: an 8-fold enhancement with ANS and a 6-fold enhancement with RH B. At lower concentrations (13-27 mM), a 4.5-fold enhancement was observed with ANS in AOT reversed micellar medium; however, a t much higher AOT concentrations (0.16 M), intensity was increased 55 times with Rh B. With ANS, the highest chemiluminescence intensity in CTAC reversed micelles was nearly twice the value found in AOT reversed micelles. However, the opposite effect was observed with Rh B. In the reversed micelles of CTAC formed in 1: (v/v) chloroform/cyclohexane mixture, the chemiluminescence intensity enhancement with ANS and Rh B was about 9and 11-fold, respectively. The greatest enhancement was also found a t concentrations of about 2 mM. No significant increase of CL intensity was observed with AOT reversed micelles formed in this solvent mixture. These results can be interpreted based on the charge interaction between micelles and fluorophors which are found to be an important parameter in normal micelle systems.17 Under basic reaction conditions (due to the presence of triethylamine), the anionic form of ANS has
0.0 ! 20
0
I
I
I
40
60
80
1
Time, sec
(b) '"
I
I
,&
-
In
0
c
d
21LI
-
CTAC
Bulk Solvent
Time.sec
Figure 2. Intensity-time profiles: (a) ANS as fluorophor; (b) Rh B as fluorophor. Bulk solvent: 1:2 (v/v) 1-butanol/cyclohexane. Final concentrations of reagents in cell: TCPO = 2.8 X 1W M fluorophor = 2.0 X le7 M; triethylamine = 2.1 X 1W M;H202 = 1.3 X 10s M;CTAC = 3.0 X 1W M; AOT = 2.0 X le2M; R = 19 for CTAC, 13 for AOT.
stronger electrostatic interaction with the cationic CTAC micelles. The polar head of anionic AOT repels ANS anions. Therefore, at the interface of reversed micelles where the CL reaction most likely takes place, the local concentration of ANS in CTAC micelles would be higher than in AOT reversed micelles, so that the chemiluminescence intensity with CTAC would consequently be greater than that with AOT. Rh B is positively charged, causing the opposite effect. Additionally, since ANS is a more efficient fluorophor than Rh B, a higher chemiluminescence intensity is obtained with ANS in both CTAC and AOT reversed micelles. The effect of R ratio on CL efficiency and reaction rate was studied in 1:2 (v/v) 1-butanol/cyclohexane bulk solvent (ANS as fluorophor, Figure 4). As the R ratio increased from 1to 30 in CTAC reversed micelles, CL intensity and the observed pseudo-first-order reaction rate constant, k, are not affected. However, in AOT reversed micellar medium, CL intensity decreases as R ratio increases, and the observed pseudo-first-order reaction rate constant, k, increases slowly. The rate constant of the CL reaction in CTAC reversed micellar medium is nearly 3 times that in AOT. In CTAC reversed micelles, the electrostatic interaction attracts most ANS and hydroperoxide anions to the cationic interface of the micelles. Increasing the size of the water pool would dilute the concentration of the species
Dan and Grayeski
Langmuir, Vol. 10, No. 2, 1994 (a) 1000.
I'
f
Concentration of Surfactant
160
,
(M)
(b) I
--i
-
0.0023
-
0.0010
-
r '
Concentration of Surfactant (M)
Figure 3. Effect of surfactant concentration on CL intensity: in CTAC and (- - -) in AOT; (b) (a) ANS as fluorophor (--)
in CTAC and (- - -) in AOT. Bulk Rh B as fluorophor (-) solvent: 1:2 (v/v) 1-butanol/cyclohexane. Reactant concentrations as Figure 2.
inside the pool more significantly than that of the species (such as ANS and hydroperoxide anions) near the pool interface. Therefore, the R ratio has little effect on the CL intensity. In AOT reversed micelles, most ANS and hydroperoxide anions are probably located well inside the water pool because of the repulsion between the anionic head group of surfactant and the similarly charged reactant species. The dilution of ANS and hydroperoxide concentrations within the water pool was more significant with the increase of water volume, resulting in the decrease of the CL intensity. It has been reported that in reversed micelles, the addition of water results in a rapid increase of the average aggregation number and the size of the water pool but a decrease of charge density of the interface.= This indicates that the micelles become less rigid with the increase of R ratio. Consequently, the mass transfer of reactants becomes a little easier and a slight increase of reaction rate constant is observed. Effect of Reactant Concentrations. The effects of triethylamine concentration were studied. In the 1:2 (v/ v) 1-butanol/cyclohexane mixture and CTAC reversed micellar medium, chemiluminescence intensity decreases as triethylamine concentration increases (Figure 5). On the contrary, in the AOT reversed micellar medium, (23) Fendler,J. H. MembraneMimetric Chemistry;Wiley: New York, 1982; Chapter 3, p 66.
I
0
5
10
15
20
.
,
25
30
R ratio
Figure 4. Effect of R ratio on CL intensity and rate constant: (a) effect on CL intensity (0)in CTAC and (0) in AOT, (b) effect in CTAC and ( 0 )in AOT. Bulk solvent: on rate constant k (0) 1:2 (v/v) 1-butanoVcyclohexane. Reactant concentrations as Figure 2. Integrated 120 s.
6
e
c m .P ;ii
-
z
b-3
Concentration of Triethylamine (M)
Figure 5. Effect of triethylamine concentrationon CL intensity (-1 in CTAC, (- - -) in AOT, and (- - -) in bulk solvent. Bulk
solvent: 1:2 (v/v) 1-butanol/cyclohexane. Final concentrations of reagents in c e k AOT = 0.09 M; CTAC = 1.0 X 1o-S M others as in Figure 2. Integrated 120 s.
chemiluminescence intensity gradually increases with an increase of triethylamine concentration, reaching a maximum a t 0.4 mM. Without triethylamine, the TCPO-hydrogen peroxide reaction proceeds slowly and the chemiluminescence
Effects of Reversed Micelles on Chemiluminescence
451 500
400
-
300
-
200
-
too
-
0
2.0
!
I
I
I
0.02
0.04
0.06
0.08
Concentration of Hydrogen Peroxide (M)
intensity is very low. The addition of triethylamine catalyzes the reaction by increasing the formation of hydroperoxide anions, which in turn increases the chemiluminescence efficiency and reaction rate (both peak high and pseudo-first-order reaction rate constant). However, the increase of triethylamine also increases the amount of hydroxide, which accelerates the hydrolysis of TCPO. In the 1:2 (v/v) 1-butanol/cyclohexane mixture and CTAC reversed micellar medium, the competitive hydrolysis reaction of TCPO overwhelmed the chemiluminescence reaction. Therefore, overall CL intensity decreases with the increase of triethylamine. In AOT reversed micelles, increasing the triethylamine concentration accelerated the formation of hydroperoxide anions more significantly than the hydroxide anions, so that CL intensity increased. It has been reported that triethylamine is also a quencher of the chemiluminescence reaction a t the higher concentrations.24 When the triethylamine concentration was over 0.4 mM, the quenching effect became more significant than the other effects; thus, CL intensity decreased. The effect of hydrogen peroxide concentration was also studied (Figure 6). With a constant R ratio, the chemiluminescence intensity remains almost unchanged in both CTAC and AOT reversed micellar media, with the hydrogen peroxide concentrations increasing from 1to 15 times that of the TCPO concentration (ANS as fluorophor). As the hydrogen peroxide concentration increases to 15 times that of the TCPO concentration, chemiluminescence intensity decreases slightly. Using Rh B as fluorophor, the change of hydrogen peroxide concentration does not affect chemiluminescence intensity significantly in either reversed micellar media. Since the hydrogen peroxide is in excess to TCPO, and a large amount of hydroperoxide anions are available around the micelle-water interface, little effect of hydrogen peroxide concentration on chemiluminescence intensity is expected. Effect of Chain Length of Cationic Surfactant. Two cationic surfactants with similar structure but different chain lengths were evaluated (Figure 7). Over the concentration range studied, the surfactant with the shorter chain length shows the greater chemiluminescence intensity. In contrast to the study of normal micelles,” this indicates that the mass transfer of reactants might be easier with shorter chain length micelles. The possible transfer process of reactants involves the diffusion of 1991,116,443. 1989,225,147.
(25) Hinze,W. L.; Igarash, S . Anal. Chim. Acta
‘1
0
Figure 6. Effect of hydrogen peroxide concentration on CL enhancement (0) in CTAC and ( 0 )in AOT. Bulk solvent: 1:2 (v/v) 1-butanol/cyclohexane. Other reagents concentration as in Figure 2. Integrated 120 a.
(24) Devasto, J. K.; Grayeaki, M. L. Analyst
\
.
1
Concentration of Surfactant (M)
Figure 7. Effect of surfactant chain length on CL intensity (-) in TBAC [CHS(CH~)~SN+(CHS)~C~H~C~-I and (- - -) in SBAC [CH~(CH~)~~+(CHS)~C~SC~-I. Bulk solvent: 1:2 (v/v) l-butanoUcyclohexane. Readant concentrations as in Figure 2 except triethylamine = 1.6 X 10-9 M; R = 10. Integrated 120 8. Table 1. Effect of Surfactant Counterions intergrated CL peak CL surfactanta intensity (AUb) intensity (AU) bulk solventc 126 19 1421 f 2.8 1180& 114 8840 f 1040 CHs(CHz)isN+(CHs)3ClCH~(CH~)~~N+(CHS)~C~H~C~498 13 7200 i 421 CHS(CNZ)IIN+(CH~)~B~ 1.3 & 0.2 130.7 & 30.9 296.4 47.9 CHS(CHZ)I~N+(CHS)SB~ 2.3 0.3
*
*
[Surfactant]= 0.5 mM, R = 20. Arbitrary units. Bulk solvent: 1:l (v/v)chloroform/cyclohexe. Other reagents concentrations as Figure 2.
TCPO into and/or fluorophor and peroxide out of the micelles. Effect of Bromide Counterions. It was reported that cetyltrimethylammonium bromide (CTAB) reversed micelles improved the sensitivity of the luminol CL method for determination of hydrogen peroxide.26 Four types of cationic surfactants with similar structures and either chloride or bromine counterions were studied (Table 1). CL intensity enhancement was observed only in the reversed micelles of surfactants with chloride counterion. The formation of reversed micelles of surfactant with bromide counterion decreased the CL intensities. This was not surprising since bromide anions are good fluorescence quenchers. Characterization of Fluorophors in Reversed Micelles. The absorption and fluorescence spectrum features of ANS and Rh B different media are summarized in Table 2. The absorption spectra of ANS and R h B in both CTAC and AOT reversed micelles are analogous to those in the bulk nonaqueous solvent, but with higher molar absorptivity. The maximum absorbance wavelengths of ANS in reversed micelles were red-shifted from those in water. In contrast, the maximum absorbance wavelengths of Rh B in reversed micelles were blue-shifted from those in aqueous media. As R increases, the absorptivity of ANS in reversed micelles decreases generally, and opposite absorbance spectral behavior has been observed with Rh B. By comparing the absorption spectra of both fluorophors in aqueous and nonaqueous media, we can anticipate that the polarity of the microenvironment of the fluorophors is between those for water and bulk organic solvent. The fluorescence emission spectra of ANS in both CTAC and AOT reversed micelles are also similar to those in the bulk nonaqueous solvent, except that the emission wavelengths are slightly blue-shifted. On the contrary, a redshifted emission spectrum of Rh B is observed only in
452 Langmuir, Vol. 10, No. 2, 1994
Dan and Grayeski
Table 2. Spectral Features of AN8 and Rh B in Aqueous, Nonaqueous, and Reversed Micelles. medium bulk solvent CTAC reversed micelle R=5 R = 10 R = 20 AOT reversed micelle R=5 R = 10 R = 20 bulk water bulk solvent CTAC reversed micelle R=5 R = 10 R = 20 AOT reversed micelle R=5 R = 10 bulk water a
UV, ,A
nm (ea, M-l cm-1)
b-1, nm
fluorescence emission bmz, nm (Im, AU)
(Zm, AU)
chemiluminescence, ,A
271 (17.5), 376 (4.19)
ANS 401 (16.6)
489 (48.2)
478
271 (33.5), 373 (8.63) 271 (36.6), 373 (7.66) 271 (30.7), 373 (7.54)
392 (26.2) 394 (26.6) 399 (9.2)
484 (54.4) 484 (52.6) 480 (18.7)
478
270 (30.4), 375 (8.71) 272 (29.9), 375 (6.66) 271 (23.4), 374 (7.17) 265 (22.2), 350 (5.76)
394 (21.5) 396 (23.0) 398 (19.6)
489 (46.9) 488 (44.2) 489 (32.0) 412 (2.5)
467
539 (7.34)
Rh B 564 (18.3)
613 (73.6)
579
540 (10.4) 540 (10.4) 540 (12.2)
565 (73.7) 565 (68.9) 565 (79.5)
612 (155) 614 (134) 611 (194)
571
544 (43.4) 546 (58.4) 553 (85.8)
571 (31.9) 573 (43.4)
610 (350) 614 (119) 639 (24.3)
576
nm
ANS: [CTAC] = 2.0 mM; [AOTI = 0.08 M. Rh B: [CTAC] = 4.7 mM, [AOTI = 0.16 M. Bulk solvent: 1:2 1-butanoUcyclohaxane.
AOT reversed micelles, and no significant difference is found in CTAC reversed micelles. The integrated fluorescence intensity is generally increased by the formation of reversed micelles except for ANS in AOT. Additionally, the relative emission intensity of ANS in water is much smaller than that in nonaqueous media, and only one blueshifted emission peak was observed. The same effect has been observed with Rh B in water and organic media, except a red-shifted smaller emission peak is found in water. The emission spectra of ANS in reversed micelles with different R ratios were consistent with previous studiesZ2 and the highest fluorescence intensity was found with the smallest R ratio. For Rh B, the 612-nm emission peak in CTAC reversed micelles was slightly red-shifted in the sequence of R of 20,5, and 10. A red-shifted and smaller fluorescenceemission peak was also found in AOT reversed micelles. These facts again imply that the microenvironment experienced by the fluorophors in the reversed micellar water pool is less polar than that of bulk water alone but greater than in that of nonaqueous solvent. The attempt was made to measure chemiluminescence spectra. Fluorophor concentration was increased to 1order of magnitude higher than that of the fluorescencespectrum in order to get adequate intensity for a scan. A single broad band was observed in the same spectrum range as one of the bands of the fluorescence spectrum. A slight shift was observed. This may be due to the significant difference between the concentrations and the rapid change in reaction rate during the scanning. Effect of Viscosity a n d Relative Fluorescence Efficiencies. It has been reported that microviscosity and macroviscosity of reversed micelles are very different from the viscosity of a bulk solvent.21p22 The effect of the viscosity of the reaction medium on CL intensity was studied by adding polyethylene glycol (M. W. 35 000) to the reaction system. The results showed that, with the increase of viscosity of reaction medium, chemiluminescence intensity was increased (Figure 8). At 0.5 mM polyethylene glycol, a CL enhancement of 52 times for ANS and 96 times for Rh B was achieved. The change of reaction medium viscosity resulted in the change of chemiluminescence efficiency, which is the
$
J
50 01
4000
'-i
,-
.:
,--
.
.
~
,i
.
,
0
0
2
4 VIscosity, cps
6
I
0
Figure 8. Effect of viscosity on CL intensity. Bulk solvent: 1:l (v/v) chloroform/cyclohexae. Final concentrations of reagents in cek TCPO = 4.5 X lo-' M, ANS = 1.5 X 10-7 M triethylamine = 3.1 X 1 V M; HzOz = 2.6 X 1W M. Integrated 120 a. Table 3. Relative Efficiency Enhancement in Micelles micellar solutiona CTAC AOT CTAC AOT
zcl(m)/zol(b)b
Im(m)/k,fi)'
AN9 7.74 4.39 Rh B 5.96 55.4
Za(m)/Za(b)d
1.07 0.67
7.21 6.60
1.07 2.10
5.57 26.3
a ANS: [CTAC] = 2.0 mM; [AOTI = 0.08 M. Rh B: [CTAC] = 4.7 m M [AOT]= 0.16 M. Bulk solvent: 1:2 1-butanoucyclohaxane. Ratio of CL intensity in micelle to that in bulk solvent. Ratio of fluorescence intensity in micelle to that in bulk solvent. d Ratio of excitation efficiency in micelle to that in bulk solvent.
*
product of fluorescence efficiencyand excitation efficiency @CL
e @FL*~=
The relative fluorescence efficiency change in reversed micellar medium and bulk solvent was measured and calculated as shown in Table 3. The fluorescence efficiencies of ANS and Rh B in both CTAC and AOT reversed micellar media (in the solvent mixture of 1:2 (v/v) 1-butanolxyclohexane) changed, but the magnitude was
Effects of Reversed Micelles on Chemiluminescence
Langmuir, Vol. 10, No. 2, 1994 453
Table 4. Analytical Parameters for Determination of Fluorophors medium:
B.S.0
analyta:
ANS 5.0 X 1V" 0.5-10
CTACb ANS 2.5 X 10-8 0.025-5.0
AO'P ANS 7.5 X 10-8 0.075-10
18.87 1.15 3.47
8.16 0.53 1.03
8.64 0.86 3.37
det, l i i i t e (M) linear rang$ (X 10-8 M) RSD % g 1.c. m.c. U.C.
B.S.0 RhB
CTACb
AOTd
5.0 X 10-8 5.0-100
Rh B 1.0 x 10-8
Rh B 1.0 x 10-7
1.0-100
0.1-100
9.03 1.65 6.08
11.6 1.04 2.65
6.66 1.13 1.17
B.S.,bulk solvent (1:2 (v/v) 1-butanol/cyclohexane). PMT voltage 900 V. b CTAC = 4.0 X 1 V M, R = 10. PMT voltage lo00 V. AOT = 6.0 X 10-2 M,R = 5. PMT voltage lo00 V. d AOT = 6.0 X 10-2 M, R = 5. PMT voltage 900 V. CLimit of detection given in molarity of 0
original analyta sample, taken as SIN = 3. f As obtained from calibration curves (log integrated intensity vs log concentration). 8 l.c., low concentration of linear range; m.c., medium concentration of linear range; u.c., upper concentration of linear range (N = 3).
relatively small (maximum 2.1-fold) compared to the CL efficiency enhancement (55-fold). The changes of excitation efficiencies are affected much more significantly than fluorescence efficiencies. An enhancement as great as 26 times is achieved. Therefore,the chemiluminescence efficiency enhancement is caused mainly by the increase of chemical excitation efficiency. Excitation efficiency a, can be expressed by energy transfer efficiency @ET (number of active intermediateshumber of molecules reacted) times effective excitation efficiency We, (number of excited moleculesInumber of active intermediates). The energy transfer process may be favored by the increase of viscosity. In a viscous medium, the proposed intermediate-fluorophor complexes will be more stable toward diffusion, a dark process. Thus the energy transfer efficiency term, @ET, may be increased. Analytical Results and Parameters. We have demonstrated that peroxyoxalate CL intensity can be increased by varying surfactant concentrations, viscosity, and surfactant chain lengths. These CL enhancements are due to a combination of changes in excitation efficiency, fluorescence efficiency, and reaction rate by the formation of reversed micelles. To determine if these factors can improve the analytical figures of merit, we evaluated the reversed micellar peroxyoxalate CL systems for potential analytical advantages in the development of assays using this reaction. Peroxyoxalate chemiluminescence detection has been shown to be a highly sensitive detection method for liquid ~hromatography.~~J' The determination of Rhodamine B labeled chlorophenols by normal phase chromatography with postcolumn TCPO-hydrogen peroxide chemiluminescence detection has been reported.2* A similar HPLC peroxyoxalate detection system has been used to determine
serum estradiol.29 Based on the results reported above, chemiluminescence enhancement using the reversed micelle systemsshould provide further improvement for such analyses. Therefore, a flow injection analysis system which mimicked the postcolumn peroxyoxalate detection part of the normal phase HPLC was used to evaluate its analytical advantages (Figure 1). The analytical figures of merit are shown in Table 4. In both reversed micellar and bulk solvent media the linearity covers the range 7.5 X 10-8 to 1.0 X 106 of ANS and 1.0 X lo-' to 1.0 X 10-4M of Rh B. The dynamic libear ranges of both fluorophors in both reversed micellar media are increased 1or 2 orders of magnitude. The detection limit (SIN = 3) of ANS is improved by factors of 20 and 7 in the reversed micellar media of CTAC and AOT. For Rh B, the detection limits are improved 5 and 50 times in CTAC and AOT reversed micelles. The precision of the measurements near the detection limit, the medium concentration, and the high concentration were also improved. This indicates that more uniform mixing was obtained with micellar media. The presence of reversed micelles increases not only the assay precision but also the sensitivity. Better mixing with a micellar medium also decreased the noise sign&, therefore, fluorophors in reversed micelles could be measured at higher detector voltages relative to the measurement in the bulk solvent (except Rh B in AOT).
(26) Imai, K. Methods in Enzymology; Deluca, M. A., McElroy, W. D.,
Conclusion We have observed the increase of TCPO-hydrogen peroxide chemiluminescence intensity by the formation of reversed micelles. These CL intensity increases are due to a combination of changes in excitation efficiency, fluorescence efficiency, and the reaction rate of the system. These factors can also be used to improve analytical figures of merit. Acknowledgment. Support of this by the donors of the Petroleum Research Fund is gratefully acknowledged.
(27) Imai, K.; Miyaguchi, K.; Honda, K. Bioluminescence and Chemiluminescence: Instruments and Applications;VanDyk, K., Eds.; CRC Press: Boca Raton, FL,1986, Vol. 11, p 65.
(28) Kwakman, p. J. M.; Mol, J. G. J.; Kamminga, D. A.; Frei, R. W.; Brinkman,U. A. Th.; De Jong, G. J. J. Chromatogr. 1988,459,139. (29) Nozaki, 0.; Ohba, Y. A d . Chim. Acta 1988,205,255.
Ede.; Academic: New York, 1986; Vol. 3, part B,p 435.
