Anal. Chem. 1087, 59, 496-504
498
solvent orderings are identical, the precipitous drop in extraction efficiency noted with 18-crown-6when the solvent was changed from chloroform to l,l,l-trichloroethane was not observed with the crown ether carboxylic acid 1. Presumably this greater sensitivity of extraction efficiency to the solvent polarity for the neutral crown ether results from the need to transfer an aqueous phase anion as well as the alkali metal cation into the organic phase during extraction. Turning now to the matter of selectivity in competitive alkali metal cation solvent extraction by the lipophlilic crown ether carboxylic acid 1, the high Na+ selectivity which is noted in chloroform is markedly diminished in all five other solvents (Figure 1 and Table I). Neither the solubility parameter 6 nor the emperical solvent polarity ET correlate with the observed extraction selectivity. Of the solvent parameters presented in Table 11, only 6h shows a clear differentiation of chIoroform from the other solvents as a group. This suggests that the hydrogen-bonding ability of the organic solvent may play an important role in determining selectivity for aqueous alkali metal cation extraction into organic solvents by lipophilic crown ether carboxylic acids, such as 1. Registry No. 1, 79519-65-6; Li, 7439-93-2;Na, 7440-23-5;K, 7440-09-7;Rb, 7440-17-7;CS,7440-46-2.
LITERATURE CITED (1) Charewlcz, W.; Heo, G. S.; Bartsch, R. A. Anal. Chem. 1982, 54,
2094-2097.
Charewicz. W.; Bartsch. R. A. Anal. Chem. 1982, 54, 2300-2303. Strzelblcki, J.; Bartsch, R. A. Anal. Chem. 1981, 53, 1894-1899. Strzelblcki. J.; Bartsch, R. A. Anal. Chem. 1981, 53, 2251-2253. Bartsch, R. A.; Liu, Y.; Kang, S. I.; Son. B.; Heo, G. S.; Hipes, P. G.; Bills, L. J. J. Org. Chem. 1989, 48, 4864-4869. Rozen, A. M. In Solvent Extraction Chemistry; Dyrssen, D., Liijenzin, J.-O., Rydberg, J., Eds.; Wiley: New York, 1967; pp 195-235. Zolotov, Y. A. Extractbn of Chelate Compounds; Ann Arbor-Humphrey Science Publishers: Ann Arbor, MI, 1970; pp 64-74. "Kirk-Othmer Encyclopsdla of Chemlcal Technology", Supplement; Interscience, 1971; pp 889-910; Wiley: New York, 1983; Vol. 21, pp
337-401.
Wakahayashi, T.; Oki, S.;Omari, T.; Suzuki, N. J. Inorg. Nucl. Chem. 1964, 26, 2255-2264. Omori, T.; Wakahayashi, T.; Oki, S.; Suzuki, N. J. Inorg. Nucl. Chem. 1984. 26, 2285-2270. Oki, S.; Omori, T.; Wakahayasti, T.; Suzuki, N. J. Inorg. Nucl. Chem. 1965, 27, 1141-1150. Stepnlak-Blenlaklewlcz, D.; Szymanowski, J. J. Chem. Technol. Botechno/. 1979. 29, 686-693. Iwachdo, T.; Mlnami, M.; Nato, H.; Toei, K. Bull. Chem. SOC. Jpn. 1982, 55, 2378-2382. Lange's Handbook of Chemistry; Dean, J. A,, Ed.; McGraw-Hili: New York, 1965. Techniques In Chemistry, Vol. I I , Organlc Solvents; RMdick, J. A,, Bunger, W. B., Eds.; Wiley-Interscience: New York, 1970. Reichart, C. Solvent Effects ln Organic Chemistry; Verlag Chernie: New York, 1979; pp 242-244. Bureli, H. I n P o / y m Handbook, 2nd ed.; Brandrup, J., Irnmergut, E. H., Eds., Wiley-Interscience: New York, 1975; p IV-337ff.
RECEIVED for review June 12, 1986. Accepted October 10, 1986. This research was supported by the Division of Basic Chemical Sciences of the United States Department of Energy (Contract DE-AS05-80ER10604).
Exploitation of Reversed Micelles as a Medium in Analytical Chemiluminescence Measurements with Application to the Determination of Hydrogen Peroxide Using Luminol Hitoshi Hoshino' and Willie L. Hime* Department of Chemistry, Analytical Micellar Institute, Wake Forest University, P.O. Box 7486, Winston-Salem, North Carolina 27109
The lumW (5-amhro-2,3-dlhydro-lYdpMhalrrzkredione)-hydrogen peroxlde-3-ambphhWe (CL) system was characterized In reversed miceWes of hexachkckk (CTAC), fernred in 6 5 (v/v) chloroform-cyclohexane. The relatlve Mecttueness of tMs medlum for analytlaal CL measurements was assessed. Whereas no Ilght emledon was observed from th6 lumlnolh y d r o p peroxlde CL reaction In bulk aqueous s d W n at mHd pH (7.8-9.1) In the absence of any added catalyst or cooxklant, intense CL was observed In the CTAC reversed mkellar system. The effeci of expdmedal varlables (Le., surfactant concentration, CorolWUzed water, luminol concentration) upon the CL InteWy was evaluated. In the CTAC reversed mkdlar medium, the lumtnol CL assay can be applkd to the andysk of hydrogen peroxlde In the 6.4 X lo-' to 6.4 X lod I# concmtratkn r a w . The predakm of the reversedmiodlrrr ~OCWBCI prooedue IS w e satisfactory, renglngfrom 0.9 to 12.3 % In t e r m of the relathre standard d e v k b over the range of peroxkle concentrations examIned. The fe88Mtty of udng the reversed mkellar effect to detemhe enzymes or rwbeQatesincoupled readJon sy$tems that produce hydrogen peroxide is discussed. Present address: D e p a r t m e n t of A p p l i e d Chemistry, T o h o k u University, Aoba, Aramaki, Sendai, J a p a n 980. 0003-2700/87/0359-0496$01.50/0
The development of new or enhanced analytical methodologies based upon the use of surfactant micellar systems is a very active area of current research (for reviews, refer to ref 1-6). For example, their use has been found to be advantageous in such analytical spectroscopic techniques as ultraviolet-visible absorption (1,6, 7), fluorescence (1,5,8, 9), phosphorescence (3,10-12), and atomic absorption (13) as well as in thin-layer (2,14) and high-performance liquid chromatographic separations (2,15,16). The success of such applications is due to the fact that micelle aggregates can be employed to judiciously manipulate the solubility and microenvironment of analytes and reagents and to control the reactivity, equilibrium, and pathway of chemical or photochemical processes among other effects (I, 2, 17). More recently, these unique properties of micelles have been utilized to facilitate analytical chemiluminescence measurements (18-23). Advantages cited include elimination of solubility problems (18, 19), improved sensitivity (19-23), increased selectively (19,22), better precision (22), and a relaxation of the usually strict pH requirements for observation of efficient chemiluminescence (19,22). Surprisingly, almost all of the micellar-enhancedanalytical procedures reported to date have utilized normal aqueous micelle systems. Consideration of so-called reversed micelles in chemiluminescencemeasurements or chemical analysis in general seems to have been overlooked. A reversed micelle 0 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987
is an aggregate formed in an organic solvent that usually contains a small amount of water. Such surfactant aggregates are termed inverted or reversed micelles since their polar groups are concentrated in the interior of the aggregate while their hydrophobic moieties extend into, and are surrounded by, the bulk apolar organic solvent (17,24,25). The interior core of the reversed micelle, i.e., the micellar interface and the inner aqueous phase, provides a unique and versatile reaction field. Depending upon its water content (which also dictates the size of the aggregate), the microscopic polarity, the local concentration (proximity), mobility of substrates (microviscosity), and/or activity of water can vary markedly, by which one can control chemical reactions as required ( I 7, 24-27). In fact, reports indicate that reversed micelles can drastically alter pK values and reactivity (much more so than that usually possible with normal aqueous micelles (17,24)) compared to that in bulk homogeneous solvents as well as effectively solubilize without a loss of activity various enzymes in their inner water pool (25-27). Consequently, the use of reversed micellar systems as a medium for chemiluminescent assays merits further investigation since their unique properties can potentially affect favorably the sensitivity and analytical performance. Previously, the use of reversed micelles to prolong the duration of chemiluminescence observed from various oxalate ester (or acid)-hydrogen peroxide-sensitizer reaction systems for application as chemical light sources has been mentioned in the patent literature (28-30). Russian chemists have reported the only analytical application of which we are aware. Namely, the use of a nonionic Brij reversed micellar medium was found to greatly simplify the luciferase bioluminescence assay for ATP as well as improve the sensitivity by over an order of magnitude (31). In this first of a sequence of papers, we describe the results of our comprehensive study of the effecta of a cationic reversed micellar system, i.e., hexadecyltrimethylammonium chloride (CTAC) in 6:5 (v/v) chloroform-cyclohexane containing specified amounts of water, upon the spectral and physical properties of the chemiluminescent reagent, luminol (5amino-2,3-dihydrophthalazine-1,4-dione or 3-aminophthalhydrazide, Lum), and its primary emitter, 3-aminophthalic acid (3-AP). Additionally, the use of the CTAC reversed micellar system as a medium for the luminol chemiluminescent assay for hydrogen peroxide was evaluated and the analytical results were compared and contrasted with those obtained in bulk water alone. The luminol(3-aminophthalate)-hydrogen peroxide chemiluminescence reaction system was selected for initial study in the reversed micelles due to its widespread use in chemical analyses (32). Its popularity in chemiluminescence assays stems from its great versatility in that a variety of possible analytes can be quantitated, e.g., (i) determination of hydrogen peroxide which in turn can be generated by either different coupled enzymatic reactions thus allowing for quantitation of the substrates or enzymes in such coupled reaction schemes (32,33)or radiation which allows its use as a radiation dosimeter (34)and (ii) quantitation of luminol as analyte in competitive binding immunoassays involving luminol (or its conjugates) as labels (32,35). Hence, any favorable reversed micellar effects on the luminol-hydrogen peroxide chemiluminescence reaction system would be of added significance and could be exploited to improve assays for a variety of analytes. The results of our study demonstrate that it is possible to obtain analytically useful chemiluminescence emission from the luminol-hydrogen peroxide reaction in the pH 7.8-9.0 region in a CTAC reversed micellar medium without the need of any added catalyst or cooxidants. The analytical utility and implications of this finding will be discussed.
497
EXPERIMENTAL SECTION Apparatus. All chemiluminescencemeasurementswere carried out with a Turner Designs Model 20-000 photometer. A Fisher Recordall Series 5000 strip chart recorder was used to record the chemiluminescence(CL) intensity-time profiles. Spectral measurements were made on a Varian Cary 219 UV-vis spectrophotometer and an Aminco-Bowman spectrofluorometer. In all fluorescence studies, both the exit and entrance slit width were 1 mm (5.5 nm band path) and no correction was made for the photomultiplier response (S-4 response). A Fisher Accumet Model 320 expanded scale pH meter was used for pH measurements. A Vortex-GenieModel K 550G mixer (Scientific Industries, Inc.) was used for agitation of the reversed micellar solutions. Reagents and Solutions. All reagents were of the highest purity grade available and used without further purification. All aqueous solutions were prepared with Fisher HPLC grade water. An appropriate amount of luminol (Sigma) to give a concentration of 0.05 M was dissolv'ed with a small quantity of 2.5 M sodium hydroxide, and then the solution was neutralized to pH 8.2 with dilute hydrochloric acid. A 0.02 M stock solution of 3-aminophthalate (3-AP) was prepared from the hydrochloride salt (Kodak) by adding dilute sodium hydroxide until the pH reached 7 or 8 before dilution to final volume. Stock solutions of hydrogen peroxide (0.10-0.0010 M) were prepared daily by diluting an aqueous 30% hydrogen peroxide solution (Sigma). The pH of these solutions was maintained at about 4.5 with dilute hydrochloric acid to minimize decomposition of the peroxide (22, 36). Acetate, phosphate, borate, and carbonate solutions (sodium salts from J. T. Baker) were used in the preparation of buffer solutions with hydrochloric acid and sodium hydroxide. A 6:5 (v/v) chloroform-cyclohexane(both Fisher HPLC grade) mixture containing appropriate concentrations of hexadecyltrimethylammonium chloride, CTAC (Kodak), was used as the reversed micellar bulk solvent throughout the study. Procedures. Reversed-Micellar Solutions. All reversedmicellar solutions were prepared by adding the 6:5 (v/v) chloroform-cyclohexane CTAC bulk solvent (5 or 10 mL) to an aqueous solution (10-200 pL) containing prescribed amounts of solutes. The solutions were then vigorously agitated for 30 s on the vortex mixer and allowed to stand for 10 min. The range of water content in the reversed-micellar solution, in which reproducible CL signals could be obtained, was 10-300 pL per 10 mL of the bulk solvent containing 0.05-0.30 M CTAC, respectively. When work was done outside of these water/CTAC ranges, the solutions became very viscous or heavily turbid. Consequently, special attention must be paid in the preparationpf these solutions in order to avoid precipitation or turbidity problems. An important parameter, the molar ratio of [H,O]/[CTAC] (hereafter designated R), and the concentrations of all solutes except for buffer components were always calculated on a final volume total solution basis. However, the pH and ionic strength of the reversed-micellary system were based on those of the aqueous buffer solution employed prior to mixing with the CTAC bulk solvent in accordance with accepted practice (37, 38). Consequently,the concentrationsof acid, base, inert salts, and/or pH buffer components are expressed on the basis of the water volume present in the reversed-micellar solution. Spectroscopic Characterization. The UV-vis absorption and fluorescence measurements were made in the conventional manner. In some experiments, attention must be paid to sample preparation in order to minimize precipitation and turbidity problems. Specifically, the cdncentrations of Lum and 3-AP should be kept as low as possible because they tend to precipitate under slightly acidic conditions even in the reversed-micellar solutions. Under some conditions employed for the spectral studies, the solutions were turbid. To eliminate the base line fluctuations caused by the light scattering, the differential absorbance method was employed for the quantitative absorption studies. The differential absorbance, Ad, is given by Ad = A - A , (1) where A is the absorbance at an adequate wavelength for the species of interest and A, is measured at a reference wavelength where there is no actual absorption by the species at any pH. The reference wavelengths were set at 470 and 480 nm for Lum and 3-AP, respectively. The apparent molar absorptivity, ea, can be
498
ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987
derived from Ad in the usual manner. Determination of Acid Dissociation Constants. In the reversed micelle microheterogeneous system, the acid diasociation constants for luminol and 3-AP (both H,L) were determined by monitoring (UV-visible and fluorescence)the concentration of the different species as a function of pH in the usual manner. The determined stepwise equilibrium acid dissociation quotients, K s , cannot be directly compared with the K , values obtained in homogeneous aqueous solutions (38-40). However, they are useful for identifying the species in the reversed-micellar solution at a given pH. Chemiluminescence Measurements. The reversed-micellar solutions of luminol and of buffer were mixed in a polypropylene cuvette, and the CL reaction was initiated by automatic injection of the reversed-micellarhydrogen peroxide solution into the cuvette with the luminometer's dispensing syringe. Typically, the volume of the solution added was 50 pL or 100 ML(total volume being 150 WLor 300 pL). In the calibration studies, 200 pL of hydrogen peroxide reversed micellar sample solutions was injected automatically into 100 pL of a luminol-borate (pH 9.1 f 0.1) mixed reversed micellar solution. All reagents and analyte concentrations reported are based on the final concentrations in the cuvette. In most cases, the delay time was 5 s with a run time of 30 s. In some instances, the entire CL intensity-time profile was obtained by using the continuous run mode. All CL intensity measurements, both peak and integrated modes, were corrected for those of the blank which was obtained using dilute hydrochloric acid at uH 4.5 containing no hvdroeen Deroxide.
RESULTS AND DISCUSSION Selection of Reversed Micelle System and Description of Its Properties. Since cationic normal aqueous micelles (Le.,cationic interface) had previously been shown to enhance the CL output for several reaction systems (19, 22, 23), we reasoned (vide supra) that even greater enhancements might be obtainable in a cationic reversed micellar medium. Of different possible cationic reversed micelle forming surfactants, the quaternary ammonium salts (e.g., hexadecyltrimethylammonium halides, CTAB and CTAC) were among the best characterized. Consequently, CTAC was selected for use in this investigation. The choice of the bulk nonpolar solvent mixture was dictated by solubility considerations. Namely, quaternary ammonium surfactants, as CTAC, are reportedly only very sparingly soluble in inert aliphatic hydrocarbons (41) but are soluble in aromatic or chlorinated aliphatic hydrocarbons, especially carbon tetrachloride and chloroform (41-44). A brief survey of the literature revealed that 1:l or 6 5 (v/v) mixtures of a chlorinated and aliphatic hydrocarbon were commonly employed as the bulk solvent for physicochemical studies involving CTAB or CTAC reversed micelles (W,45-48). Thus, a 6:5 (v/v) chloroform-cyclohexane mixture was selected as the bulk apolar solvent system since it afforded completely homogeneous solutions of CTAC reversed micelles, even in the presence of cosolubilized water and the other required chemiluminescence reagents under most conditions examined. The solubility of CTAC in this solvent mixture was found to be 20.61 M (highest concentration attempted) in the presence of 0.00-1.00 M added water a t 25.8 "C. It is important to emphasize that the concepts and structural models typically associated with surfactant aggregation in water (formation of normal micelles) are not necessarily applicable to those occurring in organic solvents (17, 41, 44-52). In fact, the aggregation behavior in organic media is complex with surfactant association occurring predominantly as a consequence of dipole-dipole and ion pair interactions between the amphiphiles. According to a classification scheme recently proposed by Muller to distinguish between two possible general modes of aggregate formation in nonpolar solvents (52), the mono-, di-, tri-, and tetraalkylammonium surfactants (like CTAC) exhibit type I association behavior (44, 52). That is, in contrast to the monomer micelle equilibrium generally observed in aqueous normal micelles, the quaternary ammonium salts seem to undergo a sequential
type of self-association in apolar solvents, Le., a smooth transiton of monomer * dimer == trimer * ... + n-mer type indefinite association (47-52). At each surfactant concentration level, there is a distribution of aggregates, and an increase in surfactant concentration leads to the formation of larger aggregates in greater percentages. These types of reversed micellar systems are typically polydisperse, their average aggregation number is generally small (3 1 1 7), they exhibit no clear-cut critical micelle concentration (cmc) as is observed in formation of normal micelles in water, and they are postulated to have lamellar structures (17, 43-52). For instance, light-scattering and infrared measurements on solutions of CTAC in anhydrous chloroform a t 24 "C indicate the formation of aggregates (reversed micelles) having an apparent number average aggregation number of approximately 3.4 to 6 or 7 over a CTAC concentration of 0.003-0.04 (in terms of mole fraction) (50, 51). In fact, aggregates can exist at very low surfactant concentrations (i.e., 10-7-10-6M (17, 47) in general and at 10.003 mole fraction of CTAC in chloroform) (50). A CTAC reversed micellar system can solubilize appreciable amounts of water; for example, approximately 1.1 M water is soluble in chloroform containing 0.20 M CTAC at 25 "C (48). Addition of increasing amounts of cosolubilized water is thought to cause an increase in both the average aggregation number and aggregate size (17,41,44-48). Consequently, due to possible variability in aggregation size with conditions, it is important to exactly specify the surfactant and water concentrations employed and to accurately prepare such solutions so that reproducible experimental data and chemiluminescence signals are obtained. Characterizationof Luminol and 3-Aminophthalate (3-AP) in CTAC Reversed Micelles. As a prelude to the CL studies, the effects of the presence of CTAC reversed micelles upon the spectral properties and ionization constants of the CL reagent, luminol, and the primary emitter, 3-AP, were determined. Such information is required in order to rationalize any observed reversed micellar effects upon the luminol CL reaction. The absorption and fluorescence data for luminol and 3-AP are summarized in Table I. Since the cations of luminol and 3-AP and the dianion (L2-) of luminol are predominantly present only under extremely acidic and basic conditions (see Table 11),respectively, the spectral data of these species were not extensively examined. The absorption spectra of luminol (HzLand HL-) in the pH, range of 4-12 in CTAC reversed micellar media are quite analogous to those in aqueous solution (54,56), except that the maximum wavelengths are slightly red-shifted (Table I). On the contrary, the emission maximum of neutral luminol (H2L)in the micellar system is blue-shifted by 15 nm and, additionally, the relative emission intensity is smaller than that in water by a factor of 2.4. Similar spectral behavior for luminol has been observed on going from an aqueous to an organic solvent; Le., ,A, = 360 nm (H,L) and 370 nm (HL-) in MezSO (60),and A,, = 413 and 410 nm in t-BuOH (61) and MezSO (60),respectively. These facts imply that the microenvironment experienced by the different luminol species in the reversed micellar water pool is less polar than that of bulk water alone. The charged luminol species have no or very weak fluorescence in both the aqueous and reversed micellar media. Increasing the R value, i.e., increasing water content of the reversed micelle water pool, caused an increase in the molar absorptivity of the monoanionic luminol a t 357 nm (Table I). In contrast, the neutral H2L species at 335 nm exhibited the opposite trend. The spectra of 3-aminophthalate a t different pHs in aqueous and reversed micellar media are shown in Figures 1 and 2. The emission wavelengths of the different 3-AP species
ANALYTICAL CHEMISTRY, VOL. 59,NO. 3, FEBRUARY 1, 1987
499
Table I. Spectral Features of 3-AP and Luminol Species in Aqueous and Reversed Micellar Media , ,A
medium
H3Lt
nm H2L
e,,
M-' cm-') ,X,[ nm]" HL-
L2-
Luminol in bulk water alone
297 (5.74) (in 6 M HC1)
CTAC reversed micelled R = 4.45
294 (6.21) [430] 350 (6.51) [432Ib 294 355 296 355 296 355
R = 8.89 R = 11.1
(6.92) (7.37) (6.35) (6.19) (6.70) [415] (5.99)
301 (7.20) 348 (7.86) 348.5' (7.72)' 357 (7.98) 357 (8.57) 357 (8.81)
3-AminophthalicAcid in bulk water alone
278 (1.15) (in 6 M HC1)
CTAC reversed micelled R = 4.45 R = 8.89 R = 11.1
325 (1.45) [451] [455]'
309 (1.82) [445] [451]'
303 (2.02) [421] 303' (2.43)' [424Ie
336 (3.32) [447] 336 (3.24) [445] 337 (3.17) [448]
330 (2.0) [445] 327 (2.3) [445] 328 (2.4) [445]
311 (2.10) [422] 310 (2.40) [422] 309 (2.90) [422]
"Maximum emission wavelength based on uncorrected spectra. bTakenfrom ref 53. 'Taken from ref 54.
from ref 55.
[CTAC] = 0.10 M. eTaken
Table 11. Apparent Acid Dissociation Quotients of Luminol and 3-Aminophthalic Acid in Aqueous and CTAC Reversed Micellar Media (25 oC)a medium CTAC reversed micelled R = 4.45 R = 8.89 R = 11.1 bulk water
f
PKlb
luminol PK2' PKsb
-0.26
6.81 6.43 6.33
13.8
1.46 1.30
6.35 6.74
15.2 14.2 15.1
2.15 3.37
6.20 6.35
3-AP
ref
56e 57, 54e 58' 59f
PKlb
PKzC
P W
0.04
3.95 3.53 3.39
6.03 5.83 5.43
4.05 3.0
5.9 5.7
1.3
ref
598 55'
"Confidence limit of fO.lO. bionic strength = 3.0. CIonicstrength = 0.3. d[CTAC]= 0.10 M. eDetermined spectrophotometrically. Determined by fluorometry. #Determined potentiometrically.
are summarized in Table I. Although the absorption maxima of 3-AP in the reversed micelles are considerably red-shifted (6-21 nm) compared with those in aqueous solutions, the emission maxima remain essentially unchanged within experimental error. An interesting spectral feature in the reversed micellar medium is that the intensity of the absorption band at 336 nm under acidic conditions (pH, 1-3), where the H2L form predominates, was twice that of the corresponding species in aqueous solution. Likewise, 3-AP fluoresces most intensely in such an acidic micellar medium. As reported by Lind et al. (55), the quantum yield of the H2L species in aqueous solution (@f = 0.10) is one-third that of the L2- form (*f = 0.30). However, in the reversed micellar medium, the emission intensity ratio of these two species (Le., I = FIHZI/ FIp) is about 2.9. The H3L+ species which is present in concentrated acid solution at pH, 10 is nonfluorescent. The emission intensity of the L2- form of 3-AP in the reversed micellar system (pH, 8-10) is approximately 1.7 times smaller than that in bulk water probably because of the diminished polarity of the reversed micelle water pool. In agreement, the addition of Me2S0 to water was reported to cause an approximately 50% reduction in the fluorescence quantum yield of the 3-AP dianion (60). Also in the CTAC reversed micellar medium, fluorescence quenching by hydroxide ion (55, 62) was observed a t pH, 112.5. The data shown in Figures 1 and 2 indicate that the possibility of a
0.21
ABSORPTION SPECTRA
WAVELENGTH, nm
Flgure 1. Absorption and uncorrected fluorescence emission spectra
of 3-AP in aqueous solutions at the indicated pH. [3-AP] = 7.98 X M and 1.94 X lo3 M for the absorptlon and fluorescence studies, respectively. The fluorescence emission spectra were obtained employing excitation wavelengths of 315,322,and 342 nm for pH 9.72, 4.90,and 2.50,respectively. dramatic shift of the excited state acid dissociation equilibria compared to the ground state ones can be reasonably ruled out. Hence, the intrinsic fluorescence of the respective species of 3-AP is depressed or enhanced by the unique interactions possible with the reversed CTAC micellar system. The chemiluminescence spectrum observed from the luminol-hydrogen peroxide reaction in the CTAC reversed micelle system is also shown in Figure 2. As can be seen, the
500
ANALYTICAL CHEMISTRY, VOL. 59,NO. 3, FEBRUARY 1, 1987
L
0 0
WAVELF.NGTH, nm
Flgure 2. Spectra of 3-AP in the CTAC reversed micellar system at various pH values. Conditions were [3-AP] = 1.00 X M and 1.96 X 10’ M for the absorption and fluorescence spectra, respectively. The uncorrected fluorescence emission spectra were obtained using an excitation wavelength of 320 nm. The CL spectrum was measured 1-3 min after initiation of the CL reaction between lumlnol (7.83X M) at pH 9.05 M) and hydrogen peroxide (6.40X (0.10M borate-bicarbonate buffer).
chemiluminescence emission in CTAC matches the fluorescence spectrum of the 3-AP dianion, L2-,observed at pH 11.50 in water (Figure 2). The 3-AP dianion has been postulated to be the primary emitter in the luminol CL reaction in bulk aqueous solutions (32,55,60)and is thus thought to also be the light-emitting product in the reversed micellar medium. The acid dissociation quotients obtained for luminol and 3-AP in this work are listed in Table 11. The values of pKl and pK3 for luminol are obviously shifted to the acidic side as compared with the correspondingK, values in water, while the pK2values are not appreciably perturbed by the reversed micellar environment. Similarly, the pKl value of 3-AP is shifted to the acidic side by 0.9 units from the aqueous pKal value. The pK3 value in CTAC, as well as the pKz, is of the same order of magnitude as that reported for 3-AP in water alone. A plausible explanation for the large pK, shift to the acidic side is that the H3L+species is being repelled from the highly charged surface of the micelles and “destabilized” in the relatively low dielectric field of the reversed micellar water pool. In basic solution, the anions of luminol, HL-, and L2are probably adsorbed at the interface of the micelle (bound to the CTAC cationic head group) where the concentration of hydroxide ion is expected to be higher than that in the central part of the water pool. Such a concentration effect at the reversed micelle interface diminishes the apparent pK, value of luminol. However, in the case of 3-AP, this local concentration effect is not operative under the slightly acidic conditions at relatively high ionic strength, I = 0.3 (in the presence of a large excess of competing anions), so that no actual pK, shift is observed for 3-AP. As seen in Table 11, the larger the R value, the smaller is the observed pK value. Such a dependenceof pK on the R value in the CTAC reversed micelles is similar to that reported for some other acid-base indicators in reversed micellar media by Nome and E l Seoud et al. (38,63,64). They explained this phenomenon in terms of an increase in the degree of dissociation of the counterions of the charged surfactant head group, i.e., the electrical potential of the interface, with an increasing R value (63).
10
t/sec
20
30
Figure 3. CL intensity-time profiles for the luminol-hydrogen peroxide reaction in (A) aqueous basic solution with [luminol] = 1.17 X M, [H202] = 5.23 X lo-‘ M, and [NaOH] = 0.025 M and in (B)the reversed micellar solution with [CTAC] = 0.10M, R = 11.1,[luminol] = 1.96X M, [H202] = 6.41 X lo4 M, and pH, 8.96(borate and phosphate each at 0.10 M).
6
7
8
9
10
11
12
PH Flgure 4. Integrated CL intensity vs. pH profiles for the luminol-hydrogen peroxide reaction in different media: curve A (O), reversed micellar system, [CTAC] = 0.10M, R = 11.1, [luminol] = 1.96 X lo4 M, and [H202] = 6.41 X M; curve B (0),aqueous system, [lurninol] = 2.01 X 10“ M, and [H202] = 6.47 X lo4 M. All CL data were collected using a delay time of 5 s and a run time of 30 s.
Luminol-Hydrogen Peroxide Chemiluminescence Reaction in CTAC Reversed Micelles. Typical time c o m e profiles of the CL production are shown in Figure 3. In the reversed micellar system at pH, 8.96 (curve B), a fast CL build-up followed by the prolonged (steady-state) emission was observed, while in basic aqueous solution (pH 12.39),the CL emission profile, curve A, showed a higher initial peak value with an ensuing steep decay to a much lower intensity level. This prolonged CL emission in the reversed CTAC micelles will give reproducible integrated signals, thus being highly advantageous for m y s based on peroxide quantitation. On the basis of data such as shown in profile B (Figure 3), a delay time for initial peak height measurements and run time for integration of light output of 5 (or 3) and 30 (or 15) 8, respectively were selected. A longer integration time at the sacrifice of saving analysis time did not actually affect the results. The CTAC reversed micellar medium caused a drastic change in the pH,-CL intensity profile of the luminol-hydrogen peroxide reaction. As shown in Figure 4 (curve A),
ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987
I
0
0.5
b
I
1.0
I
1,5
0.5 0,2
0,l
I
R
0.06
0.04
KTACI/M
[H201/M
5
501
10
I
I
I
15
0
10
I
20
Figure 5. Dependence of the CL intensity upon the R value (Le., [H,O]/[CTAC]). Condltions were [luminol] = 1.96 X lo-' M, [H202] = 6.40 X lo4 M, pH, 9.00 (0.10 M borate), delay time 5 s, and run time 30 s (for integrated signal).
the intensity was sufficiently developed under slightly basic conditions, pH, 8.0-9.0, in the CTAC reversed micelle where virtually no CL emission was produced in the aqueous system (curve B). It is of great potential significance that in the reversed micellar system the luminol CL reaction can be performed under such mild pH conditions without the need of any added cooxidants or oxidative catalysts. The use of reversed micellar media thus provides an excellent approach to minimize some problems that are frequently encountered under the strongly basic conditions required for the CL production process in aqueous solution, i.e., high background emission, autooxidation of luminol, spontaneous decomposition of peroxide or interferences from potentially reductive species present in solution (33). More importantly, the pH constraint problem (32, 65) which obstructs a biochemical assay based on the CL reaction with enzymatically generated hydrogen peroxide is expected to be successfully avoided. Specific applications demonstrating this advantage will be detailed elsewhere. The spectroscopic survey indicated that there are no significant changes in the absorption and fluorescence properties of both luminol and 3-AP around pH, 7.8-9.0 and that the chemiluminescencespectrum is well matched with 3-AP (L2form) fluorescence (Figure 2) in the reversed micellar medium. The fluorescence quantum yield of 3-AP (L2-)in the CTAC micellar medium was found to be smaller than that in the bulk aqueous solution. These results imply that the CL production in the presence of the reversed micelle (at mild pH,) is most probably due to the enhanced formation of the chemically excited 3-AP species (the primary emitter) through the luminol-hydrogen peroxide reaction. Reversed micelles had previously been shown to drastically alter the rate and pathway of other oxidation-reduction types of reactions ( 1 7, 24-26). Above pH, 10.6, the CL intensity is again increased in the reversible micelle which seemsto be consistent with the profiie seen in an aqueous system (Figure 4). It should be noted that the CL intensity in the reversed micellar media is greater at all pH values examined compared to that observed in aqueous solutions. On the basis of the Cl-pH profiles obtained in the reversed micellar system, a pH, of 9.00-9.10 (0.10 M borate buffer) was adopted for the subsequent analytical experiments. The effect of the R value (Le., water content) on the CL intensity is shown in Figure 5. Under both of the conditions adopted here, i.e., [H20]variable at 0.10 M CTAC (Figure 5A) and [CTAC] variable at 1.09 M H20 (Figure 5B), the CL intensity reached a maximum a t R = 11.1. It is important to note that the parameter R is only a concentration ratio of water to surfactant. Consequently, the CL intensity a t con-
Figure 6. Dependence of the peak (0)and integrated (0)CL intensity on CTAC and water concentrations at a constant R value of 11. l . Conditions were [luminol] = 1.97 X M, [H202]= 6.42 X lo4 M, pH, 9.05 (0.10 M borate), delay time 5 s, and run time 30 s (for integrated CL signal).
stant R is still dependent upon the absolute concentration of CTAC (or water). As shown in Figure 6, the lower the CTAC (or water) concentration, the higher is the CL intensity for solutions containing an identical total concentration of peroxide. These results could reflect the fact that the rate of the CL reaction is diminished with added CTAC or water due to a dilution of the reactants (due to increased micelles and/or water pool water). Such arguments have been employed to rationalize similar data observed for other reaction systems (66). Alternatively, the reactivity (and CL intensity) could be dramatically altered due to subtle variations in the CTAC reversed micellar structure (size/shape) brought about by increases in the CTAC (or water) concentrations. Unfortunately, there is a lack of information concerning the size/shape of CTAC reversed micelles as a function of surfactant and/or water concentration (41,44-48). Most likely, the CL reaction occurs exclusively at the surfactant-water pool interface (Le., near the quaternary ammonium head groups) of the CTAC micelles dispersed in the bulk organic solvent. In terms of analytical sensitivity, the use of a smaller water concentration in the reversed micellar system (which results in more intense CL) means that a higher dilution factor with respect to an initial aqueous peroxide test solution by the CTAC bulk solvent will result. Consequently, the actual sensitivity of an aqueous peroxide sample on a concentration basis is eventually at a comparable level over a water concentration range of 0.55-1.09 M (dilution factor of 100 or ca. 50). Therefore, the optimal reversed micellar conditions were chosen as follows: 200 1 L of an aqueous sample solution is mixed with 10.0 mL of the bulk solvent containing 0.10 M CTAC such that R = 11.1. The effect of luminol concentration upon the integrated CL intensity is shown in Figure 7. Although the CL intensity strongly depends upon the final luminol concentration, no concomitant increase in the background emission was observed. In contrast, the background CL is reported to increase as a function of added luminol in aqueous media (33). In addition, no solubility problem was observed in CTAC reversed micelles even a t the highest luminol concentration examined, 1.31 X lo4 M,which was employed for the peroxide calibration studies. For comparison,the solubility of luminol in bulk water (pH 7.0) is approximately (2.8 0.4) X lo4 M (67).
*
502
ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987
Table 111. Statistical Evaluation of Precision for the Peroxide Determination"** hydrogen peroxide concn,c 10-1 M
0.00 (blank)
run I (N = 5 ) peak integrated 33.5 f 4.1' 9.9%
2.67 f 0.47e 14.1%
6.39
CL intensityd and re1 std dev run I1 ( N = 4)
run I11 ( N = 3)
peak
integrated
peak
integrated
7.19 f 1.77 15.5% 47.0 f 9.2 12.3%
0.95 f 0.37 24.6% 3.17 i 0.25 4.9%
9.38 f 3.35 14.4% 77.5 f 4.9 2.5% 241 f 26 4.3%
3.27 f 1.41 17.4% 21.6 f 1.5 2.9% 77.5 f 5.4 2.7%
129 f 8 3.9% 197 i 18 5.8%
12.5 f 1.1 5.6% 29.4 f 1.3 2.8%
1453 f 286 3.8%
366 f 8 0.9%
19.2 32.0 63.9
168 f 17 8.1% 1005 f 40 3.2% 2060 f 88 3.4%
320 639
46.7 f 3.0 5.2% 388 f 6 1.2% 976 f 35 2.8%
"Conditions: [luminol] = 1.31 X lo4 M, pH 9.10 (0.10 M borate), [QTAC]= 0.10 M in 6:5 CHCl,-CeHG, R = 11.1,delay time 3 s, run time 15 s. *95% confidence level. 'Final concentration. dMeasuredwith kifferent instrument sensitivity settings for each run. e Impurities that caused the high blank were thought to be present in the aged bulk solvent (prepared one day prior to use) or in the HC1 solutions stored in a polyethylene bottle.
3t
01
I
-6
I
I
I
-5 log( [luminol]/l'l)
-4
-3
Flgure 7. Effect of luminol concentration on the CL intensity. Conditions were [CTAC] = 0.10 M, R = 11.1, [H202]= 6.40X lo-' M, and pH, 9.03 (0.10M borate).
Analytical D a t a a n d Figures of Merit for Determination of Hydrogen Peroxide. Typical log-log calibration plots for the CL determination of peroxide are shown in Figure 8. In this case, despite the use of the luminometer's highest sensitivity setting, the blank signal was found to be at an acceptably low level (refer to the blank level of run I11 in Table 111). In order to minimize the sensitivity loss caused by the final sample dilution, the calibration experiments were designed as follows: 200 pL of a peroxide reversed micellar sample solution was automatically injected into 100 pL of a luminol-borate buffer reversed micelle solution. For analysis employing this protocol, the linear range of the calibration graphs and their regression equations were determined to be: A (peak height): linear range (5.7-640) X M log (C1intensity) = 9.44 1.22 log [H202], r = 0.994
+
B (integrated CL): linear range (9.3-1900) X lod8M log (integrated CL intensity) = 8.97 1.24 log [H202], r = 0.997 The lower limit (detection limit) was defined as the concentration of peroxide which gives a CL signal equivalent to 3ab, where 'Tb is the standard deviation of the blank signal. The detection limits presented here in the presence of the CTAC reversed micelles are 1 or 2 orders of magnitude poorer than those obtained with catalyzed (or enzymatic) luminol oxidation
+
I
-8
I -7
I
I
-6
-5
I
I
I
-4
-3
-2
l o g ( [H202J/M)
Figure 8. log-log calibration plots for the determination of hydrogen peroxide in 0.10 M CTAC reversed micelles [line A (based on peak height) and line B (based on integrated intensity)] and in an aqueous medium [line C (based on peak height)]. Conditions were [luminol] = 1.31 X lo4 M, pH 9.0f 0.1 (0.10and 0.05M borate for reversed micelle and aqueous systems, respectively), delay time 5 s, and run time 30 s, with a R value of 11.1 for the reversed micelle system.
systems in water if based on an initial peroxide sample concentration basis (34,65, 68-70). The detection limits calculated in terms of the initial aqueous peroxide concentration are 4.4 x lo* M and 7.1 x lo4 M for the peak height and the integrated CL signal, respectively. However, in the reversed micellar media, no catalyst or cooxidant is present and the conditions are much milder compared to the usual situation in aqueous media. When calculated on a final peroxide concentration basis, the CTAC reversed micelle system enhanced the CL production by approximately 3 to 3.5 orders of magnitude compared with that in aqueous solution at similar pH conditions (compare curve C and curve A of Figure 8).
The slope of the log-log calibration plots is about 1.2 in CTAC, which is significantly greater than first order. This is not surprising since the slope has been reported to depend upon such factors as pH, luminol concentration, and type of catalyst used (33). For example, the slope varied from 1.3 to 0.94 in the Cu(I1) catalyzed system, from 1.4 to 1.1 in the microperoxidase or hematin system, and from 2 to 1 in the horseradish peroxidase system (34, 69). This reflects the
ANALYTICAL CHEMISTRY, VOL.
intrinsic complexity of the mechanism and stoichiometry of the luminol-hydrogen peroxide oxidation reaction system (33). Representative precision data that were obtained by using different instrumental sensitivity settings (run I, 11, and I11 in order of sensitivity) are listed in Table 111. The highest sensitivity setting was used for run 111. All precision data presented were calculated at the 95% confidence level. An accidentally high blank signal in the peak height was observed in run I. However, the integral signal seemed to be less influenced. The precision of the reversed micellar modified luminol method is quite acceptable ranging between 0.9 and 12.3% in terms of the relative standard deviation over the peroxide concentration range of (6.39-639) X lo-’ M. The integrated signals appear to be only slightly more precise when compared to the peak height signals (Table 111). The use of a flow injection system (69, 71)is expected to be an attractive alternative to the batch method in terms of providing high accuracy and precision at the lower peroxide concentration levels. The relatively high steady-state CL emission in the CTAC reversed micellar system as previously mentioned will undoubtedly favor such an approach.
CONCLUSION The results demonstrate that the luminol-hydrogen peroxide CL reaction can be successfully conducted under relatively mild conditions (i.e., pH 7.8-9.0) without any added catalyst or cooxidants in the presence of a cationic CTAC reversed micellar system. The reversed micellar mediated luminol procedure should be ideal for the CL detection of enzymatically generated hydrogen peroxide (particularily in view of the fact that enzymes are stable and retain their activity in such media (25-27)). Currently, the main limitation of the luminol reaction is that efficient CL is observed only at high pH while most enzymatic reactions occur at near neutral pH. Consequently, a pH mismatch exists and a sequential reaction approach under two seta of conditions is required (32,33). In the CTAC micelles, this pH mismatch between the two reactions is greatly reduced. Thus, the use of the reversed micellar medium helps to circumvent this problem and allows use of the continuous monitoring method in which the coupling enzymatic reaction and the luminol CL detection reaction are performed simultaneously. This continuous monitoring approach is attractive since it is more convenient, requiring fewer operations and addition of only a single reagent (in which all necessary reactants are combined) (32,33). Additionally, the mild pH requirements in the reversed micelle procedure should help to alleviate interference caused by the reaction of the peroxide generated with any reducing components found in the original samples (23). The use of the CTAC mediated luminol procedure for the determination of biological substrates or enzymatic activities will be described in subsequent papers (72). Registry No. CTAC, 112-02-7; H202, 7722-84-1; luminol, 521-31-3; 3-aminophthalicacid, 5434-20-8. LITERATURE CITED Hinze, W. L. Solution Chemistry of Surfactants; Mittal, K. L., Ed.; Plenum: New York. 1979; Vol. 1, pp 79-127. Armstrong, D. W. Sep. furif. Methods 1985, 14, 213-304. Cline Love, L. J.; Harbafta, J. G.; Dorsey, J. G. Anal. Chem. 1984, 56, 1132A-1148A. Pellzzetti, E.; Pramauro, E. Anal. Chim. Acta 1985, 169, 1-29. Hlnze, W. L.; Singh, H. N.; Baba, Y.; Harvey, N. G. Trends Anal. Chem. 1084. 3 , 193-199. Diaz-Garcia. M. E.; Sanz-Medel, A. Talanta 1986, 33, 255-275. Spurlln, S.; Hlnze, W. L.; Armstrong. D. W. Anal. Lett. 1977, 70, 997-1008. Slngh. H. N.; Hlnze, W. L. Analyst (Lsndon) 1982, 107, 1073-1080. Sanz-Medel, A.; Alonso, J. I. Anal. Chim. Acta 1084, 165, 159-169. Cline Love, L. J.; Grayeski, M. L.f Noroskl, J.; Weinberger, R. Anal. Chim. Acta 1985, 170, 3-12. Cline Love, L. J.; Skriiec, M.; Habarta, J. Anal. Chem. 1980, 52, 754-759. . .. Armstrong. D. W.; Hinze, W. L.; Bui, K. H.; Singh, H. N. Anal. Lett. 1081, 1 4 , 1659-1667.
59,NO. 3, FEBRUARY 1, 1987
503
Kornahrens, H.; Cook, K. D.; Armstrong, D. W. Anal. Chem. 1982, 54, 1325-1329. Armstrong, D. W.; Terrlll, R. Q. Anal. Chem. 1979, 51, 2160-2163. Armstrong, D. W.; Henry, S. J. J . Liq. Chromatogr. 1980, 3 , 657-672. Armstrong, D. W.; Ward, T. J.; Berthod, A. Anal. Chem. 1986, 58. 579-582. Fendler, J. H. Membrane Mimetic Chemistry;Wiley: New York, 1982; Chapters 3, 9, 11, and 12 and references therein. Klopf, L. L.; Nleman, T. A. Anal. Chem. 1984, 56, 1539-1542. Hinze, W. L.; Rlehl, T. E.; Singh, H. N.; Baba, Y. Anal. Chem. 1984, 56,2180-2191. Kato, M.; Yamada, M.; Suzuki, S. Anal. Chem. 1984, 5 6 , 2529-2534. Yamada, M.; Suzuki, S. Anal. Lett. 1984, 17, 251-263. Malehorn, C. L.; Riehl, T. E.; Hinze, W. L. Ana/yst (London) 1986, 117 , 941-947. Lasovsky, J.; Grambal, F. Acta Univ. Palacki. Olomu., Fac. Rerum Nat. 1980, 61-65, 57-61. Chem. Abstr. 1981, 94, 218980g. Fendler, J. H. Acc. Chem. Res. 1976, 9 , 153-161. Khmel’nitskii, Y. L.; Levashov, A. V.; Klyachko, N. L.; Marlinek, K. Russ. Chem. Rev. 1984, 53, 319-331. Reverse Micelles; Luisl, P. L., Straub, B. E., Eds.; Plenum: New York. 1984. Luisi, P. L. Angew. Chem., I n t . Ed. Engl. 1985, 24, 439-528. impexa International B. V. Neth. Appl. NL 82 01 ,713 16 Nov. 1983, 8 pp. Chem. Abstr. 1984, 100, 111995f. Cohen, M. L.; Arthen, F. J.; Tseng, S. S. Eur. Pat. Appl. EP 96,749 28 Dec 1983, 21 pp. Chem. Abstr. 1984, 100, 182970. Yeda Research and Development Co., Ltd. Israeli I L 59,263 30 Nov 1982, 10 pp. Chem. Abstr. 1984, 100, 15173r. Brovko, L. Y.; Ugarova, N. N.; Kiyachko, N. L.; LevaBalyaeva, E. I.; shov, A. V.; Martinek, K.; Berezin, 1. V. Dokl. Akad. Nauk SSSR 1983, 273, 494-497. Chem. Abstr. 1984, 100. 134856a. Seitz, W. R. CRC Crit. Rev. Anal. Chem. 1981 (Dec), 1-58, and references therein. Seitz, W. R. Methods in Enzymology; DeLuca, M. A,, Ed.; Academic: New York, 1978; pp 445-462. Armstrong, W. A,; Humphreys, W. G. Can. J. Chem. 1965, 43, 2576-2584. McCapra, F.; Beheshti, I.Bioluminescence and Chemiluminescence: Instruments and Applications; Van Dyke, K.; Ed.; CRC Press: Boca Raton, FL, 1985; Vol. I,Chapter 2, pp 9-42. Schumb. W. C.; Satterfield, C. N.; Wentwork, R. L. Hydrogen f e r oxide; Reinhold: New York, 1955. Smith, R. E.; Luisi, P. L. Helv. Chim. Acta 1980, 6 3 , 2302-2305. Ei Seoud, 0. M.; Chinelatto, A. M.; Shimlzu, M. R. J . Colloid Interface Sci. 1982, 88, 420-427. Haapakka, K. E.; Kankare, J. J.; Linke, J. A. Anal. Chim. Acta 1982, 139, 379-382. Erdey, L.; Buzas, I.; Vigh, K. Taianta 1966, 13, 463-465. Kertes, A. S.;Gutmann, H. Surface and Colloid Sicence; Matijevic, E., Ed.; Why: New York, 1976; Vol. 8, Chapter 3, p 193-295. Sunamoto, J.; Kondo, H.; Klkuchi, J.; Yoshinaga, H.; Takei, S. J . Org. Chem. 1083, 48, 2423-2424. Sunamoto, J.; Kondo, H. Inorg. Chim. Acta 1983, 79, 6. Magid, L. Solution Chemistry of Surfactants; Mlttal, K. L., Ed.; Plenum: New York, 1979; Vol. 1, pp 427-453. Hihorst, R.; Laane, C.; Veeger, C. R o c . Natl. Acad. Scl. U . S . A . 1982, 79, 3927-3930. Robinson, B. H.; Freedman, R. B.: Oldfield, C. J. Fletcher, P. D. I.; Chem. SOC.,Faraday Trans. 11985, 81, 2667-2679. Nagy, J. B. Solution Behavlor of Surfactants; Mittal, K. L., Fendler, E. J., Eds.; Plenum: New York, 1982; Vol. 11. pp 743-766. Sunamoto, J. Solution Behavior of Surfactants; Mittal, K. L.. Fendler, E. J.; Eds.; Plenum: New York, 1982; Vol. 11, pp 767-790. O’Connor, C. J.; Lomax, T. D. Surfactants in Solution; Mittal, K. L.. Lindman, B., Eds.; Plenum: New York, 1984; Vol. 3, pp 1435-1461. Kato, T.; Fujiyama, T. J . fhys. Chem. 1977, 81, 1560-1563. Kato, T.; Fujiyama, T. Bull. Chem. SOC.Jpn. 1978, 5 1 , 1328-1331. Muller, N. J . Colloid Interface Sci. 1978, 6 3 , 383-393. Auses, J. P.; Cook, S. L.; Maloy, J. T. Anal. Chem. 1975, 47, 244-249. Haapakka, K. E.; Kankare, J. J.; Linke, J. A. Anal. Chim. Acta 1982, 139, 379-383. Lind, J.; Merenyi, G.; Erlksen, T. E. J . Am. Chem. SOC. 1983, 105, 7655-7666. Kalinichenko, I.E.; Pilipenko, A. T.; Borouskii, V. A. J . Gen. Chem. USSR (Engl. Trans/.) 1978, 48, 299-301. Babko, A. K.; Lukovskaya, N. M. Ukr. Khem. Zh. 1963, 29, 479. Vigh, K. Talanta 1966, 13, 463-469. Erdey, L.; Buzas, I.; Weber, K. Chem. Ber. 1942, 75, 565. Gorsuch, J. D.; Hercules, D. M. fhotochem. Photobiol. 1972, 15, 567-583. White, E. H. Light and Life; McElroy, W. D., Glass, B., Eds.; The Johns Hopkins Press: Baltimore, MD, 1961; p 183. Selinger, H. H. Light and Life; McElroy, W. D., Glass, B., Eds.; The Johns Hopkins Press: Baltimore, MD, 1961; p 200. El Seoud, 0. A.; Vieira, R. C.; Chinelatto, A. M. J . Chem. Res. 1984, 80.
Nome, F.; Chang, S.A.; Fendler, J. H. J. Colloid Interface Sci. 1076, 56, 146-158. Bostick, D. T.; Hercules, D. M. Anal. Chem. 1975, 47, 447-450. Kon-no. K.; Kitahara. A.: Fuiiwara. M. Bull. Chem. SOC. Jon 1978. 51, 3165-3169. Babko, A. K.; Dubovenko, L. I.Ukr. Khem. Zh. 1063, 29, 479-484. Seitz. W. R.; Neary, M. P. Anal. Chem. 1974, 46, 188A.
Anal. Chern. 1987, 59, 504-508
504
(89) Olson, B. Anal. Chlm. Acta 1982, 136, 113-118. (70) Kok, G. L.; Holler, T. P.; Lopez, M. B.: Nachtrieb, H. A.; Yuan, M. Environ. Sci. Technol. 1978, 12, 1072. (71) Hwang, H.; Dasgupta, P. K. Anal. Chim. Acta 1985, 170, 347. (72) Hinze, W. L.: Igarashi, s.,submitted for publication in Anal. Lett.
RECEIVED for review May 13, 1986. Accepted September 16, 1986. This research was supported by the National Science
Foundation (CHE-8215508) and, in part, by Wake Forest University through a Research and Publication Fund Grant. We thank a referee for helpful comments. This work was presented at the Symposium on Processes in Organized Assemblies, Santiago, Chile, December 9, 1985 [Abstr. No. 31 and at the 191st National Meeting of the American Chemical Society, New York, NY, April 14,1986 [Abstr. No. ANAL 161.
Use of Specific Bacteria for the Determination of Mutagenic and Carcinogenic Compounds Stanislav MiertuEi,*' Jozef Svorc,' Ernest Sturdjk: and Helena Vojtekovii' Department of Analytical Chemistry and Department of Biochemical Technology, Slovak Polytechnical University, 812 37 Bratislava, Czechoslovakia
PosslMmles of the use of €scher/ch/a CON K-12 in the determination of mutagenic compounds have been tested. Analylkai parameters, e.g., the range of linearity of the analytical curve, the detection limit and the serrsitivlty, the accuracy, and the precision of analysls, have been evaluated for a series of nitrofurans. The detection ilmit Is In the range of lO-'-lO5 mol L-'. Anaiyses of real samples (determination of 5-nltro-2-furyiacrylic ackl in wine and nitrovin in chicken meat) have been completed without any preseparatlon.
The mutagenic and carcinogenic hazards of many chemical compounds are some of the most topical problems in the protection of the living environment. The number of such compounds is still increasing. They are spread in different areas of the living environment-in the atmosphere, water, and soil or as contaminants in food. Many of them are present in industrial processes (1, 2). Analysis of these samples by classical analytical procedures is often complicated due to the complexity of the samples as well as the low specificity and sensitivity of the methods. That is why more sophisticated methods are urgently needed. As regards the specificity of analytical methods, biological systems belong to the most specific ones. This possibility also exists in the case of sensitive microorganisms with a specific response to the mutagenic compounds. Certain bacteria, e.g., Salmonella typhimurium (3)or Escherichia coli (4-7)) have been frequently used for the detection of mutagenicity. However, there have only been a few attempts to use that systems in the analytical determination of mutagenic compounds. Karube et al. (8) have constructed a Salmonella microbial electrode and correlated the current decrease with mutagen concentration. The goal of the present work is to study the possibilities of the use of the response of biological systems, namely, bacteria Escherichia coli K-12, to the mutagenicity of chemical compounds for analytical purposes. Recently, Quillardet et al. (4-6, 9) have specifically manipulated bacteria strain E. coli K-12 to produce SOS response after DNA damage by mutagenic compounds (the so-called SOS chromotest) (Figure 1). The SOS response is manifested by increased production 'Department of Analytical Chemistry. Department of Biochemical Technology.
of enzyme /3-galactosidase. For details of genetic manipulation, see original papers of Quillardet et al. (4,6).The amount of produced enzyme correIates with the amount of mutagenic compound. The level of enzyme alkaline phosphatase is also determined. This enables detection of the proteosynthesis inhibition, which can occur at higher concentrations of mutagenic compounds (4). The direct assay consists of incubating the tester strain with increasing concentrations of the agent to be tested. After a time for protein synthesis, @-galactosidase and alkaline phosphatase are determined. The SOS chromotest has been exclusively used until now for tests of mutagenic potency and sensitivity to various compounds, namely, benzofurans, naphthofurans, nitrosamines, fungal toxins, and antibiotics (4, 6). In spite of the fact that there is quantitative relation between SOS response and amount of mutagenic compound, there has been no attempt until now to use this specific bacteria for analytical purposes, i.e., for determination of the amount of mutagenic compound. That is why we focus our attention in this paper to the study of analytical application of the SOS chromotest. We have studied in detail the following analytical parameters: (1) the linearity range of dependence between the concentration of mutagen and response of bacteria; (2) detection limits and sensitivity for different compounds; (3) accuracy and precision of the analysis. The above-mentioned tasks have been solved for 10 nitrofurans. This type of compounds was chosen for the following reasons: (i) the mutagenic potency of these compounds has not been tested by SOS chromotest; (ii) they are considered by many authors as a representative direct mutagens (10); (iii) they are still widely used in clinical and veterinary medicine and in the food industry (10, 11). We have also examined the possibilities of determination of mutagenic compounds in real complex samples without any preseparation. We have determined 5-nitro-2-furylacrylicacid (NFAA) directly in wine. This compound was used as a wine stabilizer (12). In the second example, nitrovin (used as a growth stimulator (11))is determined directly in chicken meat.
EXPERIMENTAL SECTION Chemicals. The formulas of the studied nitrofurans are shown in Table I. 5-Nitro-2-furylacrylicacid (NFAA) (1)was obtained from Slovakofarma Hlohovec (Czechoslovakia). Alkyl estersand amides of NFAA (2-9) were synthesized according to Sturdik et al. (13, 14). Nitrovin (9) was obtained from Chemapol Praha (Czechoslovakia),and nitrofurantoin (10) was purchased from Sigma (St. Louis, MO). Other chemicals of analytical grade
0003-2700/87/0359-0504$01.50/0 C 1987 American Chemical Society
