ACKNOWLEDGMENT We thank Ann Elofson for assistance with portions of the experimental work.
Received for review September 18, 1972. Accepted December 6, 1972. Financial support by the National Research Council of Canada and by the University of Albert a is gratefully acknowledged.
Selective Determination of Copper(l1) in Aqueous Media by Enhancement of Flash-Photolytically Initiated Riboflavin Chemiluminescence E. L. Wehry’
and Arthur W. Varnes2
Departments of Chemistry, University of Tennessee, Knoxville, Tenn. 37976, and lndiana University, Bloomington. Ind. 47407
Copper(l1) is determined in aqueous media (pH 6.0) by measuring its enhancement of riboflavin chemiluminescence in systems containing hydrogen peroxide and pdioxane. The chemiluminescence is initiated by flash photolysis of the reaction system. Interfering ions are Co(ll), Ag(l), Hg(l), and Hg(ll); other common metal ions do not significantly interfere. The effects of other experimental parameters (anions, organic solvents, pH, buffer composition, initiating flash energy) are evaluated. The minimum detectable quantity of copper is 30 nanograms. The sensitivity and selectivity of light- and chemically-induced riboflavin chemiluminescence methods are compared; the light-induced system is concluded to be superior. The results suggest that flash photolysis, widely employed in mechanistic photochemistry, also has useful analytical potentialities.
As research in trace-metal analysis proceeds, it is becoming increasingly apparent that selectivity, as well as high sensitivity, is a critical analytical criterion. Chemiluminescence methods have recently been recognized as a valuable approach to selective, sensitive analyses. The metal-catalyzed oxidation of luminol has received considerable analytical attention ( I ) ; approaches to selectivity in the luminol system (2) and experimental apparatus for monitoring chemiluminescence (3, 4 ) have recently been described in detail. It has been reported (5-7) that riboflavin, in the presence of hydrogen peroxide, exhibits chemiluminescence, the intensity of which is significantly enhanced by copper(I1) and cobalt(I1) but is relatively unaffected by other common metal cations. The chemilumiITo whom correspondence should be addressed a t the University of Tennessee. 2NDEA Fellow, 1965-68; NIH Fellow, 1968-69 at Indiana University; present address, St. Andrews Presbyterian College, Laurinburg, N.C. 28352. (1) A. K . Babko, Z. Ana/. Chem.. 200,428 (1964). (2) W. R. Seitz, W . W . Suydarn, and D. M . Hercules, Anal. Chem.. 44, 957 (1972). (3) R. E. Santini and H. L. Pardue, ibid., 42, 706 (1970). (4) R . Bezman and L. R . Faulkner, ibid.. 43, 1749 (1971). (5) R. H . Steele, Biochemistry. 2, 529 (1963). (6) J. E. Vorhaben and R . H . Steele, Biochem. Biophys. Res. Commun.. 19, 187 (1965). (7) J. E . Vorhaben and R. H . Steele. Biochemistry. 6, 1404 (1967).
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nescence can be initiated either by addition of a reducing agent, such as ascorbic acid (7), or by irradiation with visible light [“photoinduced chemiluminescence” (511. The purpose of the present paper is to delineate the conditions under which the metal-enhanced photoinduced chemiluminescence of aqueous riboflavin-HzOz solutions may be employed for selective, sensitive metal-ion analyses. The light source employed is a microsecond-duration xenon flashtube.
EXPERIMENTAL Apparatus. The photoinduced chemiluminescence of riboflavin is very weak when standard continuous light sources are employed, but a rather intense chemiluminescence results from flash excitation. Light from a single Xenon Corp. FP-5-100C linear xenon flashtube, fired by a Xenon “Model A” micropulser, was filtered by a sheet of borosilicate glass and then impinged upon a stemmed 1-cm rectangular spectrophotometer cell (Pyrocell No. 60054). The lamp-filter-cell assembly was contained in a plastic housing constructed in such a way that the cell stem protruded through the top of the assembly. Chemiluminescence was measured a t right angles to the direction.of the initiating flash. An electrically-activated shutter, triggered by the light flash via a Xenon Model D delay line, was opened 0.5 sec after the initiating flash; inasmuch as the flash durations used were never greater than 500 rsec, the flash had completely decayed before the shutter was opened. Beyond the shutter was placed a Baird-Atomic interference filter (nominal wavelength 540 nm; bandwidth 21 nm) and an Ealing 22-8056 collimator. Light which traversed the filter-collimator system impinged on an RCA 1P28 photomultiplier, operated a t 0.8 kV, enclosed in a Schoeffel D500 housing; the power supply and amplifier was an Aminco 10-280 “photomultiplier microphotometer.” The amplifier output was displayed as a function of time on a Tektronix 555 oscilloscope or the time base of an Aminco 1620-827 X-Y recorder. Reagents. Purification of riboflavin has been described (8). Metal perchlorate and chloride salts were purified by recrystallization of the reagent-grade material from a dilute aqueous solution of the appropriate mineral acid. Water was distilled in a borosilicate glass apparatus, then treated with Bio-Rad “Chelex 100’’chelating resin and stored in polyethylene containers until used. No detectable luminescence blank was introduced by use of the chelating resin, but it was necessary to use the water within one week of purification to avoid introduction of a significant blank (presumably from impurities leached from the plastic bottles). Dioxane (MC/B “Spectroquality”) was used as received. Hydrogen peroxide (3070, reagent grade) was treated with BioRad “Chelex 100” and stored in polyethylene containers until used. Other chemicals were reagent grade, used without additional purification. (8) A. W . Varnes, R . B. Dodson, and E. L. Wehry, J . Amer. Chem. Soc., 94, 946 (1972).
i
I
I
I
i
600
H
4 , t
Time A f t e r Flash (seconds)
in riboflavin, 0.78M in dioxane, and 0.85M in H202: , no C u ( l l ) present; - - - - - , solution 3.33 X iO-5M in
X 10-5M
CU(c104)z Procedure. In the usual procedure, 1.5 ml of a solution 1.2 X 10- 4M in riboflavin in p H 6.0 phosphate buffer (9),1.0 ml of the metal-ion solution to be analyzed, and 0.2 ml of dioxane were successively added by syringe (equipped with a Teflon (Du Pont) needle) to the cell (the stem of which was covered by a Serum cap). The cell contents were then bubbled with 0 2 for 2 min, following which 0.3 ml of 8.5M H202 was injected into the cell. Immediately following addition of the hydrogen peroxide, the light flash was fired.
RESULTS AND DISCUSSION General Chemiluminescence-Time P a t t e r n a n d Response to Cu(I1). The measured chemiluminescence-time pattern is shown in Figure 1. The maximum chemiluminescence intensity was normally observed ca. 7 sec following the initiating light flash, followed by prompt decay to zero intensity over a total period of 20-25 sec. The intensity-time behavior is generally similar to that reported by Steele and coworkers (5-7) for steady-state illumination, except that the duration of the flash-induced chemiluminescence is much shorter. The peak chemiluminescence intensity is enhanced by a factor of at least lo3 when initiated by a 250 J , 35 psec flash, compared to that observed for activation via continuous irradiation with a 150-W xenon lamp. In the presence of Cu(II), the general appearance of the intensity-time profile was unchanged (except for a slightly slower decay to zero intensity), and the peak intensity was greatly increased (Figure 1). A linear relationship existed between the logarithm of the "corrected" peak chemiluminescence intensity [i.e., the chemiluminescence peak height for a sample minus that for a blank treated identically but containing no Cu(II)] and the logarithm of the Cu(I1) concentration, as shown in Figure 2. The useful linear range of the curve in Figure 2 was from 5 x 10-7~1-2 x 1 0 - 4 ~ . Effects of Other Metal Ions. Table I lists the influence of other metal ions X n + (added as the perchlorate) upon the peak chemiluminescence intensity, of solutions 6.0 X 10-5M in riboflavin, 0.78M in dioxane, 0.85M in H202, 3.33 x lO-5M in Cu(C104)2, and 3.33 x lO-5M in X(C104),. In general, the chemiluminescence peak height is remarkably insensitive to other metal ions. A slight, but significant, enhancement is noted for Fe(I1) and a more significant enhancement is produced by Co(I1). The ions Hg(I), Hg(II), and Ag(1) all seriously inhibit the chernilu(9) H . T S. Britton. "Hydrogen Ions." Chapman and Hall, London, 1929, pp 182-84
1
I
i
4
5
6
7
-Log [Cu(II)I
Figure 1. Chemiluminescence intensity vs. time for solutions 6.0
-_
i
3
Figure 2. Relationship between "corrected" chemiluminescence peak intensity and Cu(ll) concentration. I denotes peak intensity in presence of Cu; l o denotes blank intensity. Concentration values refer to those for C u ( l l ) solutions prior to injection into reaction cell
Table I. Cation Interference Tests Peak intensity (arbitrary units) ~
With C u ( l l ) "
Without C u ( l i ) b
550 545 550 550 545 560 545 525 545
45 47 44 48 45 43 50 42 44
585
51
535 640 540 550 560 535 230 565 460 380
45 110
46 45 44 43 39 46 38 17
Performed by adding solution 1.00 X 10-4M in Cu(C104)2 and 1.00 X 10-4M in X(C104)n to riboflavin-dioxane solution (see Experimental). Performed by adding solution 1.00 X 1 0 - 4 M in X(CI04), Id reaction mixture. Q
minescence. The mechanism for production of chemiluminescence in the riboflavin-H202-Cu(II) system is still unclear, though rather strong evidence has been presented for involvement of a complex of Cu(1) with riboflavin (or an 0 2 or HzO2 adduct of this complex) (10, 11). It is noteworthy that all ions which significantly influence the chemiluminescence intensity [except Co(II)] have been demonstrated to be among the very few metal cations which complex riboflavin in aqueous media (12, 13). The (10) M. 0. Stone, J. E . Vorhaben. and R. H . Steele, Biochem. Biophys. Res. Commun., 36,502 (1969). (11) M. 0.Stone and R. H . Steeie, Biochemistry. 9,4343 (1970). (12) P. Hemrnerich. F. Muller, and A. Ehrenberg, in "Oxidases and Related Redox Systems," Vol. 1 , T. E. King, H . s. Mason, and M . Morrison, Ed., Wiley. New York, N . Y . . 1965, p 157.
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Table II. Effect of Added Solutes on Peak intensity Added solutea
Table 111. Chemiluminescence Peak Intensities in pH 6.0 Buffersa
Peak intensity (arbitrary units)
Cu(Cl04)2 only
550 545 540 550 51 5 490 70 535 555 530 520 95 460 510 560 420 535 455 20 65 340
NaC104 NaC2H302 NaF NaCl NaBr Nal NaN03 NazS04 Na2S03 NaSCN
NaCN NaN02 K103
KC103
Potassium oxalate NaHC03 "3
EDTA 1 ,lo-Phenanthroline
8-Hydroxyquinoline
4
5
i
I
PH
6
7
I
I
1
I
1
I
500L
.- 4 0 0 1 n
480 475 380 260 175
550
*
700 600
Peak intensity (arbitrary units)
a All solutions were 6.0 X 1 0 - 5 M in riboflavin, 0.78M in dioxane, 0.85M in H 2 0 2 , and 3.33 X 1 0 - 5 M in C U ( C I O ~ ) ~Recipes . given in Refs. 9 and 15.
a Solution 1.0 X lO-'M in Cu(C104)2and 1.0 X 10-3M in solute added to reaction mixture as described in Experimental.
r
Buffer componentsb
Na2HP04-NaH2P04 NaH2P04-borax NazHP04-citricacid Potassium hydrogen phthalate-NaOH Sodium acetate-acetic acid Succinic acid-sodium succinate
i
noted in Figure 3, the nominal flash energy has surprisingly little effect upon the observed chemiluminescence, though a t very high flash energies the chemiluminescence decreases sharply [presumably because intense flashes destroy a substantial portion of the riboflavin (1411. Data of the type shown in Figure 3 cannot be easily applied to other laboratories, because the wavelength distribution of the flash, the geometry of the lamp-filter-cell assembly, and the cell thickness all influence the effective rate of light absorption of the flashed solution. The data demonstrate a plateau region in which the chemiluminescence intensity is effectively independent of flash energy; hence, small fluctuations in lamp output (which are virtually impossible to control in high-energy flash discharges) do not necessarily introduce corresponding imprecision in the measured chemiluminescence intensities. Effect of pH. The influence of p H (Figure 3) is similar to, though less dramatic than, that reported by Steele (7). The optimum pH range is 6.0-7.0, though precipitation of metal hydroxides may render it necessary to employ pH < 6 for some samples. As shown in Table 111, the nature of the buffer components (15) also influences the peak intensity, with the H2P04--HP042- buffer clearly the one of choice for maximum sensitivity. Influence of H 2 0 2 Concentration. Figure 4 shows the influence of H202 concentration upon chemiluminescence peak intensity. The H202 concentration routinely employed was 0.85M. Effects of Organic Solvents. Steele (7) has reported that addition of organic solvents decreased the peak intensity of chemiluminescence initiated by continuous exposure to light, whereas Stone and Steele (11) noted an enhancement of thermally-activated chemiluminescence when solvents such as dimethyl sulfoxide and dioxane were added to solutions containing riboflavin, H202, Cu(II), and ascorbic acid. We have found that several organic solvents (most notably dioxane, tetrahydrofuran, or acetone) reproducibly enhance the peak intensity of flashinduced chemiluminescence, provided that they are added to the reaction mixture prior to injection of hydrogen peroxide. The data are listed in Table I\'. Dioxane was routinely added to solutions (see Experimental) in order to exploit its enhancement effect. Effect of Oxygen. While quantitative studies were not performed, it was clear that the peak chemiluminescence intensity was enhanced by saturating the riboflavinCu(I1)-dioxane solutions with oxygen prior to injection of H202. Conversely, bubbling the solutions with N2 prior to addition of H202 decreased the peak intensity by a factor of 6-15, depending upon the sample composition. Precision. Because the peak intensity is dependent upon a plethora of variables, it is necessary to ascertain I
100
c
01 0
100
I 200
300
I 400
I / 500
Nominal Flash Energy (Joules)
Figure 3. Variation of chemiluminescence peak intensity with nominal flash energy (lower abscissa, 0 ) and with p H (using citrate-phosphate buffers (75);upper abscissa, 0 )
effect of Co(II), initially noted by Vorhaben and Steele (61, cannot now be rationalized, for Co(I1) interacts only weakly with excited states of riboflavin (8) and no interaction whatsoever between Co(I1) and the ground-state riboflavin molecule has been detected (8, 12). In view of the fact that all entries in Table I pertain to equal concentrations of Cu(I1) and the interfering ion, the selectivity of the phenomenon for copper is quite remarkable. Influence of Anions and Complexing Agents. As noted in Table 11, C104-, F-, acetate, SO42-, and C103- have no significant effect upon the peak intensity. Most other ions exert an inhibitory effect, most notably I - , NOz-, and CN-. Species capable of chelating Cu(I1) also inhibit the chemiluminescence. Influence of Initiating Flash Energy. Because the chemiluminescence is triggered by a light flash, possible effects of the flash intensity are of critical importance. As (13) I. F. Baarda and D. E. Metzler, Biochim. Biophys. Acta, 50, 463 (1961).
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(14) M. Green and G. Tollin, Photochem. Photobid.. 7,129 (1968) (15) C.Long, "Biochemists' Handbook," Van Nostrand, New York, N.Y., 1961, pp 30-41,
I
I
I
I
600
Table I V . Influence of Organic Solvents upon Peak Chemiluminescence Intensity Solventa
None Dioxane Tetrahydrofuran Acetone Dimethyl sulfoxide Acetonitrile Dimethylformamide Methanol Triethanolamine
Peak intensity (arbitrary units)
370 550 51 5 480 425 360 260 225 20
a Added. in 0 2-ml volume. to reactton mixture as described in Experimental.
Effect of H202 concentration upon chemiluminescence peak intensity. All solutions were 6.0 X 1 0 - 5 M in riboflavin, 0.78M in dioxane, and 3.33 X 10-5Min C U ( C I O ~ ) ~ Figure 4.
that: the precision is adequate when the experimental parameters are controlled as closely as possible. In the most demanding test, fifteen successive measurements, using aliquots of a sample solution 1.00 x 1 0 - 4 ~in Cu(C104)2 and 1.00 x 10-4M in NaC104 (following the procedure outlined in Experimental), yielded a peak intensity of 545, with a standard deviation of f18,arbitrary units. It is evident that, if buffer composition, order of reagent addition, and quantity of organic solvent are carefully reproduced, the procedure exhibits quite satisfactory precision. Sensitivity. As noted previously, the useful linear range of a logarithmic corrected intensity us. Cu(I1) concentration plot is 2 X 10-4-5 X 10-7M. Inasmuch as a sample volume of 1.0 mi is employed, the effective detection limit for Cu(II) is approximately 30 nanograms. The sensitivity of the riboflavin chemiluminescence procedure is substantially less than that of chemiluminescence methods for metal ions based on the luminol reaction (1, 2). However, it appears rather difficult to render the luminol system selective for Cu(I1) in the presence of other cations (1). Standardization. Because sample constituents (especially anions and complexing agents) can influence the peak intensity, one must empirically determine the relationship between intensity and Cu(I1) concentration. This is most conveniently accomplished by a “standard addition” procedure wherein a known quantity of Cu(C104)2 is added to an aliquot of the sample solution. This aliquot, and an “unstandardized” aliquot of the same sample, are then treated identically in successive runs, with the “copper response factor” being computed from the difference in peak intensity recorded for the two solutions. The riboflavin is destroyed in the reaction sequence; hence, each solution can be subjected to only one flash. [The riboflavin is destroyed as a result of chemical reactions initiated or accelerated by the flash, not by the flash itself. Destruction of riboflavin is also observed in thermally-induced chemiluminescence experiments (16). ] Finally, it must be noted that thermally induced chemiluminescence in systems containing riboflavin, H202, and (16) R. D. Towner, H . A. Nsufeld, and P. 6. Shevlin, Arch. Biochem Biophys., 137, 102 (1970).
reductant such as ascorbic acid or 2-mercaptoethanol is also enhanced by the presence of Cu(I1) in .a rather specific manner (7). Despite its substantially greater experimental simplicity, we do not find the thermally-initiated chemiluminescence to be as analytically effective as the photoinduced chemiluminescence for Cu(I1) determinations, for four reasons. First, the thermally-activated chemiluminescence is, in our hands, at least a factor of 102 less intense than the flash-induced chemiluminescence. Second, for reasons which are presently unclear, enhancement of the photoinduced chemiluminescence intensity is much more selective for Cu(I1) than is the darkinduced luminescence. Third, an “incubation” period of several hours is needed to achieve maximum intensity in the riboflavin-H~0~-Cu(II)-ascorbicacid system (1I ) . Finally, our studies indicate that the inhibitory effects of anions (especially Br-, I-, and NOz-) are substantially greater in the thermally-induced system. In view of the exceedingly complex series of events which occur in both the thermally- and photo-induced chemiluminescence systems ( I l ) , no convincing rationalization of these observations can now be advanced. It is, however, clear that the photoinduced chemiluminescence procedure exhibits by far the greater potential for selective, sensitive determinations of Cu(I1). Finally, it is noteworthy that, although steady-state photochemical procedures are experiencing increased analytical use (17), the present study appears to be the first application of flash photolysis to analytical chemistry. The results obtained in the riboflavin-Cu(I1) system strongly suggest that flash photolysis may be broadly useful as a method of initiating reactions for chemical analysis. a
ACKNOWLEDGMENT We thank P. Stoyanov for use of the electrically-actuated shutter system. Received for review October 18, 1972. Accepted December 14, 1972. Presented in part a t the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 9, 1973. Financial support by the National Science Foundation (Grants GP-8705 and GP26109) is gratefully acknowledged. (17) J. M. Fitzgerald, “Analytical Photochemistry and Photochemical Analysis,” Marcel Dekker. New York, N.Y., 1971.
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