can be obtained for 4 x 10P8M T1 (equivalent to 1 x 10-8M P b or Cd) by a 4-minute plating at a flow rate of 8.4 ml/minute. This requires a sample volume of 33.7 ml to go through the TMCGE. The same sensitivity can be obtained by plating for 6Y2 minutes at 2.1 ml/minute or 12 minutes a t 0.42 ml/minute requiring 13.7 and 5 ml; respectively. Interferences. In sea water, lead and cadmium strip out of mercury at potentials close to the stripping peak potential for thallium. EDTA was added to eliminate possible lead and cadmium interferences. The logarithmic formation constants for Pb(II) EDTA, Cd(I1) EDTA, and Tl(1) EDTA are 17, 16, and 5.8, respectively (14). In 45% ( 1 4 ) L. G . Sillen and A. E. Martell, "Stability Constants," Spec. Publ. 17, The Chemical Society. London, 1964.
No.
sea water, 5% 0.2M EDTA, no peak is observed for lO-5M P b or Cd plating for 15 minutes at -1.05 volts at a flow rate of 8.4 ml/min. A small peak was observed for lO-5M copper under these conditions, but the peak appeared around -0.4 volt, sufficiently positive not to interfere with thallium.
Received for review September 25, 1972. Accepted December ll, 1972. One of the authors (R.J.) acknowledges support from NSF Undergraduate Research Participation Grant No. GY-8791. Use of trade names does not imply endorsement by the Environmental Protection Agency or the Southeast Water Laboratory.
Determination of Phenols and Aromatic Amines by Direct Titration with Bromine in Propylene Carbonate Richard D. Krause and Byron Kratochvil
Department of Chemistry, Universityof Alberta, Edmonton, Alberta, Canada J6G 2G2 Propylene carbonate is used as a medium for bromine substitution reactions. A series of aromatic amines and phenols were determined with accuracies of about 1% and precisions of a few ppt. A base such as pyridine must be present to accept protons released in the substitutions. Advantages include rapidity of the reactions, solubility of reactants and products, and convenient standardization of bromine with solutions of bromide. The log formation constant of Br3- in propylene carbonate at zero ionic strength is 7.37.
Bromine substitution reactions are widely used for the quantitative determination of phenols and aromatic amines (1). The procedure commonly involves addition of excess bromate-bromide reagent to an acidified aqueous solution of the sample, addition of iodide after a suitable reaction time, and titration of the liberated iodine with standard thiosulfate solution. Alternatively, a strongly acidified solution of the sample can be titrated with standard bromate-bromide reagent until free bromine is observed visually f2), spectrophotometrically (3), or by a spot reaction on starch-iodide paper. The bromate-bromide method requires use of aqueous solutions, which limits its applicability to water-soluble phenols and amines. Ingberman, investigating glacial acetic acid as solvent, found that bromination of phenol was complete in 20 minutes if catalytic amounts of pyridine were added ( 4 ) . Huber and Gilbert titrated several phenols in 90% glacial acetic acid-10% pyridine directly with 0.15M bromine in glacial acetic acid (5). End points were determined by constant-current potentiometry. Ashworth, "Titrimetric Organic Analysis," Vol. I , Interscience, NewYork, N . Y . , 1965, p 135; Vol. I I , p214.
(1) M. R. F.
(2) /bid., Vol. I , p 119. (3) P. B. Sweetser and C. E. Bricker,Anal. Chem., 24, 1107 (1952). (4) A. K. Ingberrnan,AnaLChem., 30, 1003 (1958). (5) C. 0. Huber and J. M. Gilbert, Anal. Chem., 34, 247 (1962).
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ANALYTICAL CHEMISTRY, VOL. 45, NO. 6 , M A Y 1973
This paper describes the ure of propylene carbonate (PC) as a solvent for a number of analytical bromine substitution reactions. Among the advantages of PC are its resistance to chemical attack by halogens (6) and its high dielectric constant, 65, which increases reaction rates. It has a wide liquid range (-49 to +242 "C), is colorless, odorless, and nontoxic, and is not appreciably hygroscopic. Its principal disadvantages are that it hydrolyzes fairly rapidly in the presence of strong acids or bases, and that it has a moderately high viscosity (2.5 cP). Further information on properties and reactivity is available from the supplier (6). Most phenols and aromatic amines, as well as their bromination products, are soluble in PC. In many instances, a base must be present to accept protons displaced by bromine in the substitution step. Several unusual reaction stoichiometries were observed.
EXPERIMENTAL PC (Jefferson Chemical Co.) was distilled at a pressure of 0.01 mm mercury on a 48- by 1-in. vacuum-jacketed column packed with nichrome helices (Podbielniak size-C Heli-Pak), The vacuum-jacketed still head contained a solenoid-operated glass valve set a t a 1O:l reflux ratio. The purity of the distilled PC was monitored by ultraviolet spectroscopy; the fraction kept had an absorbance of less than 0.3 at 250 nm in a 1-cm quartz cell, measured against a distilled water blank. In a typical distillation, the first 800 and last 200 ml of a 2000-ml charge were discarded. Liquid chromatography (Chromasorb W packing) of the fraction retained revealed one unidentified small impurity peak just ahead of the major peak. Solid phenols and aromatic amines were sublimed once at reduced pressure and stored in a desiccator. Aniline and p-phenetidine were distilled on a 30-cm Vigreux column at atmospheric pressure. Tetraethylammonium bromide was recrystallized from ethanol and dried 12 hr at 90 "C under vacuum. Other chemicals were reagent grade. Titrations were performed on a Metrohm Model E436 automatic titrator equipped with a 5-ml buret. Bromine titrant was stored in a low-actinic glass flask fitted with a Teflon (Du Pont) stopper. Venting the flask to the atmosphere only during withdrawal of ti(6)
Propylene Carbonate Technical Bulletin, Jefferson Chemical Compan y , Houston, Texas.
Table I. Stoichiometries in Titration of Phenolic Compounds with Bromine in Propylene Carbonate as a Function of Amount of Pyridine Presenta
>
Ratio of moles pyridine to moles phenolic compound Compound titrated
Phenol 2-Naphthol p-Nitrophenol Salicylic acid Methyl salicylate p-Cresol Thymol Resorcinol
2:l
3.86 3.93 3.77 4.04 3.73 3.79 3.84 4.19 =Table values are moles of the end point.
6:1
4.1
3.1
8:1
W
1O:l
5.85 5.97 5.99 5.99 5.92 4.00 3.98 3.98 4.00 3.98 3.96 3.99 4.00 3.96 3.99 4.61 4.67 4.54 4.49 4.43 4.13 4.05 ... 4.02 ... 5.78 5.97 5.98 5.99 6.01 5.74 5.97 5.98 5.99 5.98 4.86 7.68 9.56 10.01 10.02 bromine per mole of phenolic compound at
trant minimized bromine loss through volatilization. The rate of titrant delivery was about 2 ml per min; this rate was automatically decreased in the end-point region. The titration cells were 4- by 10-cm cylindrical glass weighing bottles with machined Teflon covers. A 1-cm2 platinum flag was used as the indicating electrode. The reference electrode, a silver wire in 0.01M silver perchlorate in PC, was placed in a glass tube sealed to a short length of porous Vycor rod, and the rod immersed in a bridge solution of 0.1M lithium perchlorate in PC. The bridge was separated from the solution being titrated by a ' 0.9- to 1.4-micron glass frit. Magnetic stirring was used. Samples typically ranged in size from 0.3 to 1 millimole, and were dissolved in approximately 25 ml of PC. Values for E"' and the conditional formation constant were obtained from bromine-bromide titrations performed at 25 f 0.5 "C. Potentials were measured with an Orion Model 801 digital pH meter.
RESULTS AND DISCUSSION Bromine-Bromide Equilibrium in PC. Bromine solutions in PC are conveniently standardized by titration of bromide. Titration of tetraethylammonium bromide with bromine in PC yields a potential break a t a 1:l mole ratio corresponding to formation of tribromide ion. Formal potentials of the tribromide-bromide and bromine-tribromide couples in PC, obtained from three titration curves, are Eo'(Br3-/Br-) = -0.31 V (std dev = 0.01) and Eo'(Br2/Br3-) = 0.32 V (std dev = 0.01), both measured against a silver-0.01M silver perchlorate in PC reference electrode. The conditional equilibrium constant for the reaction Br- + Br,
__
Br,-
200
E.
t
100
Ly
W Y
Ly Ly
0 0)
Q > y1
-100
20
40
60 80 [BrZ] 1 [PHENOL]
100
12 0
Figure 1. Effect of varying [pyridine]/[phenol] ratios on the potentiometric titration of phenol with bromine in propylene carbonate
PC and 250 ml of 0.01M H N 0 3 in deionized water. The titrant, 0.5M bromine in PC, was standardized daily by titration of 25-ml aliquots of this standard bromide solution. The relative standard deviation of replicate titrations of this system on the automatic titrator was 2 ppt or better. When stored in a closed system to minimize bromine volatilization, and protected from light, the titer of 0.5M solutions of bromine in PC decreased less than 0.3% per day. In contrast, solutions exposed to normal laboratory illumination decreased on the order of 2% per day. Solutions prepared from commercial PC as received also decreased in titer more rapidly than solutions prepared from distilled material. Bromination of Phenols. Addition of excess bromine to phenol in P C does not result in bromine substitution because PC is not a sufficiently strong base to accept the protons released in the reaction. On addition of 3 or more equivalents of a base such as pyridine to each equivalent of phenol in the solution, however, formation of tribromophenol is rapid and complete. The stoichiometry is 6 to 1, owing to the stability of tribromide ion in PC. The overall reaction in the presence of pyridine can be written
PH
?H
(1)
was calculated by the expression 2 log K , = LE"'(Brz/Br,-) - E " ' (Br3-/Br-)] (3) (0.059 1) (2) Values for log K f of 7.19, 7.28, and 7.31 were obtained a t 25 "C a t ionic strengths of 0.05, 0.025, and 0.017 (ion pairing assumed negligible). Extrapolation of these values to zero ionic strength gave a thermodynamic log K f value of 7.37. The enthalpy of reaction of bromine with bromide in PC was determined from the temperature dependence of K,. Titrations a t 0, 25, and 50 "C a t an ionic strength of 0.06 yielded log K , values of 7.39. 7.23, and 7.09, giving a value for AH of -2.42 f 0.05 Kcal per mole. Standardization and Stability of Solutions of Bromine in PC. The exact concentration of a 0.05M solution of tetraethylammonium bromide in P C was determined by precipitation of silver bromide in a mixture of 25 ml of
where Py is pyridine and PyH+ is pyridinium ion. Stoichiometries in titrations of representative phenolic compounds are listed in Table I, along with the effect of varying amounts of pyridine. Figure 1 shows the effect of pyridine on the shape of the potentiometric titration curve of phenol with bromine. The decrease in potential after the end point with increasing amounts of pyridine appears to be caused by formation of a pyridine-bromine complex. When less pyridine is present than is required to combine with the replaceable protons of the phenol being brominated, the titration break occurs after addition of 2 moles of bromine per mole of pyridine. Thus bromination stops after all the pyridine present is protonated. The ratio of bromine to pyridine is 2: 1 because of formation of one tribromide ion for each proton released in the bromination. Bromine uptake for brominations of phenol, 2-naphthol. p-nitrophenol, and methyl salicylate is as expected. In the presence of excess pyridine, phenol consumes 6 moles and ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 6, M A Y 1973
845
Table 11. Titrations of Aromatic Amines with Bromine in Propylene Carbonate First end point Compound titrated
[Br~]/[amine]ratio
p-Nitroaniline Anthranilic acid Anthranilic acidb Aniline Anilineb
rn-Phenylenediamine rn-Phenylenediamineb p-Toluidine p-To1uidine* ,+Phenetidine p-Phenetidineb
Second end point
Approx. break, rnV
Midpt. pot., mV
[Br*]/[amine] ratio
4-150
...
...
150
+110 -150
4.1
50
-250
2.00a
150
4.00" 3.00"
50 50
1.07 2.00" 1.4-1.6 0.6-0.8 1.0-1.1 1.14
500
-325 -300
2.6
300
-400
6.00a
-300 -275 -350 -350
1.7-1.8 4.00" 1.8
0.47-0.52 150 0.9-1.1 100 0.46-0.4 7 200 1 .OO" 200 End point recommended for analytical use. Excess pyridine present.
*
the other three compounds 4 moles of bromine. Thus, all free positions ortho or para to the hydroxyl group are brominated stoichiometrically. Salicylic acid consumes about 15% more bromine than the expected 4 equivalents. This may be the result of partial substitution of the carboxylic acid group by bromine, a common side reaction in the bromination of hydroxybenzoic acids (7). Reduction of the temperature during titration to -20 "C reduced the overconsumption of bromine to about 8%, but further reduction of the temperature gave poorly defined titration curves. Titration stoichiometries for bromination of p-cresol, thymol, and resorcinol were also higher than expected. With the assumption that the unoccupied ortho and para positions are the only ones brominated, the first two compounds should consume 4, and resorcinol 6, moles of bromine. Instead the amounts are 6 and 10. The additional uptake of bromine can be explained by assuming replacement of the hydroxyl-group protons of these compounds by bromine. For example, the reaction for resorcinol could be written OH
-"eBr OBr
+
OH
10Brl
+
5Py
+
H
5Br;
+
5PyH*
(4)
OBr
irr
These products would be analogous to the tribromophenol bromide (CeHZBr30Br) formed in aqueous solutions of phenol containing excess bromine (8). Thus, compounds that have electron-releasing substituents on the aromatic nucleus in addition to the first hydroxyl group can undergo substitution of the hydroxyl proton. Indirect evidence of interaction of the hydroxyl groups is given by disappearance of the 0 - H stretching band a t 3380 cm-1 when excess pyridine and bromine are added to a solution of resorcinol in PC. Phenolic compounds that contain a proton-accepting site can be titrated in PC without addition of pyridine. For example, 8-hydroxyquinoline consumes exactly 2 equivalents of bromine. Though the stoichiometry of the reaction is 2 : l as in aqueous solution, the reaction products are tribromide ion and monobrominated 8-hydroxyquinolinium ion, not the dibromo derivative. To illustrate (7) I . M. Kolthoff and R. Belcher, "Volumetric Analysis." Vol. I l i , Interscience, New York, N.Y.. 1957, p 535. (8) W. M. Lauer, J. Amer. Chem. Soc.. 48, 442 (1926)
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ANALYTICAL CHEMISTRY, VOL. 45, NO. 6, M A Y 1973
Approx. break, mV
Midpt. pot., mV
none none
...
0
.
...
50
+10
250 100
+50
50
+loo
50
0
300 150 200
0 0 4-50
none
...
-30
Table 111. Analysis of Aromatic Amines by Titration with Bromine in Propylene Carbonate End point ratio Compound titrated
Aniline Aniline" A n t h r a n iI i,c acid Anthranilic acid" p-Toluidine"
[amine]
Approx sample size, mmoies
2
0.6
5
4 2
0.3 0.6
4 4
99.0 99.5 99.4
8
4
0.3
5
103.9
6
0.3
4
99.5
A
@I/
4 Excess pyridine present.
No. of titrns
AV analysis, %
Re1 std dev. PPt
1 6
the reproducibility of this reaction, seven titrations of 0.6-millimole samples of 8-hydroxyquinoline with bromine in PC gave an average of 99.870, with a relative standard deviation of 1ppt. Water in small amounts does not affect any of the brominations significantly, but a t levels of 0.1M or so interferes. The effect of water on titration plots suggests that the stability of the tribromide complex is reduced greatly by water, presumably through bromide ion solvation by water. Bromination of Aromatic Amines. The amino group, like the hydroxyl, is activating and ortho-, para-directing in electrophilic aromatic substitution. Because primary amines are proton acceptors, some amine brominations are possible in PC without addition of a separate base. Results of titrations of several aromatic amines, both with and without the addition of excess pyridine, are shown in Table 11; analytical data are summarized in Table 111. In titrations of p-phenetidine, p-toluidine, and aniline, the potential remains below -400 mV ( u s . the silver0.01M silver perchlorate in PC reference electrode) until the addition of 1h equivalent of bromine if no pyridine has been added to the solution or until the addition of 1 equivalent of bromine if excess pyridine is present. An estimate of the ratio of bromide to tribromide present before the first break can be made from the relation
with the assumption that the potential in this region is controlled by the bromide-tribromide couple. The results
indicate that essentially no tribromide ion is produced in the first portion of these titrations. The potential break a t a ratio of l/~ equivalent of bromine per equivalent of amine in the absence of pyridine may be explained by the reaction
A
A
A
Since a monobrominated aromatic amine is a weaker base than the nonbrominated compound, protons produced in the substitution step should be preferentially accepted by the nonbrominated species. Subsequent bromination of the protonated amine would not be expected to occur readily because the protonated amino group is deactivating toward electrophilic substitution. With excess pyridine present, however, the first potential break does not occur until after the addition of 1 equivalent of bromine since, under these conditions, none of the amine is deactivated by protonation. The first reaction, then, is
A
On the other hand, p-phenetidine and p-toluidine, probably because of less-favorable acid-base equilibria, consume only 1.7 to 1.8 equivalents of bromine under the same conditions. Aniline also can be titrated quantitatively with bromine in PC in the presence of excess pyridine. Again two breaks are seen, the first after the addition of about 1, and the second after the addition of 4 moles of bromine per mole of aniline. The overall reaction under these conditions corresponds to
A
Addition of this first bromine proceeds readily, and tribromide is formed only after all the aromatic amine has been either monobrominated or protonated. The introduction of an electron-withdrawing bromine onto the aromatic nucleus renders further bromination more difficult, enabling the formation of tribromide ion to compete. A second potential break in the titration of aniline in the absence of pyridine occurs after the addition of 2 moles of bromine per mole of aniline. The overall reaction is
p-Toluidine behaves similarly. Introduction of a third bromine atom into the remaining open ortho position of aniline appears to be difficult, although after the addition of sufficient bromine to produce tribrominated aniline plus 3 moles of tribromide ion (6:lmole ratio), a very small potential break appears. The size and sharpness of this break is enhanced to some extent by reducing the speed of the titration. Thus some substitution of a third bromine appears to take place, but slowly. Addition of pyridine prior to titration of anthranilic acid with bromine results in a very gradual potential break a t a mole ratio of bromine to acid of 2 : l and a sharper break a t a mole ratio of somewhat more than 4: 1. Neither of these breaks is analytically useful. The overall reaction corresponds to
Analysis of m-phenylenediamine by titration with bromine is possible both with or without pyridine. When no pyridine is present, a small potential break occurs after the addition of 3 moles of bromine. The reaction is postulated to be
BI
The bromination product in this instance was confirmed by comparison of the PMR spectrum of a 2 : l mixture of bromine and aniline in PC with that of a PC solution of p-bromoanilinium bromide. The first inflection in the titration of aniline with bromine is greater than 0.5, probably owing to a proton exchange equilibrium between p-bromoaniline and anilinium ion:
Br
Br
The pKb values of aniline and p-bromoaniline in water, 9.34 and 10.06 (9), are sufficiently close for some free aniline to form, resulting in additional bromination. The sharp break in potential seen a t a ratio of bromine to aniline of 2 : l appears to be enhanced by this equilibration. (9) "Dictionary of Organic Compounds," 4th ed.. Vol. I , I. Hedbron. Ed., Eyre and Spottiswoode. Ltd , London, 1965, pp 243 and 418.
Br
Br
When excess pyridine is present, 6 moles of bromine are consumed. In this case the reaction may be "2
Here as elsewhere, the reactions appear rapid, and the rate does not seem to be affected by the amount of pyridine prepent. In summary, an aprotic solvent such as PC can provide a useful medium for bromine substitution. Many reactions are rapid, and the scope appears to be broad. Unusual stoichiometries are observed with some phenols and amines, and the formation in many instances of tribromide doubles the sensitivity of the method. ANALYTICAL CHEMISTRY, VOL. 45, NO. 6, M A Y 1973
047
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).
848
ANALYTICAL CHEMISTRY, VOL. 45, NO. 6, MAY 1973
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).
