procedure I1 because it appeared to be more widely applicable than procedure I. This investigation indicated that Cu(I1) cannot be tolerated without interference. Copper is reduced with antimony at both -0.21 and -0.35 volt us. S.C.E., and it is also reoxidized with the antimony. The presence of moderate amounts of As(V) and Pb(I1) apparently do not affect this titration to any large extent although there appears to be a small negative error in the reduction of Sb(V) to Sb(II1) a t -0.21 volt us. S.C.E. when Pb(I1) is present. Tin interferes in the reduction steps when present either in the stannic or the stannous state, but total antimony can be determined by reoxidation with very good precision. The interferences of Fe(III), Ni(II), and U(V1) are eliminated by reduction a t -0.21 volt us. S.C.E.; total antimony is then determined by reduction a t -0.35 volt us. S.C.E.Bismuth(V) does not interfere in the reduction of Sb(V) to Sb(II1) at -0.21 volt us. S.C.E.However, it is reduced with Sb(II1) at -0.35 volt us. S.C.E., so that only Sb(V) can be determined in the presence of Bi without interference.
LITERATURE CITED
Table V. Effect of Cations on Coulometric Titration of Antimony by Procedure II
[Each value is average of a t least 3 trials. Antimony(V) taken in each titration, 4.76 mg] Cation Sb Found, Error, 071 Added, Mg. blg. /O 2 . 5 $effi 4.730 -0.6 4.77b $0.2 3 Bif6 4.744 -0.4 $10.8 2 . 5 cu+2 5.3Zb 4,776 +0.2 2.5 Fe+3 +0.4 4.78b 5 Fe+3 5 Ni+2 +0.6 4.79b -1.3 4.70a 2 . 5 Pbf2 +0.4 4.7@ 5 Pb+2 -0.8 4.773 4.79b +0.6 4.7% +0.4 2.5 Sn+2 +0.8 4 . 8OC 5 Sn+2 +0.4 4.7P 2.5 Sn+4 +1.1 4.8lC 5 Snt4 $0.2 4.776 3 u+o $0.4 4. 78b 6 U+6 a Value obtained by reduction a t -0.21 volt us. S.C.E. *Value obtained by reduction a t -0.35 volt us. S.C.E. c Value obtained by oxidation at -0.21 volt us. S.C.E.
(1) Diehl, H., “Electrochemical Analysis
with Graded Cathode Potential Control,” p. 45, G. Frederick Smith Chemical Co., Columbus, Ohio, 1948. (2) Hayakawa, H., Bunseki Kagaku 7, 360 (1958). (3) Jones, H. C., “Automatic Coulometric Titrator, ORNL Model Q-2005, Electronic, Controlled-Potential,” Methods 1 003029 and 9 003029 TID-7015,See. 1, -4ugust 17, 1959. ( 4 ) Kelley, M. T., Jones, H. C., Fisher, D. J., ANAL. CHEY.31, 488, 956 (1959). (5) Kolthoff, I. M., Lingane, J. J., “Polarography,” 2nd ed., pp. 545-50, Vol. 11, Interscience, New York, 1952. (6) Lingane, J. J., Nishida, Fumio, J . Am. Chem. SOC.69, 530 (1947). 17) Norwitz,, G.,, -4XAL. CHEX 23, 386 (1951). (8) Reynolds, S. A., U.S.Atomic Energy Comm. Rept. ORNL-1557(1957). (9) Schleicher, A., Toussaint, L., Chem. Ztg. 49,645 (1925). (10) Shults, M7. D., “Uranium, Automatic Controlled-Potential Coulometric Titration Method,” Methods 1 219225 and 9 00719225; TID-7015,Sec. 1, January 29, 1960. (11) Tanaka, M., Bunseki Kagaku 7, 296, 631 (1958). RECEIVED for review November 17, 1961. Accepted January 17, 1962. Fifth Conference on Analytical Chemistry in Nuclear Reactor Technology, Gatlinburg, Tenn., October 12, 1961.
Spectrofluorometer Calibration in the UIt raviolet Region C. A. PARKER Admiralty Materials Laboratory, Holfon Heafh, Poole, Dorsef, England
b A simple method is described for calibrating a fluorescence spectrometer so that corrected fluorescence emission spectra in the ultraviolet region can be determined. The method makes use of a fluorescent screen monitor which was originally designed to allow direct recording of corrected fluorescence excitation spectra. Corrected fluorescence emission spectra and relative fluorescence efficiencies of anthracene, naphthalene, phenol, and benzene are presented.
M
SPECTROFLUOROMETERS record “apparent” excitation and emission spectra which are a function of the particular instrument used. Such apparent spectra are often grossly distorted versions of the true spectra, and before they can be compared with results obtained with other instruments, they must be corrected to give the true spectra. Methods for determining true fluorescence excitation spectra throughout the visible and quartz
502
OST
0
ANALYTICAL CHEMISTRY
ultraviolet region have been described (5, ’)I but a convenient method for correcting fluorescence emission spectra in the ultraviolet region has not previously been reported. The true fluorescence emission spectrum of a solution is a plot of fluorescence intensity, measured in relative quanta per unit frequency interval, against frequency ( 7 ) . When the fluorescence monochromator is scanned a t constant slit width and constant detector sensitivity, the curve obtained is the apparent eniission spectrum. To determine the true spectrum, the apparent curve has to be corrected for changes of the sensitivity of the multiplier phototube, the band width of the monochromator, and the transmission of the monochromator with frequency. Thus, if dQ/dv represents the fluorescence intensity a t any frequency v , the observed multiplier phototube output, A , , which corresponds to the apparent emission spectrum, is given by: AD =
yz)
PYBYLY =
rz)
SU
where
P,
output per quantum of the multiplier phototube at frequency Y B,, = band rvidth in freauencv ~. . ” units at frequency v L , = fraction of light transmitted by the spectrometer a t frequency v =
The quantity S, is the sensitivity factor of the monochromator/multiplier phototube combination; the true emission spectrum is calculated from the apparent emission spectrum by dividing it, point by point, by 8,. The simplest method of determining S,as a function of frequency in the visible region of the spectrum is to take multiplier phototube readings when the entrance slit of the spectrometer is illuminated by a tungsten lamp of known spectral distribution ( 7 ) . For wavenumbers greater than 2.5 p - 1 (400 mp), the intensity of light from a tungsten lamp falls off rapidly. Such a lamp is therefore not suitable for calibration a t the higher wavenumbers. Unfortunately, since discharge lamps with accurately known spectral distribution in the ultraviolet’
, 4.0
3.5
3.0 RECIPROCAL
Figure 1 . Arrangement for calibrating fluorescence spectrometer Xenon arc lamp Excitation monochromator Silica plate beam splitter 0.5-Mm. silica optical cell containing fluorescent screen solution Monitoring multiplier phototube Screen coated with M g O Fluorescence monochromator Fluorescence multiplier phototube
region are not readily available, the user must calibrate his own ultraviolet lamp (hydrogen or xenon arc) or multiplier phototube by comparison with a thermopile or with the ferrioxalate actinometer ( 2 , 6 ) . With either method, the transmission of a monochromator as a function of frequency, L,, has to be determined unless i t is assumed constant. The advantage of the method of calculation to be described is that it makes use of a fluorescent screen monitor to measure directly the product, POLv,as a function of frequency. The sensitivity factor, S,, is then obtained by multiplication by the band width,
B”. APPARATUS
The spectrofluorometer was similar to that described previously (4, 5 ) . It employs a 375-matt Xenon arc lamp as a source of visible and ultraviolet light, and a fluorescent screen and multiplier phototube to monitor the quantum intensity of the beam of exciting light isolated by the first monochromator. I n normal use, the operation of the fluorescent screen monitor is as follows. The sample cuvet (not shown in Figure 1) is placed at the focus of the beam of e x i t i n g light from the monochromator, M I , and the fluorescence monochromator, Jf2, is situated with its entrance slit opposite the sample-Le. , nearer to flf, by a distance X than the position shown in Figure 1. Before reaching the sample, the beam of exciting light passes through a clear silica plate, B , which reflects a small proportion of the beam on to the front surface of the fluorescent screen, F . This consists of a silica cell, 5-mm. optical depth, containing a suitable fluorescent solution, the fluorescence of which is viewed through the back face of the cell by the monitoring multiplier phototube, P I . ilfter amplification, the outputs of P I and P2 are passed to a ratio recorder.
2.5
2.0
MICRONS
Figure 2. Spectral sensitivity curve of quartz monochromator with 6 2 5 6 multiplier phototube 0 By comparison with fluorescein monitor X By measurements with calibrated tungsten lamp
To measure a n excitation spectrum, a suitable band of fluorescence light from the sample is selected by the monochromator M2. The excitation monochromator, Mi, is then scanned, and the slits are adjusted so as to maintain the output of P1 approximately constant. If the contents of F have been properly chosen ( 5 ) , the output of P2 can be made proportional to the quantum output of Xl over a wide frequency range. The recorded ratio, P2/Pl, then represents the true fluorescence excitation spectrum of the sample ( 5 ) . For the present experiments, a 4.4 x. 1 0 - ~ 3solution of fluorescein in a mixture of sodium carbonate and bicarbonate, both 0.1A7, n.as used for the fluorescent screen F . The relative fluorescence yield, p v , of this solution was determined by measuring the dose rates of ultraviolet light received by the contents of the sample curet, using the ferriosalate actinometer (6, 6 ) , and
comparing these with the corresponding readings of P I . Results are shown in Table I. To use the fluorescence screen monitor to calibrate the fluorescence monochromator/multiplier phototube combination ( A 4 1 - P2),the following procedure was adopted. The fluorescence monochromator, M2, was moved to a distance X (Figure 1) from the focus of the beam of exciting light so that X was much greater than the distance, V , of this focus from the lens. A magwas placed in nesium oxide screen, front of the entrance slit of the fluorescence monochromator and situated in the center of the diverging beam of exciting light. Thus, assuming t h a t the reflectivity of the magnesium oxide was independent of frequency, the entrance slit of the fluorescence monochromator was illuminated with a n intensity proportional to the total quantum intensity of the beam of exciting light. Changes in intensity due t o changes in the focal length of the lens with frequency were negligible because the distance X was large compared with the distance Ti. The entrance slit of the fluorescence monochromator was set to 0.06 mm., while the exit slit was set fully open (1.75 mm.). The
s,
Table 1. Calculation of Spectral Sensitivity of Fluorescence Spectrometer Wave- Reciprocal length, Microns, R,? = s, = mfi. P-1 R* ‘pv R”VV B” RlJR, 490 2.04 7.64 1.20 9.17 0.117 1.07 465 2.15 9.63 1.17 11.27 0.110 1.24 448 2.23 9.99 1.15 11.49 0.106 1.22 418 2.39 11.27 1.11 12.51 0,100 1.25 385 2.60 11.76 1.07 12.58 0.091 1.14 357 2.80 11.11 1.05 11.67 0,082 0.96 333 3.00 10.38 1.04 10.80 0,075 0.81 312 3.20 9.20 1.04 9.57 0.069 0.66 294 3.40 7.72 1.04 8.03 0.062 0.50 278 3.60 6.20 1.05 6.51 0.056 0.36 263 3.80 4.72 1.06 5.00 0.053 0.26 250 4.00 3.08 1.08 3.33 0,050 0.17 Rv = Multiplier phototube readings of light reflected from magnesium oxide screen (with constant output from the fluorescent screen monitor in beam of exciting light), corrected for variation of coefficient of reflection of magnesium oxide. q v = Relative efficiency of monitor. Rvl = Multiplier phototube readings corrected to constant quantum intensity of exciting light. B , = Band width of fluorescence spectrometer at constant slit width. S , = Spectral sensitivity of spectrometer multiplier phototube combination.
VOL 34, NO. 4, APRIL 1962
503
I
I
A
ford and coworkers ( I ) and varies by less than 8% over the range 2.0 to 4.0 1-l (500 to 250 mp). The reported values of the coefficient were used to correct the observed multiplier phototube readings R, before calculating S, as described above. This calculation is shown in Table I; the results obtained for S, by this method are plotted in Figure 2 along with results obtained in the visible region using a calibrated tungsten lamp ( 7 ) . APPLICATION
3 5
2.5
3.0 RECIPROCAL
MICRONS
Figure 3. Corrected fluorescence emission spectra of some simple compounds in oxygen-free ethyl alcohol Instrumental sensitivity settings refer to solutions having absorption per cm. of 0.2 a t the excitation wavenumber of 4.03 p - l ( 2 4 8 mp) Slit width of fluorescence monochromator corresponded to half-band width of 0.01 9 p - l (3.0 mp] a t
(400 mp)
2.5
p-l
a.
Anthracene X 10 Naphthalene X 1 8 Phenol X 1 8 Benzene X 1 8
b. c.
d.
Table II.
Substance Anthracene
Fluorescence Data for Spectra Shown in Figure
Fluorescence Efficiency -4pproxiin mate Quenching DeoxyFactor for genated Saturation Ethyl Concentration, Alcohol, with Air, pg. Per RI1. (VI (P/Va) 0.32 [O. 271 1.2 12
0.19
6.0
Phenol
100
0.19
1.3
Benzene
170
0.042
2.0
Tiaphthalene
widths of the slits of the excitation monochromator were kept equal to one another but were adjusted as the frequency setting of the excitation monochromator was changed so as to keep the output of the monitoring multiplier phototube, P I , constant. The slit settings of the excitation monochromator never exceeded 0.8 mm.-Le., the sum of the slit widths on MI was always less than the width of the exit slit on M 2 . Hence, the band width passed by the excitation monochromator, MI, was always less than the band width accepted by the fluorescence monochromator, X 2 ,when the frequency settings of the two monochromators coincided. At each frequency setting of the excitation monochromator, the fluorescence monochromator was scanned across this frequency so as to obtain the maximum reading of P2. Since the fluorescence monochromator accepted the full band 504
ANALYTICAL CHEMISTRY
3
Fluorescence Sensitivity Index at Main .4bsorption M axinium , 1.2 at 3.97 p-1 (232 mp) 0.029 at 3.64 p - l (275 nip) 0.015 at 3.66 p - l (273 mp) 0.00046 at 3.93 p-' (254 m p )
of frequencies a t each setting, the masimum output of P z was proportional to L,PJ, where I , was the quantum intensity of the exciting light a t each frequency setting. Since a t each setting the output of the fluorescence screen monitor was constant, I , was inversely proportional to the relative fluorescence efficiency of the monitor solution (py). Thus, if R, represents the maximum readings of the fluorescence multiPL plier phototube, R, = z ' ; a n d hence, (a*
the sensitivity factor S, equals R,p,B,. The magnesium oxide screen was prepared by holding a piece of thin aluminum sheet in the smoke from a burning magnesium ribbon until the latter had become uniformly covered with a thick coating of the oxide. The coefficient of reflection of magnesium oxide in the visible and ultraviolet regions has been determined by Ben-
The values of S , shown in Figure 2 were used to determine the corrected fluorescence emission spectra and relative fluorescence efficiencies of solutions of anthracene, naphthalene, phenol, and benzene in ethyl alcohol using a n excitation wavenumber of 4.03 p - 1 (248 mp) isolated from a 1kilowatt high-pressure mercury lamp by means of the excitation monochromator. The concentrations were adjusted so that all four solutions had a n absorbance of 0.2 per em. a t 4.03 k-l (248 mp). The solutions were deaerated before and during measurement to eliminate oxygen-quenching of the fluorescence, which was appreciable in all cases and especially with naphthalene. The corrected emission spectra are shonm in Figure 3. The areas under these curves, after division by the corresponding instrumental sensitivity values, are proportional to the total number of quanta of fluorescence emitted. Since the proportions of e x i t ing light absorbed by all the solutions vere the same, the areas are also proportional to the fluorescence efficiencies of the compounds ( 7 ) . Assuming a value of 0.27 reported by Rlelhuish (5) for the fluorescence efficiency of anthracene in ethyl alcohol, the efficiencies, (o, of the other compounds were calculated and are given in Table 11. The fluorescence sensitivity indices (7) TI ere calculated from ' *where , : p
A,,,
= =
H
=
fluorescence efficiency absorbance per centimeter at the principal absorption maximum for a concentration of 1 pg. per ml. half-band width of the corrected fluorescence emission spectrum. H is the area under the corrected spectral curve divided by the maximum value
The value of benzene is very low compared with those of well known strongly fluorescent substances. This, together with the fact that excitation requires frequencies in the middle ultraviolet where lamp outputs are low, is probably the reason rvhy fluorometric methods
have not been used for determination of this substance. During the course of the measurements, quenching factors for saturation with air were determined. These are only approximate because they depend on the solubility of oxygen in ethyl alcohol and are thus critically dependent on temperature which was not accurately controlled for these measurements. The fluorescence of naphthalene, normally regarded as weak, is increased by a factor of six by deaeration of the solution. This emphasizes again the neressity to test for
oxygen quenching when the fluorescence of a new compound is being investigated. CONCLUSIONS
The attachment of a fluorescent screen monitor to a spectrofluorometer allows the direct recording of true fluorescence excitation spectra, compensates for fluctuations in the intensity of the exciting light when a fluorescence emission spectrum is recorded, and can be used to calibrate the fluorescence spectrometer/multiplier phototube in the visible and ultraviolet regions.
LITERATURE CITED
(1) Benford, F., Lloyd, G. P., Schwarz, S.,J. Opt. SOC.Amer. 38, 445,964 (1948). (2) Hatchard, C. G., Parker, C. 4.,Proc. Roy. SOC.A235, 518-36 (1956). (3) hlelhuish, W. H., J . Phys. Chem. 65,
229-35 (1961). (4) Parker, C. A., Analyst 84, 446-53 (1959). (5) Parker, C. A , , iVature 182, 1002-4 (1958). (6) Parker, C. A., Proc. Boy. SOC.A220, 104-16 (1953). (. 7,) Parker. C. A , . Rees. W. T.. Analust 85, 587-600 (1960). ‘
RECEIVED for review October 20, 1961. Accepted Januarx 9, 1962.
A Comparison of Methods for Spot Test Detection and Spectrophotometric Determination of Glyoxal EUGENE SAWICKI, THOMAS R. HAUSER, and RONALD WILSON Robert A. Taft Sanitary Engineering Center, Cincinnati 26, Ohio
b The 1,2-dianiIinoethane, 2,3-diaminophenazine, 2-aminobenzenethiol, 2,4-dinitrophenylhydrazine, and 4nitrophenylhydrazine procedures for the determination of glyoxal are compared. The last three are new methods for the determination of glyoxal. The 2-aminobenzenethiol and the 1,2-dianiIinoethane procedures are the most highly selective for the determination of glyoxal, while the 2,3diaminophenazine procedure is more selective than the 4-nitrophenylhydrazine method. O f all the methods, the 3-methyl-2-benzothiazolone hydrazone and the 2-hydrazinobenzothiazole plus p-nitrobenzenediazonium fluoborate procedures have the poorest selectivity for glyoxal. The Dechary, Kun, and Pitot diaminophenazine method is about 2 0 times more sensitive than the 2-aminobenzenethiol and 1,2dianilinoethane methods. The 4-nitrophenylhydrazine procedure is, b y far, the most sensitive method of all; however, pyruvaldehyde and biacetyl also react with this reagent.
G-
can be readily detected (6,Ia) or determined ( I S ) with 1,2 dianilinoethane. 2,3 - Diaminophenazine can also be used for the determination of glyoxal ( 2 ) . Other reagents that hare been used with fairly good selectivity in the detection of glyoxal are 2,4-dinitrophenylhydrazine ( d ) ,4-nitrophenyl hydrazine (4,5),2-aminobenzenethiol, 2,3-diaminonaphthalene, and 2-hydrazinobenzothiazole ( 6 ) , salicylalhydrazone, and 2-hydroxy-lLYOXAL
naphthalhydrazone (9). The last three methods are fluorescence spot tests. EXPERIMENTAL
1,2-Dianilinoethane dihydrochloride was obtained from Aldrich Chemical Co., Inc., MilTI aukee, Vis. 2-Aminobenzenethiol was obtained from Laboratory Services Inc., Cincinnati 9, Ohio. 2,4Dinitrophenylhydrazine, 4-nitrophenylhydrazine, 10% aqueous tetraethylammonium hydrouide, and 1,2phenylenediamine dihydrochloride were obtained from Distillation Products Industries, Rochester, K. Y.; 29y0 methanolic tetraethylammonium hydroxide was obtained from Southwestern Analytical Chemicals, Austin, Tex. Glyoxal sodium bisulfite (K & K Laboratories, Jamaica, N. Y.) was crystallized from 40% aqueous ethyl alcohol and then dried over phosphorus pentoxide in a vacuum for 3 days. dnalysis by the Galbraith Laboratories, Inc., Knoxville, Tenn., showed the molecular structure to be (CHO)*.2 NaHS03.H 2 0 . Preparation of 2,d-Diaminophenazine. Steigman’s procedure (11) was simplified in the following manner. One hundred milliliters of 4oyO aqueous ferric chloride solution was poured into a stirred solution containing 33.5 grams of 1,2-phenylenediamine dihydrochloride in 100 ml. of water a t room temperature. A red-brown precipitate was quickly formed. The mixture was then brought to the boiling point, and approximately 200 ml. of hot water was added to bring the precipitate into solution. The hot solution was filtered and allowed to cool. The blue-black needles were collected and washed with ether. Nine Reagents.
grams (%yoyield) of the hydrochloride salt was obtained and used in the qualitative and quantitative procedures. Calculated for C12H10N4. HC1. H 2 0: K, 21.2; C1, 13.2. Found: N, 21.5; C1, 13.4. If necessary, the compound can be purified by Fischer and Hepp’s procedure (3). Reagent Solutions. 2-.4n11Ko~EKZEKETHIOL. d 1% aqueous solution containing 6 ml. of concentrated hydrochloric acid per 100 ml. of solution. The solution is stable for at least 24 hours. 1,2-DIANILISOETHANE DIHYDROCHLORIDE. A 1% aqueous solution. 2,4-DIKITROPHENYLHYDRAZIKE. A O.lyo solution in dimethylformamide containing 1% concentrated sulfuric acid. The solution is stable for a t least 24 hours. 4-NITROPHENYLHYDRAZINE. A 0.10/, solution in dimethylformamide containing lyGconcentrated sulfuric acid. The solution is stable for a t least 2 days. 2,3-DIAMINOPHEXAZINE HYDROCHLORIDE. A 0.02y0solution in 50% acetic acid. The solution is stable for a t least 1 week. Apparatus. Cary recording spectrophotometers Models 11 and 14 with 1-cm. cells rvere used. Spectrophotometric Procedures. 2ANIKOBENZENETHIOL. A mixture of 3 ml. of aqueous test solution, 3 ml. of reagent, and 6 ml. of concentrated hydrochloric acid is heated in 3 boiling n-ater bath for 2 minutes. Then 1 ml. of 1.65% sodium nitrite solution is added. After the mixture is cooled to room temperature under the tap, it is diluted to 25 ml. with concentrated hydrochloric acid. The absorbance of the blue solution is read a t 600 mp within 10 minutes. VOL. 34, NO. 4 , APRIL 1962
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