2184
Anal. Chem. 1980, 52, 2184-2189 Ohnesorge. W. E.; Rogers, L. B. Anal. Chem. 1958, 26, 1017-1021. Ellinger, P.; Holden, M. Biochem. J . 1944, 38, 147-150. Ziporin, Z.Z.;Beier, E.; Holland, D. C.; Bierman. E. L. Anal. Biochem. 1982, 3, 1. Mark, H. B., Jr. Talanfa 1973, 20, 257-268. Wilson, R. L.;Ingle, J. D., Jr. Anal. Chim. Acta 1976, 83, 203-214. Dyke, S.F. "The Chemistry of the Vitamins"; Interscience: New York, 1965, Chapter 2. Weast, R. C., Ed. "Handbook of Chemistry and Physics", 53rd ed.; Chemical Rubber Co.: Cleveland, OH, 1967; p C-508. Kavanagh, F.; %&win, R. H. Arch. Biochem. 1949, 20, 315-324. Kawasaki, C. Modified Thiamine Compounds, in Vitamins and Hormones"; Harris, R . S., Ed.; Academic Press: New York, 1963; Vol. 21. Wostman, B. S.;Knight, P. L. €xperientia 1980, 16, 500. Sykes, P.; Todd, A. R. J . Chem. SOC. 1951, 534-544. Fujiwara, M.; Matsui, K. Anal. Chem. 1953, 25, 810-812. Holman, W. I.M. Biochem. J . 1944, 38, 388-394. Edwin, E. E.; Jackman, R.; Hebert. N. AnaVst(London) 1975, 700, 689-695. Morita, M.; Kanaya, T.; Mlneska, T. J . Vitaminol. 1989, 75, 118-125. Prokhovnik. S. J. Analyst (London) 1952, 77,257-259. Risinger, G.E.; Pell, F. E. 8iochim Biophys. Acta 1985, 707, 374-379. Holzbecher, J.; Ryan, D. E. Anal. Chim. Acta 1973, 64, 333-338. Ryan, M. S.Ph.D. Thesis, Oregon State University, Corvallls, OR, 1980. Maier, G. D.; Metzler, D. E. J. Am. Chem. SOC.1957, 79. 4388-4391. Nesbitt, P.; Sykes, P. J . Chem. SOC. 1954, 4585-4587. Gero, E. C . R.Hebd. Seances Acad. Scl. 1952, 235, 397-399. Tanaka, A. Bhamin 1988, 33,497-502; Chem. Abstr. 1986, 65.3869d; 1988, 6 4 , 8178d; 1988, 6 4 , 97190. Cotton, F. A.; Wilkinson, G. "Advanced Inorganic Chemistry, A Comprehensive Text"; Interscience: New York, 1972; pp 517-519. Lenz. G. R.; Martell, A. E. Biochemistry 1984, 3. 745-760. Penttinen, H. K. Acta Chem. Scand., Ser. 6 1978, 830, 659-663. Waston, H. A. Cereal Chem. 1948, 23, 186-187. Satiidarma, K. S u r a Pharm. 1966, 79, 1-10; Chem. Abstr. 1966, 65, 15 164c. Ingle, J. D., Jr.; Crouch, S . R. Anal. Chem. 1971, 43, 697-701. Laldler. K. J. "Chemical Kinetics"; McGraw-Hill: New Y d , 1965; pp 13, 19. Hall, K. J.; Quickenden, T. I.; Watts, D. W. J . Chem. Educ. 1976, 53. Matsui. K.; Honda. M. Bitamin 1958, 15, 654-659; Chem. Abstr. 1982 57, 754h. De Ritter, E.; Rubin, S. H. Anal. Chem. 1947, 19, 243-248. Ryan, M. A.; Miller, R. J.; Ingle, J. D., Jr. Anal. Chem. 1978, 5 0 , 1772-1777.
Table V. Determination of Thiamine in Synthetic Multivitamin-Mineral Preparation Solutions by the Standard Method and the Kinetic MethodQ
method U.S.P.
kinetic
a
thiamine concn obtained, no. of PM runs 2.40 2.40
RDS,
%
7c
error
3
3
3
1
5 2.5 2.35 5 2 2.45 3 2 The solutions were prepared to contain 2.5
-4 -4
2.47
TM .
-1
-6 -2 X
M
TM. This sample was prepared as before except that the p H 2 sample solution was spiked with a standard T M solution a t the final dilution such that its concentration was 2.5 X lo4 M T M . The results for both methods were obtained from a calibration curve. Table V shows that both methods gave accurate results. Variations in turbidity of the butanol and the formation of a very small amount of TC in the USP method blank gave a DL of 7 X M T M for the standard method. Since the rate method does not involve extractions, the rate method is faster than the standard method in analysis and glassware cleanup time and less expensive due to the use of less chemicals and glassware.
LITERATURE CITED "Methods of Vitamin Assay"; Association of Vitamin Chemists, Inc.; Interscience: New York, 1966; Chapter 6. Strohecker. Rolf; Henning, Heinz M. "Vitamin Assay-Tested Method"; Verlag Chemie: Weinheim/Bergstr., Germany, 1966. Nicheison, 0.; Yamamoto, R. S. I n "Methods of Biochemical Analysis"; Glick, D., Ed.; Interscience: New York, 1958; Vol. 6, pp 191-257. Wilson, R. L.; Ingle, J. D., Jr. Anal. Chem. 1977, 4 9 , 1066-1070. Wilson, R. L.; Ingle, J. D., Jr. Anal. Chem. 1977, 4 9 , 1060-1065. Official Methods of Analysis of the Association of Official Analytical Chemists"; Horwitz, W., Ed., AOAC: Washington, DC, 1970. United States Pharmacopeia", 19th ed.; United States Pharmacopeia Convention, Inc.: Rockville. MD, 1975; p 629. Pippin, E. L.; Potter; A. L. J . Agric. Food Chem. 1975, 23, 523. Gassmann, G.;Janicki, J.; Kaminski, E. Int. Z . Vitaminlorsch. 1963, 33, 1-17.
RECEIVED for review January 26,1979. Resubmitted July 14, 1980. Accepted August 26,1980. Acknowledgment is made to the NSF (Grant No. CHE-76-16711 and CHE-79-21293) for partial support of this research, and M.A.R. gratefully acknowledges an NSF graduate fellowship. Presented in part a t the 1979 Pittsburgh Conference, Cleveland, OH.
Luminescent Quantum Counters Based on Organic Dyes in Polymer Matrices Krishnagopal Mandal,
1.D. L. Pearson, and J. N. Demas"
Department of Chemistty, University of Virginia, Charlottesville, Virginia 22903
New luminescent quantum counters composed of laser dyes dispersed in either poly(viny1 alcohol) (PVA) or poly(vinylpyrrolidone) (PVP) solid matrices are described. The PVA matrix is more compatible wlth water-soluble dyes and the fllms detach easily from the supporting glass substrate. The PVP fllms are more compatible with dyes soluble in organic solvents and form extremely tenacious films which can only be removed by dissolution. A variety of xanthene and coumarin dye quantum counters are reported. Relative spectral flatness and sensitivity as well as usable wavelength range are dlscussed. Several new systems wlth superior properties are presented as well as the considerations involved In the proper selection of a quantum counter.
The measurement of light intensities plays a crucial role for many spectroscopists, photochemists, analytical chemists, photobiologists, and physicists. Accurate measurements are usually made with thermal detectors or quantum counters (1-5).
Thermal detectors (e.g., thermopiles, bolometers, and pyroelectrics) are energy flat detectors that give equal output for equal incident energies a t different wavelengths. They generally suffer from low sensitivities, slow response, and/or small areas ( 4 ) . Most spectroscopic measurements are concerned with photon fluxes rather than beam powers. Only if the spectral distribution is known, however, can the photon flux be calculated directly from the power.
0003-2700/80/0352-2184$01.00/0 0 1960 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 52, NO. 13, NOVEMBER 1980
Quantum counters, on the other hand, can have nearly perfect quantum flat responses over a very wide wavelength range. The first quantum counters were developed by Bowen (6) on the basis of an earlier idea by Vavilov (7, 8). In only slightly changed form, this basic design coupled with improved materials is still used today. The standard quantum counter consists of an optically dense, highly luminescent target screen which is viewed by an optical detector. If the emission spectrum and photon yield of the screen are wavelength independent, then the response of the screen-detector system is quantum flat in the region of total absorption. Quantum counters find wide use in the spectral calibration of optical detectors and excitation monochromator systems and in the determination of photon yields (1-4). For example, many of the top of the line corrected commercial spectrofluorimeters use a quantum counter for correction of excitation spectra. Since its introduction 25 years ago (9),the most widely used quantum counting material has been Rhodamine B (RhB) dissolved in different alcohols (10-14). I t has a useful range from the UV to -600 nm. When used with a photomultiplier tube (PMT), such a combination yields appreciably higher sensitivities than thermal detectors. Current quantum counters are not, however, ideal. The strong self-absorption and self-quenching at quantum counting concentrations of RhB appreciably reduce the available signal. RhBs deep red response is gained only by red shifting the emission which further reduces sensitivity. Thus,workers who need sensitivity t o only 500 nm are wasting potential sensitivity by using a quantum counter with deeper red response. Further, they may need a more expensive red-sensitive PMT. Thus, ideally, a range of quantum counting materials should be available for different wavelength ranges. Further problems arise because all currently available counters are solutions of highly fluorescent dyes. These systems suffer from the need for expensive cuvettes, possible leakage and contamination of instruments with highly luminescent materials, and orientation problems associated with leakage or interference of the optical paths from bubbles. There has been a brief mention of a counter using RhB in solid poly(viny1 alcohol) films (14). Ghiggino subsequently reported (15) that a film containing 17 wt % of RhB in PVA was relatively quantum flat in response (*6%) over the range 250-530 nm. Marx and Schiller have reported luminescence (16, 17),polarization effects (18) and self-quenching of dyes in PVA films (19). No further work appears to have been done in this area. Because of the obvious potentiality of film quantum counters, we undertook a comprehensive study of a variety of potential quantum counting dyes dispersed in PVA and poly(vinylpyrro1idone) (PVP). We have discovered several exceptionally flat and intensely luminescent rhodamine and coumarin dye f i i systems. Some systems have shorter cutoff wavelengths but far greater sensitivities than the conventional RhB counters. Our results permit workers to select the optimum counter system on the basis of dye availability, required sensitivity, usable wavelength range, spectral flatness, and polymer film characteristics.
EXPERIMENTAL SECTION Materials. Rhodamine 6G chloride (Rh6G) from Eastman Kodak was purified by three passes through a column filled with Sephadex LH-20 using a methanol eluent. The remaining laser dyes were all from Exciton Chemical Co. and were used without further purification. The dyes and the abbreviations used here are as follows: Rhodamine 19 (Rh19), Rhodamine B chloride (RhB), Rhodamine 101 perchlorate (RhlOl), Rhodamine 110 chloride (RhllO), Coumarin 7 (C7), Coumarin 102 (ClOZ), and Coumarin 153 (C153). The poly(viny1alcohol), 99-100% hydrolyzed (PVA),was from Cellomer Associates. The poly(vinylpyrro1idone) type NP-K9O
2185
Figure 1. Cell holder used for preparing quantum counter films. The body is Teflon and the quartz plate snaps into the bottom.
(PVP) with a molecular weight of 360 000 was a gift from GAF Chemical Corp. Both polymers were used without further purification. Quantum Counter Comparator. The quantum counter comparator is an automated microcomputerized version of the high accuracy manual quantum counter comparator of Taylor and Demas (12). The excitation system was described earlier, and the beam switcher and computer interface are described elsewhere (13).
The reference quantum counter material was Exciton RhB (5 g/L) in methanol. All relative quantum yields were corrected for the known relative response of this dye system using the tabular data of Taylor and Demas (11). Irradiation of the reference counter was kept to a minimum and the solution was stored in the dark and replaced every 4-6 weeks depending on usage. Preparation of Polymer Films. Polymer films were prepared by dissolving weighted amounts of polymer and dye in an appropriate solvent (water or methanol) and evaporating them in the cell quartz plate holder shown in Figure 1. For the water solvent drying was performed in an air convection oven set at 40 "C. Drying usually took 6-12 h for 10 mL of solvent. With the higher vapor pressure methanol solvent, better films were obtained by a slower evaporation at room temperature. Attempts to build up thick layers by evaporating some of the solution to dryness and adding a second aliquot failed; the second aliquot caused separation of the film from the quartz plate with loss of optical quality. After evaporation, the f i s were i n s p e d for crystallization. Optical quality was never perfect. Some ripples and distortion of objects viewed through the plate were always present especially with the PVA films. The PVP films, particularly thin ones, were of better optical quality, but with some distortion around the edges. Since our excitation beam was large (15 mm X 2 mm) at the sample, averaging of small imperfections should have largely eliminated errors from this source. The seal between the quartz plate and the Teflon in the cell of Figure 1 was sufficiently tight that little or no leakage of the high viscosity polymer solutions resulted. The cell holder which was close to the diameter and thickness of our standard disk shaped cuvettes was used directly in the quantum counter comparator without removing the Teflon support plate. Absorption and Emission Measurements. Absorption measurements were made on a Carey 14 recording spectrophotometer. All measurements were corrected for absorbance and reflection losses from the support plate by running a clean quartz plate for the blank. The samples were mounted as close to the detector as the sample compartment permitted so as to minimize offsets associated with imperfections in the film. With the pure P W films, film quality was good enough so that little contribution from the sample distortion arose. For the more wavy appearing PVA f i ,a near constant offset was observed through the visible even though the color of the film was near water white. Thus, we attributed this offset to transmission imperfections and subtracted this value from the data at all wavelengths. Luminescence spectra were measured on a microcomputercontrolled SLM Model 8000 DS spectrofluorimeter. All reported spectra were corrected for the spectral sensitivity characteristics of the emission detection system. Correction factors were obtained by means of a standard tungsten lamp (1, 3 ) . Lifetime Measurments. A limited number of lifetime measurements were made on the films. A Photochemical Research Associates Model 2000 nanosecond single photon counting system
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 13, NOVEMBER 1980
0.38 0.2s
0
1
u
1.0
c 10
W
z
i0.20 m
2 z
E< a. is
(L
0.6
w
2 I-
a. i a
0.4
< LL1 -1
0. 0s 218
250
298 330 370 WAVELENGTH (nm) Flgure 2. Absorption spectra 14 mg/cm2 PVA (A) and PVP (6) films.
Table I. Composition of Polymer-Dye Quantum Counter Systems Studied PPV film film concn, concn, mg dye/ mg dye/ wt % d y e cmz wt % d y e cm2
0. 0 570 630 690 750 WAVELENGTH (nm) Flgure 3. Corrected back-viewed luminescence spectra of the quantum counter PVP films (from left to right): (A) C102; (B) C7; (C) RH19; (D) RhllO; (E) Rh6G; (F) RhB; (G) RhlOl. 450
PVA
RhB
13.Ba 7.4a
Rhl9 Rh6G RhlOl RhllO c7 c102 C153
1.6 1.6
13.Bb
1.6
3.6a 1.2b
0.4 1.6 1.1 1.1
C
13.8a C
C C C C
1.6
Lob
0.6gb 2.5 0.60b
0.6 1.6 1.0
a Films prepared with water as the solvent. Films prepared with methanol as the solvent. The dye is insoluble in water.
was used. The lamp was a triggered hydrogen-filled lamp. All data reduction was performed on a dedicated DEC MINC minicomputer using a least-squares deconvolution algorithm (20). Data could be fitted to either a single exponential or a sum of two exponentials. RESULTS F i l m Properties. Figure 2 shows the absorption spectra of the pure polymer f i s . The PVA was virtually transparent down to 210 nm. The PVP film, however, exhibited a strong absorption a t
