600
Anal. Chem. 1984, 56,600-601
Reduction of Background Emission in Room-Temperature Phosphorescence David L. McAleese and R. Bruce Dunlap*
Department of Chemistry, University of South Carolina, Columbia, South Carolina 29208 Solid surface room-temperature phosphorescence (RTP) is a well-established luminescence technique for the determination of important organic and biological compounds. Molecules exhibit the phenomenon when adsorbed and dried on a suitable solid substrate. RTP is recognized as a method of superior selectivity since relatively few compounds display this luminescence compared to fluorescence or low-temperature phosphorescence (LTP). In addition, solid surface RTP analyses are simpler in design and require lower cost than LTP or solution RTP methods. The technique has been successfully applied to the determination of compounds in pharmaceutical preparations (1-3),synthoil(4), and air particulates (5) without any prior separation steps. Thus, RTP is gaining a reputation as a rapid screening method for the identification of compounds in complex mixtures. The major limitation of the RTP technique is the presence of background phosphorescence in virtually all substrates which induce significant analyte phosphorescence signals. Cellulose filter and chromatographic papers are credited as the most applicable RTP supports to date (6, 7). Impurities in the supports generate broad, featureless bands of excitation spectra from 200 to 400 nm with a maximum near 300 nm and phosphorescence spectra from 375 nm to 625 nm with a maximum near 500 nm. For a large number of organic substances absorbing and emitting light in this spectral region, the phosphorescence detection limits increase depending on the magnitude of the background signal. RTP has not received wide acclaim as a particularly sensitive technique, partly attributable to the significant background intensity. The background emission is also a source of spectral interference for compounds yielding weak phosphorescence. As with other analytical methods, RTP detection limits can be improved by (1) increasing the analyte signal, (2) minimizing the background noise, or (3) quenching the background signal. A previous attempt at decreasing RTP detection limits by reducing the background emission of paper only produced a 2-fold improvement (8). In that investigation, filter paper was treated with a variety of solvents by a rinsing or chromatographic method. Rinsing the supports with acetone or demineralized water reduced the background intensity by 30-50% without adversely affecting the analyte signals. Heating the supports at 120 "C or 250 "C for various lengths of time quenched the background and analyte signals concomitantly. The substances generating the background emission evidently must be either strongly adsorbed or bound to the cellulose matrix, since chromatography with several solvents failed to eliminate the impurities. We have previously observed that the analyte phosphorescence signals decreased with continuous exposure of the samples to excitation light in the sample compartment of the phosphorimeter (9). The rate of phosphorescence quenching varied among several different compounds but was slightly suppressed for samples protected from moisture with sodium citrate. Further drying of the samples only partially regenerated the signals. Hence, moisture quenching of the samples, probably caused by air turbulence inside the rotating can chopper, was a minor contributing factor to the dissipation of RTP intensity. It was unclear though whether photolytic degradation of the phosphors or some other triplet quenching mechanism was primarily responsible. In any event, it was our contention from these preliminary experiments that illumination of the paper supports might permanently diminish
the background emission as well.
EXPERIMENTAL SECTION Apparatus. Room-temperature phosphorescence intensity measurements and excitation/emission spectra were obtained on an Aminco-Bowman spectrophotofluorometer equipped with a 150-W xenon arc light source, a phosphoroscope, a laboratory constructed sample holder (7),and a 1P21 photomultiplier tube detector. Sample Preparation. Circles of Whatman 3 MM chromatography paper, 6.4 mm diameter,were dried for 2 h in a glovebag attached to the sample compartment of the instrument (10). The supports were mounted on the sample holder with a plate that covered all but a 5 mm X 3 mm section of the cellulose. The vertical image of the excitation beam was broadened to irradiate the entire exposed surface. This was accomplished by an optical modification of the excitation monochromator (IO).
RESULTS As illustrated in Figure 1,the background emission at 495 nm decreased 87% during the first 3 h of illumination with 285-nm light from the 150-W xenon lamp. The rotating can chopper operated continuously during the 3 h of illumination. The chopper was then removed from the sample compartment to allow continuous illumination of the supports. A previously dried paper circle was mounted on the sample holder and irradiated with 285-nm light in the sample compartment for 24 h. The sample holder was then moved to the glovebag chamber while the chopper was reinstalled. After a 2-h desiccation period of the sample compartment, the phosphorescence intensity of the irradiated support and a control were measured. The background intensity at 495 nm decreased from 96.0 nA to 7.8 nA, corresponding to a 12.3-fold reduction. Approximately the same decrease was observed at the other background emission wavelengths. The excitation and emission spectra of the background before and after 24 h of illumination are shown in Figure 2. The maximum excitation wavelength shifted from 285 nm to 300 nm following exposure to 285-nm light, while the emission maximum shifted from 495 nm to 485 nm. However, no additional insight was gained into the spectral identification of substances responsible for the background emission. The spectrum of the quenched support was also broad and featureless. In a batch experiment, several supports were subjected to illumination by white light from the xenon lamp. After 24 h of exposure, the paper circles were dried in the glovebag for 2 h and analyzed. Compared to the controls, the background intensities were reduced by an average of 10.3-fold. The experiment was also a success in terms of precision as the quenched intensities were reproducible to within 3.5%. The excitation and emission spectra of the supports irradiated with white light were essentially identical with those obtained with 285-nm illumination. Since the quenching process appeared to be more efficient for samples illuminated in the dry state, a support was mounted and irradiated continuously in the sample compartment with 285-nm light for 57 h. The intensity dropped from 86.2 nA to 3.8 nA, correspondingto a substantial 25.4-fold reduction, Upon closer inspection of the irradiated supports, the cellulose matrix appeared slightly discolored. We suspected that the substances causing the background emission were photolytically degrading. To ensure that the light was not damaging the paper, thereby altering the support-phosphor interactions necessary for induction of RTP,
0003-2700/84/0356-0600$01 .50/0 0 1984 American Chemical Society
Anal. Chem. 1984, 56,601-602
: tloo\ 74\ W
g
o
2
1
3
TIME, hours
Flgure 1. Phosphorescence intensity at 495 nm plotted as a function of time for paper exposed to 285-nm light.
-
W I-
r W
601
batchwise well in advance of actual RTP analyses. Since RTP is a suitable detection method for compounds chromatographed on paper, entire sheets of paper could be illuminated prior to chromatography. Although no other cellulose supports were investigated, effective photolytic quenching is anticipated among different types of paper. We have found that the papers yield similar spectra, suggesting that the same impurities may be inherent in these papers. The importance of the background reduction is manifested in lower RTP detection limits for a vast number of phosphors absorbing and emitting light in the same spectral region. Recently, we implemented a sample drying technique and an instrumental modification (10) for RTP analysis. With those developments, the detection limit of p-aminobenzoic acid adsorbed on Whatman 3 MM chromatography paper was 12.9 pg. By utilization of the illuminated support described herein, the calculated detection limit has been reduced to approximately 0.5 pg or 200- to 20000-fold lower than previously reported on solid supports. With over an order of magnitude reduction in the background intensity, RTP has now matured into a more viable quantitative analytical technique.
?
LITERATURE CITED
4I W K
300
200
400
500
600
W A V E L E N G T H , nm
Flgure 2. Phosphorescence excitation and emission spectra of paper before (---) and after (-) quenching with 285-nm light for 24 h. These spectra were recorded on the 100 nA and 10 nA scales, respectively.
previously illuminated supports were spotted with solutions of p-aminobenzoic acid and 4-biphenylcarboxylic acid. The samples were analyzed after 2 h of desiccation, and the analyte intensities were found to be identical with those obtained from the control samples.
DISCUSSION These results indicate that the background emission of cellulose papers can be diminished without the use of laborious techniques. The paper circles can be conveniently prepared
(1) Bateh, R. P.; Winefordner, J. D. J. Pharm. Sci. 1983, 72, 559-560. (2) Bateh, R. P.; Winefordner, J. D. Anal. Left. 1982, 15 (B4), 373-383. (3) Von Wandruszka, R. M. A.; Hurtubise, R. J. Anal. Chem. 1978, 48, 1784-1708. (4) Vo-Dinh, T.; Gammage, R. B.; Martinez, P. R . Anal. Chim. Acta 1980, 118, 313-323. ( 5 ) Vo-Dinh, T.; Gammage, R. B.; Martinez, P. R. Anal. Chem. 1981, 5 3 , 253-258. (6) Parker, R. T.; Freedlander, R. S.; Dunlap, R . B. Anal. Chim. Acta 1980, 119, 189-205. (7) Parker, R. T.; Freedlander, R. S.; Dunlap, R. B. Anal. Chlm. Acta 1980, 120, 1-17. (8) Ward, J. L.; Yen-Bower, E. L.; Winefordner, J. D. Talanta 1981, 28, 119-120. (9) McAleese, D. L.; Freedlander, R. S.; Dunlap, R. B. Anal. Chem. 1980, 52, 2443-2444. (10) McAleese, D. L.; Dunlap, R. B. Anal. Chem., in press.
RECEIVED for review September 6,1983. Accepted November 28,1983. These investigations were supported by NIH Grant CA 15645 from the National Cancer Institute. R. Bruce Dunlap is the recipient of a Faculty Research Award (FRA144) from the American Cancer Society.
Kinetic-Spectrophotometric Determination of Trace Levels of Iron Henri Rooze Universitd Libre de Bruxelles, Facultd des Sciences Appliqudes, 1050 Brussels, Belgium During a study of the kinetics of chlorite disproportionation (1,2) we observed that this process is catalyzed by traces of
Table I. Iron Content in Reagents 1.63 X 2.5 x T = 25.0
iron. o-Tolidine (biphenyl, 4,4’-diamino-3,3’-dimethyl-) is oxidized by intermediates of the reaction so that the overall stoichiometric reaction is given by eq 1. Here, o-tolidine is HClOz
+ 2NHzRNHz
?
M NaClO, M o-tolidine 0.2 “C amt of Fe, ppb
Fe(II1)
+
2NHRNH HC1+ 2H20 (1) represented by NH2RNH2and the imine resulting from its oxidation by NHRNH. In acid medium (pH
