Anal. Chem. 1900, 52, 2443-2444
2443
Elimination of Moisture and Oxygen Quenching in Room-Temperature Phosphorescence David L. McAleese, Richard S. Freedlander,' and R. Bruce Dunlap" Department of Chemistry, University of South Carolina, Columbia, South Carolina 29208
Room-temperature phosphorescence (RTP) is attracting considerable attention as a new means of trace analysis for important organic and biological compounds (1-6). A variety of compounds exhibit the phenomenon when adsorbed on a suitable solid substrate. In the past, phosphorimetric methods have suffered from experimental difficulties associated with low-temperature techniques; however, the advent of roomtemperature phosphorimetry promises to alter this situation (7, 8). Despite several advantages of R T P over more traditional types of analysis, this technique is highly susceptible to oxygen and moisture quenching (8-10). Investigators have attempted to circumvent these quenching problems through the use of sophisticated drying apparatus (11,121, cumbersome sample preparation (13), and expensive, time-consuming drying procedures ( I , 2,8). Herein we describe a simple, rapid, reproducible, and economical technique for R T P sample preparation which alleviates moisture and oxygen quenching. EXPERIMENTAL SECTION Phosphorescence Apparatus. Phosphorescence intensity measurements and excitation/emission spectra were obtained on an Aminco-Bowman spectrophotofluorometer equipped with a phosphoroscope accessory (14). A 150-W xenon arc light source and a 1P21 photomultiplier tube detector were used. Instrumental slits were set at 4 mm throughout the study. Chemicals. All compounds were reagent grade and were utilized without any further purification. Acetone solutions of the various analytes were stored in the dark for several days. Sample Preparation. Our technique simply involved spotting the phosphor on a paper substrate. Five microliters of an acetone solution containing the analyte was applied to a 6.4-mm diameter Whatman No. 1 filter paper circle, followed by the application of 3 pL of a 1 M aqueous sodium citrate solution at pH 7 . Samples were subsequently dried in an evacuated vacuum desiccator for 1 h and then transferred one at a time to the phosphorimeter and analyzed. For comparison purposes, similar RTP samples were prepared by spotting water in place of sodium citrate. These were the appropriate control samples since RTP intensities can be affected by the choice of solvents originally applied to the paper supports. However, the phosphors were irreversibly quenched during the transfer from the vacuum desiccator to the phosphorimeter. For exclusion of oxygen and moisture, phosphors were, instead, spotted on mounted paper supports which were immediately placed in the cell compartment to dry. Maximum signals were produced by flushing dehumidified argon over the samples for 30 min at a flow rate of 500 mL/min. Sample Irradiation. All phosphors were susceptible to degradation by continuous exposure to xenon light in the cell compartment. Therefore, the light was only permitted to impinge upon the samples for 5 s when recording intensites. Controls were run for samples exposed to light intermittently. Sample Quenching. The sample holder was removed from the cell compartment to expose the cell-dried RTP samples to humidified air. Vacuum desiccated samples were exposed to humidity after removal from the desiccator, during the mounting process, and while immobilized in the sample holder. Oxygen quenching studies were performed by flushing dehumidified oxygen in the cell compartment at 125 mL/min. RESULTS R T P samples prepared with water were sensitive to oxygen and moisture quenching as indicated by a rapid decrease in intensity for all phosphors exposed to humidified air (Table I). However, samples prepared with sodium citrate appeared Present address: IC1 Americas, Inc., Biological Research Center,
P.O. Box 208, Goldsboro, NC 27530.
0003-2700/80/0352-2443$01 .OO/O
to be insensitive to immediate oxygen and moisture quenching when exposed to a relative humidity of 60% or less. The quenching curves from the model phosphor, 4-biphenylcarboxylic acid, illustrate this point (Figure 1). A comparison of the quenching percentages from several compounds applied t o paper and to our mixed support demonstrates the protection from oxygen and moisture afforded by the mixed support (Table I). Furthermore, our technique provided the same good analytical reproducibility as the paper-only samples with an average relative standard deviation of less than 3% from triplicate measurements of several phosphors (Table 11). Noteworthy was the fast analysis time of 1 min/sample after a 1-h drying period. The sodium citrate impregnated paper did not prevent immediate oxygen and moisture quenching at relative humidities greater than 60%. For example, a t a relative humidity of 72 % , the intensity from 4-biphenylcarboxylic acid quenched 5 % during the first minute and 36% after 2 min. On the other hand, samples exposed t o relative humidities less than 40% for several hours failed to quench at all. T o illustrate the capability of the mixed support to discourage oxygen diffusion alone, we generated quenching curves for 4-biphenylcarboxylic acid adsorbed on paper and sodium citrate impregnated paper (Figure 2). Previous work indicated that oxygen quenching increases concomitant with rising humidity (8). The phosphorescence intensity of sodium citrate treated samples was maximized by optimizing the amount of impregnating agent, the drying time, and the spotting order for three compounds, 4-biphenylcarboxylic acid, l-naphthalenesulfonic acid, and o-aminobenzamide. The addition of 3 pmol of sodium citrate to a paper sample containing 5 nmol of phosphor produced the highest intensity. A 3-pL delivery of the nearly saturated 1 M sodium citrate solution covered the entire paper surface without leakage of sample off the edge. Further vacuum desiccation of samples beyond 1 h did not significantly affect the intensities, but better reproducibility resulted with consistent drying times. Solutions of the analytes deposited on previously dried sodium citrate impregnated paper yielded very low intensities. Samples prepared by spotting sodium citrate first, immediately followed by the phosphor or by spotting both together, appeared to exclude oxygen and moisture to the same degree as spotting sodium citrate immediately after the phosphor. Again a consistent method was required for good reproducibility. DISCUSSION The preservation of R T P intensity from the quenching effects of moisture and oxygen appears t o depend on the nature of the mixed support system. Niday and Seybold suggest that impregnating agents may plug channels and interstices within the matrix thereby decreasing oxygen permeability and its inherent quenching effect (15). Similarly, we can postulate that the impregnating agent's ability to decrease moisture permeability explains the inhibition of moisture quenching. A series of protective agents is currently under investigation in this laboratory, and sodium citrate is the best thus far examined. Sodium citrate impregnated paper facilitates the rapid analysis of samples at relative humidities of 60% or less. This humidity limit allows a 2-min exposure to air during the mounting procedure before any quenching is apparent; however, samples can be mounted and analyzed in less than 1 min. 0 1980 American Chemical Society
2444
ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980
Table I. Paper and Sodium Citrate/Paper Supports Na citratelpaper
paper
intens quenchedb 7%
phosphor a
7% humidity
re1 intense
58 2 1 100 0 4-biphenylcarboxylic acid 58 800 0 1-naphthalenesulfonic acid 0 1-naphthoic acid 50 2 380 0 1-naphthol 50 410 0 o-aminobenzamide 52 1000 51 8 000 0 p-aminobenzoic acid 0 p-hydroxybenzoic acid 50 1310 Results from a 2-min exposure to humidified air, a 5 nmol of phosphor. sities.
Table 11. Sodium Citrate/Paper RTP Support excitation/ emission re1 76 % h ~ phosphor" h (nm) intensb RSDC midity 4-biphenylcarboxylic acid l-naphthalenesulfonic acid 1-naphthoic 1-naphthol o-aminobenz-
v)
z
quenched intensb
7% intens quenchedb
1620 86 610
18 000
1530 3 900 6 20 1620
91
94 84 86
84
260 84 7 000 260 96 1260 76 94 Impregnating agents d o affect relative inten-
l
o
o
h
7s-
Y
289/482
21 004
2.1
58
2961514
796
4.0
58
-cz
2971518
2 305
1.5
50
E
3081520 3371412
341
2.9
992
4.6
50 52
2861422
7 963
2.2
51
2641406
1282
1.3
50
acid
re1 intens
W
50-
c 4 -1 W
2s-
c
amide
p-aminobenzoic acid p-hydroxyben-
0
zoic acid a 5 nmol of phosphor. background subtracted.
Average of triplicate samples, Relative standard deviation. ,.
100
u
-0
e 5 z Y c
-
75-
SO-
? c 4
3
0
4 TIME,
6
8
1
io
min.
Figure 2. Room-temperature phosphorescence intensity plotted as a function of quenching time for 4-biphenylcarboxylic acid adsorbed on paper (W) and sodium citrate impregnated paper (0). Quenching was accomplished by flushing dehumidified oxygen in the cell compartment at 125 mL/min.
LITERATURE CITED
2s-
E
",
2
is not limited by solubility or solvent incompatibility since R T P analyses are conducted upon samples in the solid state. It should be stressed that R T P is a nondestructive means of chemical analysis, so samples can be reanalyzed at a later date or eluted from their substrates and further analyzed via other procedures. With the development and maturity of R T P as an analytical tool, promising applications utilizing this technique can be anticipated.
t
Y
0
1
2 3 TIME, rnin.
4
5
Figure 1. Room-temperature phosphorescence intensity plotted as a function of quenching time for 4-biphenylcarboxylic acid adsorbed on paper ( 0 )and sodium citrate impregnated paper (0). Quenching was accomplished by exposure to air at a relative humidity of 58 YO.
During a 2-h period, a maximum of 60 samples can be dried and analyzed by using sodium citratelpaper supports vs. four samples for the paper-only supports. Besides the innovation of rapid sample analysis without the interference of moisture and oxygen quenching processes (utilizing our sample preparation technique), R T P analysis is attractive to the analytical chemist for several other reasons. On the basis of the inherent advantages of phosphorescence analysis, this technique has proven to be selective and sensitive. Since nanogram quantities of phosphors are detectable, it is a convenient technique for handling samples containing highly toxic components. In addition, R T P analyses are simple in design and require minimal costs. This technique
Aaron, J.; Kaleel, E. M.; Winefordner, J. D. J. Agric. FoodChem. 1979, 27. 1233-1237. Parker, R. T.; Freedlander, R. S.; Shulman, E. M.;Dunlap, R. B. Anal. Chem. 1979, 57, 1921-1926. Ford, D. D.; Hurtubise, R. J. Anal. Chem. 1979, 5 1 , 659-663. Yen-Bower, E. L.; Winefordner. J. D. Anal. Chim. Acta 1978, 101, 3 19-332. de Lima, C. G.; Nicoh, E. M. de M. Anal. Chem. 1978, 50, 1658-1665. Vo-Dinh, T.; Yen-Bower, E. L.; Winefordner, J. D. Anal. Chem. 1976, 48, 1166-1188. Paynther, R. A.; Wellons, S. L.; Winefordner, J. D. Anal. Chem. 1974, 4 6 , 736-738. Shulman, E. M.; Parker, R. T. J . Phys. Chem. 1977, 81, 1932-1939. Shulman, E. M.; Walling, C. J . Phys. Chern. 1973, 77,902-905. Yen-Bower, E. L.; Winefordner, J. D. Anal. Chim. Acta 1978, 102,1-13. Vo-Dinh, T.; Walden, G. L.; Winefordner, J. D. Anal. Chem. 1977, 49, 1126-1 130. Yen-Bower, E. L.; Winefordner, J. D. Appl. Specfrosc. 1979, 33, 9-12. Von Wandruszka, R. M. A.; Hurtubise, R. J. Anal. Chem. 1976, 4 8 , 1784-1 788. Parker, R. T.; Freedlander, R. S.; Dunlap, R. B. Anal. Chim. Acta, in press. Niday, G. J.; Seybold, P. G. Anal. Chem. 1978, 50, 1577-1578.
RECEIVED for review June 16, 1980. Accepted September 2, 1980. These investigations were supported by NIH Grant No. CA 12842 from the National Cancer Institute. R. Bruce Dunlap is the recipient of a Faculty Research Award (FRA144) from the American Cancer Society.
