I .061
t
CEMENT
5
000-MEAN
(94000 counts1
t
0 94.
CEMENT
4
i 1
0 9 6 r
4
I
tn
i
1
i
0961
2
lA06
1
I
0 92
0 92
J
I 5
5 Length
of
fusion , minules
Figure 7. Variation of Ca fluorescence intensity as a function of
melting time and agitation for a cement sample Agitation. 0 manual, 10 sec every 5 min. 0 continuous
CONCLUSION Continuous or intermittent agitation during fusion yields beads with the same quality (homogeneity and absence of bubbles) if the melting time is sufficiently long. The minimum melting time is about 5 minutes with continuous agitation and about 10 to 15 minutes with occasional agitation for the samples examined. These minimum values are expected to increase when heating temperature is lower or when less reactive samples are fused. If some security factor is allowed for this, the melting time is not changed significantly with automatic agitation but it may become prohibitive with manual agitation.
Automatic agitation also has some additional advantages: the radiation effects on arms and face of the operator are minimized; the operator is free to do other work during fusion; and the productivity is increased.
ACKNOWLEDGMENT The authors wish to thank Regent Marcoux for his contribution to the experimental part of this study and the Minister of the Department of Natural Resources for the permission to publish this manuscript. Received for review June 25, 1973. Accepted November 16, 1973
New Method of Analysis Based on Room-Temperature Phosphorescence R. A. Paynter, S. L. Wellons, and J. D. Winefordner' Department of Chemistry, University of Florida, Gainesville, Fla. 32601 Until recently, with few exceptions, strong phosphorescence of organic molecules had been observed only in the gas phase, in rigid media, or a t liquid nitrogen temperatures (2-3). Room temperature triplet state emission has recently been reported by Walling and Schulman (4, 5 ) from ionic organic molecules adsorbed on a variety of supports, including silica, alumina, paper, asbestos, and oth-
A u t h o r t o w h o m r e p r i n t requests s h o u l d b e sent.
(1) M. Zander, "The Application of Phosphorescence to the Analysis of Organic Compounds." Academic Press, New York, N.Y.. 1968. (2) J. D. Winefordner, P. A. St. John, and W. J. McCarthy, Chapter on "Phosphorimetry" in "Fluorescence Assay in Biology and Medicine, Vol. 1 1 , " S. Udenfriend, Academic Press, New York, N.Y., 1969. (3) J. D . Winefordner, "Phosphorimetry," in "Accuracy in Spectrophotometry and Luminescence Measurements," in Proceedings of a Conference held at NBS, March 22-24, 1972, NBS Spec. Pub/. 378, Washington, D.C., 1973. (4) E. M . Schulman and C. Waliing, Science, 178, 53 (1972): (5) E. M. Schulman and C. Walling, J. Phys. Chem., 77, 902 (1973).
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ANALYTICAL CHEMISTRY,
VOL. 46, NO. 6 , M A Y 1974
ers. Also, phosphorescence a t room temperature, involving either particularly rigid molecules (6) or the use of a spin forbidden transition enhancer such as dimethylmercury (7), has been observed in fluid media. In these previous reports, room temperature phosphorescence (RTP) was simply reported, and no analytical applications of the phenomena were dealt with. In the present note, the authors developed and applied the phenomena observed by Walling and Schulman ( 4 , 5 ) toward analytical usefulness. As this report will further illustrate, RTP appears to offer a fast, economical, and convenient method of analyzing a variety of molecules, many of biological interest.
EXPERIMENTAL Apparatus. I n t e n s i t y measurements a n d e x c i t a t i o n / e m i s s i o n spectra were d e t e r m i n e d w i t h a n A m i n c o - B o w m a n Spectrophotofluorometer (SPF) w i t h a phosphoroscope a t t a c h m e n t (phospho-
(6) R. 8. Bonner. M. K . DeArmond, and G. H. Wahl, J. Arner. Chem. SOC.. 54, 988 (1972). (7) E. Vander Donckt, M. Matagne, and M. Sapir. Chem. Phys. Lett.. 20, 81 (1973).
Table I. Phosphorescence Characteristics of Filter Paper
Companya-Type
Curtin-7760 E-D-613 E-D-615 Reeve Angel-201 Reeve Angel-202 S S-604 S-P--F-2402 S-P--F-2406
+
Whatman-1 Whatman-30 W hatman-40 Whatman-41 Whatman-42 Will-13021 Will--13061
Excitatioqb nm
Emission, nmb
300 (316, 460) 294 (320) 316 317 310 292 295 290 290 294 (316) 294 (320) 294 (316) 300 (322) 295 (320, 390) 316
494 487 499 517 505 490 485 485 49 1 490 480 486 495 494 501
I Relative phosphorescence signals'
3.0 1.0 4.8 5.0 1.5 3.0 1.8 3.0 2.6 5.0 2.0 3.5 1.6 10.
9"
11.
Key: Curtin = Curtin Scientific; E-D = Eaton Dikeman; S + S = 'Schleicher and Schuell; S-P = Scientific Products; Will = Will Scientific. Excitation and emission peak wavelengths. Excitation wavelengths in ( ) are shoulders. All signals taken with respect to E-D-613, the filter paper used in the present studies. a
roscope was used but not the quartz dewar flask). The source and photodetector were a 150-W xenon arc lamp and a IP21 photomultiplier tube with S4 spectral response, respectively. Reagents. All chemicals were reagent grade. The solvent for all RTP studies was 1M NaOH. Procedure. Because no other reports of this phenomenon as an analytical tool have been made, great care was taken in developing a reproducible method of preparing and testing the samples, A thorough study of the phosphorescence background intensities of a variety of filter papers was performed and, of the filter papers studied (see Table I), Eaton Dikeman 613 yielded the lowest phosphorescence background and had excitation and emission peaks located a t more preferable wavelengths-Le., wavelengths a t which some of the analytes studied were not greatly excited or greatly emitting. The filter paper was cut into %-in. circles using a conventional paper hole puncher. The paper was then suspended vertically by small alligator clips in a clothesline fashion. The sample solution was allowed to drain slowly from the tip of the microliter syringe when it came into contact with the filter paper circle; 5 p1 of sample was placed on each filter paper circle, because this volume resulted in an even and reproducible distribution of the solution. Drying of the sample was essential; it had been both previously reported by Walling and Schulman ( 4 , 5 ) and further shown by us that moisture on the paper caused radiational (phosphorescence) quenching. After applying the 5 p1 of solution to the filter paper, the filter paper was air dried for a t least 1 hr. The samples were then removed from the clips and placed in a desiccator for a t least 80 min for final drying. Hot air blowers and other methods of drying, including ovens, were tested and, contrary to the previous reports ( 4 , 5 ) , air drying followed by a period in a desiccator provided more reproducible as well as more intense phosphorescence measurements. After drying, the sample was placed in a long spindle-like holder (see Figure l), and placed into the sample compartment of the spectrophotofluorometer with phosphoroscope attachment (but without the quartz dewar flask). Dry air was passed through the cell to keep the chamber moisture-free and also to allow for a final drying stage of the sample in case it collected any moisture in transit from the desiccator to the cell ( 2 min was allowed for this step),Afterwards, the phosphorescence signal was measured. It should be stressed that the filter papers with the samples must be carefully handled to minimize contamination and/or loss of sample. Work is currently in progress to decrease the extensive drying times.
!5/64'DIAM.
Figure l a . Schematic diagram of filter paper cell system for room temperature phosphorescence studies. (All dimensions to scale unless otherwise stated. Only the hole size of 15/64 in., the inside diameter of part C, and t h e distance of 6.45 in. from t h e bottom of the cap A and the center of the46''' in. hole are really critical). It should also be noted that this sample cell unit fits into the standard Aminco phosphoroscope accessory instead of the normal cap and cylinder used for t h e dewar flask assembly A . Threaded brass bushing to hold B. B. Threaded stainless steel rod. C.
Brass cylinder (mounts on standard Aminco phosphoroscope accessory). D. Flat brass filter paper holder. Filter paper slides between D and flat end of rod B. Spacers of '/32 in. used to separate D from flat end of Rod B. E . Filter paper circle, '14 in.-diameter. filter paper slid into piace by means of tweezers. The filter circle is positioned within inwhole; the amount of analyte not excited is negligible. After placing filter paper under hold, it is pressed slightly with tweezers to assure that it is aligned
SIDE V I E W
I
E +--
FRONT V1EW
?dk-
RESULTS AND DISCUSSION The present s t u d y indicates that room-temperature phosphorescence (RTP) is possible from a wide variety of ionic organic molecules and that RTP could h a v e q u a n t i -
I5/6d'DI AM.
Figure 1b. Detailed drawing of part D A N A L Y T I C A L C H E M I S T R Y , V O L . 46, NO. 6, M A Y 1974
737
~~
~~
Table 11. R o o m - T e m p e r a t u r e Phosphorescence Characteristics of Several Ionic Organic Molecules Adsorbed o n Filter Paper. Molecule
2-Amino-1-naphthalene sulfonic acid Eosin Y Naphthalic anhydride 2-Naphthalene sulfonic acid Diphenic acid Naphthalic acid, sodium salt Naphthalene-2-sulfonic acid 1-Naphthalene sulfonic acid 2-Amino-6-methylmercaptopurine
Excitation peak, nrn
Emission peak, nm
352 523e 285 286 277 333 286 293 331
526 56ge 543 511 491 541 518 513 487
Limit of detection rg/mlc
Range of iinearityb
420 3,000
Limit of detection,
wd
0.4
2. 0.1
0.02 0.6 0.2 100. 17. 28.
1,000
>5,000 10
14 15 12
3. 1.
500. 73. 140. 55. 29.
11.
6.
22
All molecules in 1 M NaOH. Range of linearity extends from detection limit to upper concentration where deviation from linearity is 1%. The ranges for the last five molecules is of order of 10 because of the rather high detection limits compared to the first four molecules (the high detection limits of the last five are a result of the high phosphorescence background at the respective excitation wavelengths). Limit of detection (in rg/ml) is the concentration of analyte resulting in a signal-to-noise ratio of 3. Limit of detection (in ng) is the amount of analyte resulting in a signal-to-noise ratio of 3; it is calculated from the concentrational value by using the sample volume of 5 pl. e The luminescence of Eosin Y a t room temperature is a combination of E-type delayed fluorescence and phosphorescence.
77'K,SOL. 297'K,PAPER
3-00
ZUO
...
, 4'00
I*
.
500 WAVELENGTH,m
600
loo
I
Room temperature (297 O K ) and low temperature (77 phosphorescence of 2-amino-1-naphthalene sulfonic acid (in 1M NaOH). Room temperature measurements are performed with the analyte adsorbed on filter paper and the low temperature measurements are performed with sample in 1M NaOH solvent and measured with a conventional Aminco spectrophotofluorometer with phosphoroscope attachment Figure 2.
O K )
tative and qualitative identification purposes. In Table 11, a summary of analytical figures of merit of the samples tested so far is given. These samples are representative of a wide variety of polynuclear, carboxylic or sulfonic acids, phenols, and amines. Virtually any salt of these compounds can be expected to phosphoresce a t room temperature. Continuing studies have begun on a wide variety of compounds of biological interest. The phosphorescence spectra of samples a t room temperature adsorbed on filter paper were compared with the phosphorescence spectra of the same samples a t liquid nitrogen temperature (77 OK); some typical spectra are shown in Figures 2 and 3. These spectra compare favorably-ie., excitation and emission peaks are almost identical-although there is slight band broadening and a slight shift of emission peaks toward the red for RTP. The low wavelength excitation peak was a result of phosphorescence background of the filter paper. Our findings on the compounds tested indicate that this
738
ANALYTICAL CHEMISTRY, VOL. 46, NO. 6, M A Y 1974
.-' KJO
:i1 x-
300
_-
400
500
\, ,'. 600
I
700
Room temperature (297 O K ) and low temperature (77 phosphorescence of 2-amino-6-methylmercaptopurine (in 1 M NaOH). Also see comment in caption of Figure 2
Figure 3. O K )
method provides a sensitive means of identifying molecular aromatic species which are ionic. Although there is no strong theoretical explanation for this phenomenon, it is believed that the ionic state of the molecule results in great molecular rigidity via adsorption to the substrate, which reduces radiationless decay due to collisional deactivation. It appears that there should be many analytical applications of this method, particularly for biological and pollution samples. Studies to be carried out in the near future include the investigation of other substrates-e.g., thin layer materials, the effect of sample temperature, and possibly the use of a heater in the filter paper holder, the influence of pH on the phosphorescence signals, and analysis of species in blood serum, urine, and air pollution particulates. Finally, it would seem that time-resolved phosphorimetry should provide additional selectivity in measurements of real samples (3). Received for review September 10, 1973. Accepted December 10, 1973. Research was carried out as a part of a study on the phosphorimetric analysis of drugs in blood and urine, supported by U S . Public Health Service Grant No. GM-11373-10.
