an entirely different Raman spectrum, e.g., Irish et al. (9) studied the effect of pH on the Raman spectrum of SO4*-. Carbonate and NO2- anions are lost in acid solutions. This caustic requirement is seldom a disadvantage in our laboratory because nuclear waste materials are highly caustic to begin with. It is because of this caustic character that we are able to use peak heights instead of peak areas as recommended by Irish and Chen (1).

Samples are not turbid as were those experienced by Marston, but filtering to remove colloidal particles is occa- sionally necessary. This is a relatively minor disadvantage.

ACKNOWLEDGMENT The author acknowledges the work of T. J. Hanson in

performing the nitrate and sulfate calibrations, investigating fluorescence effects in waste liquors, and establishing the sample filtering technique.

LITERATURE CITED (1) D. E. Irish and H. Chen, Appl. Spectrosc., 25, 1 (1971). (2) hbkoto Ozeki and bid0 Ishii, BUnsekiKiki, 14, 153 (1976): Chem. Abstr.,

85, 096761b (1976). (3) K. M. Cunningham, M. C. Goldberg and E. R. Weiner, Anal. Chem., 49,

70 (1977). (4) D. E. Irish and J. D. Riddell. Appl. Specfrosc., 28, 461 (1974). (5) A. L. Marston, Nucl. Techno/., 25, 576 (1975). (6) W. I. Winters, USAEC, ARH-SA-110, (1972). (7) W. M. MacNevin and E. A. Hakkila, Anal. Chem., 29, 1019 (1957). (6) R. J. Moolenar, J. C. Evans, and L. D. McKeever, J . Phys. Chem., 74,

3629 (1970). (9) D. E. Irish and H. Chen, J. Phys. Chem., 74, 3796 (1970).

RECEIVED for review March 17, 1977. Accepted August 15, 1977. Presented a t the 173rd National Meeting, American Chemical Society, New Orleans, La., March 1977. This work was performed for the US. Energy Research and Development Agency under Contract EY-75-C-06-2130.

Lead or Thallium Salts as External Heavy Atoms for Room Temperature Quantitative Phosphorescence

I v a n M. Jakovljevic

The Lilly Research Laboratories, Indianapolis, Indiana 46206

The use of lead or thallium salts as external heavy atoms is described as a new technique to enhance room temperature phosphorescence. I n the case of cinoxacln, a clnnollne derivative, lt Is possible to quantltate as low as 50 pg, the total amount of sample needed for measurement.

The phosphorescence of organic molecules is usually ob- served in gas media or in liquid nitrogen cooled solutions. Room temperature triplet state emission of ionic organic compounds is a relatively new technique.

Kasha (1) was first to interpret the external heavy atom (ethyl iodide) effect in terms of a collisional perturbation of spin-orbital coupling. Hood and Winefordner (2) also em- ployed ethyl iodide to provide an external heavy atom to increase the sensitivity of phosphorescence at liquid nitrogen temperature. Vander Donckt et al. (3) showed that the addition of dimethyl mercury to solutions at room temperatwe increases the phosphorescence yields of several aromatic compounds. McGlynn et al. (4-6) in a series of papers about external heavy atom spin-orbital coupling have studied the physical significance of that effect. Schulman and Walling (7) reported strong triplet-state emission at room temperature from salts of many polynuclear carboxylic and sulfonic acids adsorbed on paper, silica gel, and other supports. This phosphorescence technique requires thorough drying but is not sensitive to oxygen. Paynter e t al. (8) reported room temperature phosphorescence on '/4-inch filter paper circles thoroughly dried after the samples (sulfonic acids, phenols, etc.) were applied. Seybold and White (9) proved that the presence of sodium iodide in 2-naphthalene sulfonate used on filter paper strongly decreased the fluorescence signal and increased the phosphorescence intensity without altering the transition energies. Tuan Vo Dinh et al. (10, 11) used sodium

iodide and silver nitrate as external heavy atoms perturbers in the room temperature phosphorescence. Boutilier and O'Donnell used the same two salts to increase the phos- phorescence quantum yields (12). von Wandruszka and Hurtubise (13) used an adsorption technique on pure sodium acetate crystals (crystal surface phosphorescence). Before dry sample technique was developed, careful degassing of solutions before forming the rigid glasses was necessary to prevent collisional quenching by oxygen or impurities in the organic molecules.

In this article a very simple and reproducible quantitative technique which increases room temperature phosphorescence is described. A filter paper is impregnated with lead tetra- acetate or thallous acetate solution prior to applying the sample. These two neighboring elements help increase the phosphorescence of some organic species to the extent tha t only 50 pg of total amount of sample is required.

EXPERIMENTAL Apparatus. Phosphorescence excitation and emission spectra

are determined with an Aminco-Bowman spectrophotofluo- rometer. The source is a 150-W xenon arc, and the detector a 1P21 photomultiplier tube. The phosphorescence attachment is a Cold Finger phosphoroscope (Aminco Catalogue No. 54-82361, and a dry sample holder (Figure 1) which faces the entrance slit at an angle of about 35'.

Disposable micropipets, 1 gL, Drummond Microcaps. Reagents. Methanol, distilled in glass, Burdick & Jackson,

Laboratories, Inc. Lead tetraacetate, G. Frederick Smith Chemical Co., Eastman

Kodak Co., or Aldrich Chemical Co., Inc., have been found satisfactory.

Lead tetraacetate solution: One gram of lead tetraacetate is dissolved in 100 mL of methanol in a glass-stoppered Erlenmeyer flask. The initial light yellow color of the solution changes in a few minutes to dark brown, then becomes colorless in 3-4 h. The solution is prepared one day in advance and is stable for about

2048 ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977

Figure 1. Dry sample holder with a paper strip inserted. The dot in the center of the window marks the application point

four months at room temperature conditions. Thallous acetate, Fisher Scientific Co. Thallous acetate solution: One gram of the salt is dissolved

in 100 mL of methanol to give a clear, colorless solution. Cinoxacin, l-ethyl-l,4-dihydro-4-0~0[ 1,3]dioxolo[4,5-g]cinnoline

3-carboxylic acid is a potent antimicrobial drug synthesized at the Lilly Research Laboratories (14, 15).

Preparation of Filter Paper Strips. Whatman No. 42 filter paper is cut in strips 3.3 x 1.1 cm. The paper strips are handled carefully to prevent contamination, especially to the area des- ignated for sample application. A strip is placed in the dry sample holder and a dot is made with a pencil (Figure 1).

To apply the lead tetraacetate or thallous acetate solution, the paper strip is picked up with tweezers at the end opposite to the dot and dipped into the reagent solution for about half of its length for 5 s. The paper strip is then dried for 10-15 s with an air blower at room temperature and kept until needed in a desiccator. This paper strip can be stored in a desiccator for weeks without loss of activity.

Application of the Sample. One microliter of a solution which contains, according to the phosphorescence intensity of the compound between 0.00005 and 0.01 pg, is applied in a single spot on the marked dot of the paper strip previously treated with lead tetraacetate or thallous acetate. After allowing it to stand for about 1 min on a watch glass at room temperature, the paper strip is placed in an oven at 105 O C for 10 min, then transferred to a desiccator for 10 min before measuring phosphorescence.

RESULTS AND DISCUSSION Filter Paper St r ips . Whatman No. 42 filter paper gives

good reproducibility. This is probably due to the proper characteristics such as thickness of that paper which allows 1 p L of the sample solution to spread evenly as a small circle (4-mm diameter) which fits the window (6 X 6 mm) in the dry sample holder.

The paper strip is inserted with the dot facing the analyst. If the paper strip is inserted in the reverse manner, the readings are about 30% lower.

Lead Tetraacetate Solution. No difference was observed by using 0.5% or 1% solution of the lead tetraacetate, bu t a 0.1% solution caused a 30% reduction in phosphorescence intensity.

The brown color of the solution observed immediately after lead tetraacetate was dissolved is probably due to a transient reduction of Pb(1V). The reagent can be discolored instantly by adding a few drops of hydrogen peroxide, but in this case its efficacy is reduced to one fourth. The fact that the reagent still worked in the abundance of free oxygen is a proof that the treatment with lead tetraacetate makes the phos- phorescence no more sensitive to collisional quenching by oxygen.

Table I. Phosphorescence Intensities of Cinoxacin Using Different Metal Salts (Lead Tetraacetate = 100)

Atomic No. 81

82

4 11 12 13 20 21 28 29 30 31

38 40 47 48 49 50 51 53 55 56 58 59 63 78 80 83 90

Salt Thallic acetate Thallous acetate Thallous fluoride Thallous sulfate Lead tetraacetate Lead subacetate Lead borate Lead chloride Beryllium acetate Sodium acetate Magnesium acetate Aluminium acetate (basic) Calcium acetate Scandium chloride Nickel chloride Cupric acetate Zinc acetate Gallium nitrate Gallium ammonium chloride Strontium acetate Zirconium acetate Silver acetate Cadmium acetate Indium acetate Tin chloride Antimony chloride Potassium iod ide Cesium acetate Barium acetate Cerous acetate Praseodymium acetate Europium acetate Platinum chloride Mercuric chloride Bismuth subnitrate Thorium nitrate

Intensity 50 90 100

1

100 80 42 10

5 5 8 0 8 4 0 0 3 0 0 4 0 0 3 2 1 6 3 7 7 4 0 0 0 0 4 10

Many other solvents were tried for dissolving lead tetra- acetate and found unsatisfactory. In addition to methanol, only 2,4pentanedione was suitable. It produces a clear, yellow solution which becomes colorless in several minutes. Methanol is the preferred solvent because of its greater volatility.

Table I shows the comparison of relative phosphorescence intensities (lead tetraacetate = 100) with several other metal salts used to soak paper strips. If the acetate of a particular element was not available, another salt was used. If a salt was insoluble or partially soluble in methanol (strontium or barium acetates), the paper strip was dipped into the suspension. Paper strips were dipped in the 1 % solution or suspension of the salts listed, dried as described, and then 1 pL, equivalent to 0.001 pg of cinoxacin was applied and the phosphorescence measured.

If a sufficient amount of sample is applied (about 0.1 pg of cinoxacin) to a paper strip not treated with lead tetra- acetate, a light blue luminescence can be detected if observed under 366-nm exciting radiation. Only if the paper strip is first impregnated with 0.1 N alcoholic solution of sodium hydroxide and then cinoxacin applied and dried, will the phosphorescence phenomenon be seen clearly: after the UV source is cut off, phosphorescence effect will be seen (mean lifetime 0.45 s).

If the sample is applied to a paper strip previously treated with lead tetraacetate or thallous acetate, a bright yellow luminescence appears. The treatment with these two salts makes the mean lifetime much shorter.

Quantitation. Cinoxacin proved to be an excellent aro- matic molecule for the study. Amounts as low as 0.000 05 pg

ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977 2048

: T i t -

P

Figure 2. Fluorescence(- - -), and phosphorescence (-) spectra of cinoxacin. Excitation (A) and emission (A') spectra of cinoxacin on paper strip treated with lead tetraacetate. Excitation (B) and emission (B') spectra of cinoxacin on plain paper. The real ratio of A to B is about 80

Table 11. Room-Temperature Phosphorescence Characteristics. Cinoxacin Compared with Several Other Compounds Adsorbed on Paper Impregnated with Lead Tetraacetate

Excitation Emission Compound peak, nm peak, nm

Cinoxacin, l-Ethyl-1,4-di- hydro-4-oxo-[ 1,3]dioxolo [4,5-g]cinnoline-3- carboxylic acid 290, 370 51 5

Diphenyl 27 5 470 Diphenylacetic acid 320 5 00 4-Diphenylcarboxylic acid 300 490

1-Naphthalene sulfonic acid 290 490, 515 2-Naphthoic acid 290, 300 490, 515 p-Aminobenzoic acid 300 430 Quinine sulfate 335 510 2-Fluorobiphenyl 280 460

are measured successfully. Linear relationship was observed for relative intensity vs. pg of cinoxacin per paper strip over the range of 0,002-0.01 pg. For that particular case the in- strument settings were: excitation monochromator: entrance (xenon), 3 mm; exit, 3 mm; slit slide, 2 mm; emission

4,4'-Diphenyldisulfonic acid 290 485

monochromator: entrance, 3 mm; exit (PMT), 3 mm; slit slide, 3 mm; percent full scale, 0.3; sensitivity, 50; excitation, 370 nm; emission, 510 nm.

The background luminescence of paper itself is a limiting factor; when excited a t 310 nm, the emission is a t 505 nm. Therefore, whenever the instrument settings are at very high sensitivity, a blank consisting of a paper strip dipped in the reagent solution should be used for the necessary correction.

The phosphorescence of cinoxacin (Figure 2) does not show any change in intensity for the first 25 min if the paper strip is kept at room temperature and in the normal laboratory light. After 18 h, the phosphorescence is still 65% of the initial value. Since the phosphorescence is relatively stable, there is no need to flush the sample chamber of the phosphoroscope with dry air or nitrogen as required in many procedures. The 10-min heating period does not enhance the phosphorescence very much but makes the results become more reproducible.

This initial communication relates observations involving the surface adsorption with enhanced triplet state formation where lead or thallium salts have a profound role. The variation of the excitation and emission wavelengths observed (Table 11) also indicates its qualitative applicability.

In the next paper, the methodology will be applied to a larger number of molecules in an attempt to classify them, and to try to explain the special effect of lead and thallium salts on the phosphorescence.

LITERATURE CITED (1) M. Kasha, J . Chem. Phys., 20 , 71 (1952). (2) L. V. S. Hood and J. D. Winefordner, Anal. Chem., 38, 1922 (1966). (3) E. Vander Donckt, M. Matagne, and M. Sapir. Chem. Phys. Lett., 2 0 ,

81 (1973). (4) S . P. McGlynn, R. Sunseri, and N. Christodouleas, ./ Chem. Phys., 37,

1818 (1962). ( 5 ) S. P. McGynn, J. Daigre, and F. J. Smith, J , Chem. Phys., 39, 675 (1963). (6) S. P. McGlynn and 1. Azumi, J . Chem. Phys., 40, 507 (1964). (7) E. M. Schulman and C. Walling, J . Phys. Chem.. 77, 902 (1973). (8) R. A. Paynter, S. L. Welions, and J. D. Winefordner, Anal. Chem., 48,

736 (1974). (9) P. G. Seybold and W. White, Anal. Chem., 47, 1199 (1975).

(10) Tuan Vo Dinh, E. Lue Yen, and J. D. Winefordner. Anal. Chem., 48, 1186 (1976).

(1 1) Tuan Vo Dinh, E. Lue Yen, and J. D. Winefordner, Taknta, 24, 146 (1977). (12) G. T. Boutilier and C. M. O'Donnell, Anal. Chem., 46, 1508 (1974). (13) R. M. A. von Wandruszka and R. J. Hurtubise, Anal. Chem., 48, 1784

( 1 97 6). (14) W. A. White, Ger. Patent, Offen., 2005 104 (1970). (15) W. A. White, U.S. Patent, 3 669 965 (1972).

RECEIVED for review June 13,1977. Accepted August 29,1977.

2050 ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977