the heating time from 2 to 10 minutes did not affect the difference between sample and blank. Because the method described here measures only free Cr(III), it cannot be directly applied to samples where the value for total Cr is desired. The method also cannot be directly compared with existing methods for Cr because none of them measures only free Cr(II1).
RECEIVED for review October 20, 1971. Accepted January 11, 1972. This work was supported in part through funds provided the University of Georgia by PHS, N I H Research Grant No. G M 17913-01 from the National Institute of General Medical Sciences. Use of trade names does not imply endorsement by the Environmental Protection Agency or the Southeast Water Laboratory.
Influence of Solvent Matrix upon Phosphorescence Signals R. J. Lukasiewicz, J. J. Mousa, and J. D. Winefordner’ Department of Chemistry, University of Florida, Gainesville, Fla. 32601 The influence of methanol-water mixtures and of sodium chloride, sodium bromide, and sodium iodide aqueous solutions of several organic molecules at 77 O K upon phosphorescence signals is studied. Only a few per cent by weight of methanol in water, sodium iodide in a methanol-water mixture, and sodium chloride, sodium bromide, or sodium iodide in water resulted in phosphorescence signals at 77 O K several orders of magnitude greater than the phosphorescence signal for the same analyte in pure water solution. The salt solutions at 77 O K resulted in even larger phosphorescence signals than the methanolic solutions due to the additional effect of the heavy atom (iodide being the most effective of the three halides). The optimum aqueous solvent for routine analytical phosphorimetric measurements appears to be a 5-30% aqueous solution of sodium iodide. All studies are carried out with an open rotating quartz capillary cell.
APPLICATIONS OF MOLECULAR PHOSPHORESCENCE in chemical analysis have been limited since phosphorimetry was first reported as an analytical technique about fifteen years ago. Although phosphorimetry has been shown to be an extremely sensitive analytical method (Z-3), of considerable value in trace analysis of molecules which can achieve appreciable excited triplet state populations relative to the excited singlet states, technological problems in sample handling and choice of solvent have been the main obstacles to its wide-spread use. In the past, phosphorimetry has been limited almost exclusively to nonaqueous solvents and solvent mixtures which formed clear, rigid glasses at low temperatures. This requirement is indeed very troublesome because many factors both physical and chemical can cause serious cracking of the glasses and lead to poor analytical results. However, initial studies on the analytical utility of intensely cracked and snowed matrices in phosphorimetry were encouraging ( 4 ) . While a small degree of cracking is difficult 1
Author to whom reprint requests should be 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, “Fluorescence Assay in Biology and Medicine,” Volume 11, S. Udenfriend, Ed., Academic Press, New York, N.Y., 1969. (3) W. J. McCarthy and J. D. Winefordner, “Fluorescence Theory, Instrumentation and Practice,” G. G. Guilbault, Ed., Marcel Dekker, New York, N.Y., 1967. (4) R. Zweidinger and J. D. Winefordner, ANAL.CHEM.,42, 639 (1970).
to control and leads to poor results, intense cracking and snowing of the matrix can be achieved for some solvents and excellent analytical results can be obtained ( 4 , 5 ) . Snowed and crystalline matrices often undergo considerable changes in structure on cooling, which cause severe stress on sample containers often shattering them completely. Use of a new quartz capillary cell for phosphorimetry in predominately aqueous, snowed matrices has been previously described (5). Aqueous solutions of phosphorescent molecules form on freezing, a translucent severely cracked glass, which gives greatly reduced phosphorescence signal intensity. However, addition of small amounts of lower chained alcohols to aqueous solutions produces a snowed matrix which gives excellent phosphorescence signals ( 5 ) . Similar results have been reported in hydrocarbon solvents (6, 7). The physical and chemical nature of the matrix and its effect on phosphorescence signals are not completely understood. Although appreciable molecular aggregate formation could occur in aqueous solutions and result in reduction of the phosphorescence signal oia triplet-triplet annihilation (8), molecular aggregation is apparently minimized by addition of alcoholic solvents, and the solutions studied are too dilute for appreciable triplet-triplet annihilation. Therefore, the mechanism accounting for the effect of the matrix upon the phosphorescence signals seems to rely upon diffuse reflection of incident exciting light within the crystalline matrix ( 9 ) resulting in multiple scattering at the boundaries of the individual particles ; multiple scattering enhances the probability of an absorptive process and thus increases the intensity of phosphorescence signal emitted. We wish to report on recent studies involving the influence of the matrix upon phosphorescence signals inasmuch as the matrix affects analytical phosphorimetry, and to discuss the development of new analytically useful aqueous solvents for phosphorimetry. Previous studies (5) have demonstrated the utility of mixed alcohol-water solvents in phosphorimetry. A more detailed study of phosphorescence signals as a function of solvent composition has revealed a close correlation between the physical and chemical structure of the (5) R. J. Lukasiewicz, P. Rozynes, L. B. Sanders, and J. D. Winefordner, ANAL.CHEM., 44, 237 (1972). (6) H. H. Richtol and F. H. Klappmeier, J. Amer. Chem. Soc., 86, 1255 (1964). (7) R. A. Keller and D. E. Breen, J . Chem. Phys., 43, 2562 (1965). (8) H. Sternlicht, G. C. Nieman, and G. W. Robinson, J . Chem. Phys., 38, 1326 (1963). (9) W. W. Wendlandt and J. G. Hecht, “ReflectanceSpectroscopy,” Interscience Publishers, New York, N.Y., 1966, p 46. ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
963
-w v)
U
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30
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METHANOL /WATER
Figure 1. Phosphorescence signal as function of solvent composition (methanol-water) 0 2.0 X 10-6M 3-indole acetic acid A 4.7 X 10-6M hippuric acid 1 1.7 X 10-GMsulfacetamide
matrix and the phosphorescence Observed. These results were obtained for both aqueous alcoholic solvents and aqueous solutions of alkali halide salts as solvents. Phosphorescence background studies aqueous solvents will be presented. EXPERIMENTAL
Apparatus. An Aminco-Bowman spectrophotofluorometer with an Aminco-Keirs phosphoroscope attachment, a 150watt xenon arc lamp, and a potted RCAlP28 photomultiplier were used for all studies (American Instrument Co., Silver Spring, Md.). The basic instrument was arranged as previously described ( 4 , 5 ) with the following exceptions. A Keithley Model 244 high voltage supply (Keithley Instruments, Cleveland, Ohio) was used to provide power to the photomultiplier tube. A rotating sample cell apparatus ( 4 , 10) consisting of a Varian A 60-A High Resolution Nuclear Magnetic Resonance Spectrometer Spinner Assembly (Varian Associates, Palo Alto, Calif.) was modified for use with a quartz capillary sample tube. The sample tubes were made from T21 Suprasil capillary tubing (Amersil Inc., Hillside, N.J.), having 5 mm 0.d. and 0.90 mm i.d. Reagents. Reagents used without further purification were : sulfacetamide; 3-indole acetic acid; and hippuric acid (all from Nutritional Biochemical Corporation, Cleveland, Ohio) ; sodium chloride, sodium bromide, sodium iodide (Fisher Scientific Co., Fair Lawn, N.J.); and spectroquality methanol (Matheson, Coleman and Bell, East Rutherford, N.J.). Deionized water was obtained directly from a commercial ion exchange column. Procedure. Phosphorescence spectra for the molecules studied were obtained in various aqueous solvents using the quartz capillary sample tube suspended in liquid nitrogen (10) H. C . Hollifield and J. D. Winefordner, ANAL.CHEM..40, 1759 (1968). 964
in a quartz window dewar flask. Fluorescence spectra at room temperature were obtained using a 1-cm square conventional fluorescence cell. Light scattering data were obtained at 77 OK using the capillary sample tube as described above, but with the rotating can phosphoroscope removed. The slit arrangement used for all studies was 3,4,3,3,4,3 (in mm, corresponding to 17 and 22 nm spectral half-band pass). Spectra are not corrected for instrumental response. A 0.5-second time constant was used in the nanoammeter. Stock solutions of 10-3M of each reagent in water were prepared. More dilute aqueous solutions of each reagent with various concentrations of sodium chloride, sodium bromide, or sodium iodide and/or methanol were prepared. Phosphorescence signals were measured as a function of solvent composition. All results were taken as the average of three measurements. The final concentration of the phosphorescent molecule in each case was approximately 10-6M. Sample handling procedures were followed as previously described (5). The rate of cooling is critical for analytes in pure water or in low weight-per cents (
