Improvements in Instrumentation for Phosphorimetry of Organic Molecules in Aqueous Solution at 77°K R. J. Lukasiewicz, J. J. Mousa, and J. D. Winefordner. Department of Chemistry, Unicersity of Florida, Gainesuille, Fla. 32601
THEANALYTICAL UTILITY of aqueous solvents for phosphorimetric analysis of polar organic molecules has been recently demonstrated ( I , 2 ) . An open end quartz capillary tube requiring only about 20 p1 of sample was used as a sample cell. The relatively thick walls of the capillary tube prevented shattering of the sample cell by expansion of frozen aqueous solutions. Limits of detection were extended to the subnanogram region. Precision of replicate determinations was about lo%, with the major cause for poor precision being nonreproducible sample cell positioning in the quartz dewar flask. A study of the influence of the solvent matrix upon phosphorescence signals has revealed that for predominately aqueous solutions, a close correlation between the physical structure of the matrix and phosphorescence signal exists (2). Addition of small amounts of low chained alcohols or alkali halide salts to pure water solutions of phosphorescent molecules results in a n enhancement of signals by more than two orders of magnitude. Additional enhancement of signal may be achieved for many molecules via the external heavy atom effect (3) when sodium iodide solutions are used as solvents. The use of aqueous solvents and major improvements in sample preparation and handling should make phosphorimetry more attractive as a n analytical method, especially in the area of drug analysis. In this communication, we wish to present improvements in instrumentation and procedures which result in lower limits of detection and vastly improved precision. EXPERIMENTAL Instrumentation. An Aminco-Bowman spectrophotofluorometer with a n Aminco-Keirs phosphoroscope attachment, a 150-watt xenon arc lamp with elliptical condensing system, and a potted R C A 1P28 photomultiplier tube were used for all studies (American Instrument Co., Silver Spring, Md.) Basic instrumental components were arranged a s previously described (4). A Keithley Model 244 high voltage supply (Keithley Instruments, Cleveland, Ohio) was used to provide power t o the photomultiplier tube. A rotating sample cell apparatus ( 5 ) consisting of a Varian A60-A High Resolution Nuclear Magnetic Resonance Spectrometer Spinner Assembly (Varian Associates, Palo Alto, Calif.) was used with the quartz capillary sample tubes (Amersil Inc., Hillside, N.J.). Capillary tubes were made from T21 Suprasil quartz, having 5 mm 0.d. and 0.90 mm i.d. Glan Thompson polarizers previously used to reduce phosphorescence emission from the sample tube ( I ) were replaced by thin film quartz
Author to whom reprint requests should be sent (1) R. J. Lukasiewicz, P. Rozynes, L. B. Sanders, and J. D. Winefordner, ANAL.CHEM.,44, in press (1972). (2) R. J. Lukasiewicz, J. J. Mousa, and J. D. Winefordner, ibid.,
submitted. (3) S. P. McGlynn, T. Azumi, M. Kinoshita, “Molecular Spectroscopy of the Triplet State,” Prentice-Hall, Englewood Cliffs, N.J., 1969. (4) R. Zweidinger and J. D. Winefordner, ANAL.CHEM., 42, 639 (1970). (5) H. C. Hollifield and J. D. Winefordner, ibid., 40, 1759 (1968).
plate ultraviolet transmitting polarizers (Polacoat Inc., Cincinnati, Ohio). Reagents. Reagents used without further purification were : DL-tryptophan ; sulfapyradine ; 3-indole pyruvic acid; 2-thiouracil; (all from Nutritional Biochemical Corporation, Cleveland, Ohio); and sodium iodide (Fisher Scientific, Fair Lawn, N.J.). Deionized water was obtained directly from a commercial ion exchange column. Procedures. Phosphorescence spectra and relative intensity measurements for all molecules studied were obtained in 5 % WjW aqueous sodium iodide (2) using the quartz capillary sample tube suspended in liquid nitrogen in a quartz dewar flask. A thin film ultraviolet transmitting polarizer was placed in the excitation radiation beam and adjusted so as to minimize emission from the quartz capillary sample cell. The slit arrangement used for all spectra was 3, 4, 3, 3, 4, 3, (all in mm, corresponding t o 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 readout. Stock solutions of compounds studied were 10-3M in pure water. Analytical curves were prepared from successive dilutions of the stock solutions. All phosphorescence signals were taken as the average of three measurements. Introduction of sample into the capillary tube and other sample handling procedures have been previously described
(0.
Measurement of phosphorescence signals below the total background emission from the sample tube and the solvent was accomplished by using the blank subtract provision on the nanoammeter. At the limit of detection, the signal-tonoise ratio was n o smaller than 3 t o 1. A minimum of five replicate determinations was taken a t the limit of detection. RESULTS AND DISCUSSION
A summary of the analytical data obtained is given in Table I. In each case, the matrix formed upon freezing was completely snowed as previously described ( I , 2 ) . The use of aqueous sodium iodide as a solvent system has been demonstrated t o be analytically advantageous. Aqueous sodium iodide, however, may not be the optimum solvent system for all compounds. A few molecules show a small reduction in phosphorescence signal. This reduction can be due either t o formation of charge transfer complexes in the excited state (7) or a large reduction in the phosphorescence decay time, in which case signal losses due to phosphoroscope delay time (8) may be important. The instrumental system used in this study had several improvements over the instrumentation previously used ( I ) . These improvements were : the rotating sample cell (Varian N M R spinner), which resulted in greater precision and accuracy of measurement, was simpler to use and provided a more rapid procedure for changing samples; a better quality (6) T. Truong, R. Bersohn, P. Brumer, C. K. Luk, and T. Tao, J . Bioi. Chem., 242, 2970 (1967). (7) R. Sahai, R. H. Hofeldt, and S . H. Lin, Trum. Furuduy Soc., 67, 1690 (1971). (8) J. D. Winefordner, P. A. St. John, and W. J. ,McCarthy, “Fluorescence Assay in Biology and Medicine,” Volume I I , S. Udenfriend, Ed., Academic Press, New York, N.Y., 1969. ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, JUNE 1972
1339
Table I. Phosphorescence Characteristics of Molecules Studied Concentration Linear range of near Slope of correlation Limit of Limit of linearity linear portion coefficient detection, pg/rnlc detection pg/mld 104 0.964 0.9995 2 . 4 x 10-3 2.0 x 10-3 104 1.111 0.9771 1 . 3 x 10-3 1 . 0 x 10-4
Numbere of grams Compound at limit of detection m-Tryptophan" 4 . 7 x 10-11 Sulfapyradineb 2.5 x 10-11 3-Indole pyruvic acid* 104 0.893 0.9954 2 . 6 x 10-3 ... 5 . 1 x 10-11 a Solvent 5 % WjW sodium iodide:water, adjusted to pH 13 with sodium hydroxide (6). Solvent 5 % WjW sodium iodide:water. c Limits of detection in this study. Best previously reported limit of detection. e Volume of sample retained in capillary tube (calculated as 19.7 pl) for the present study. The sample used in previous studies was about 500 pl.
(Suprasil) quartz capillary tube, which gave a lower background signal; and a thin film polarizer which has a better transmittance in the ultraviolet than the Glan-Thompson previously used. A single polarizer in the excitation radiation beam, was used for two reasons: first, the absorption and luminescence spectra of the quartz capillary tube are polarized (1) and can be minimized by proper orientation of the polarizer; second, the absorption and luminescence of molecules in snowed frozen matrices are essentially depolarized by virtue of diffuse reflectance within the snowed matrix (4,and should be independent of polarizer orientation (the polarizer parameter which most affects the luminescence signal is the ultraviolet transmittance). The range of linearity and limits of detection reported in Table I, compare favorably with those previously obtained in clear rigid glasses. Because of the small volume of sample solution retained in the capillary tube, the total number of grams of solute necessary for a determination is in the picogram region. This should be of particular importance in biological and clinical applications where sample size is often limited. The average slopes of the analytical curves (logarithm of phosphorescence signal us. logarithm of analyte concentration) reported in this study and those reported in a previous study (1) are 0.94 which approaches the ideal slope of unity for phosphorescence signals in snowed matrices (4). Both positive and negative deviations from a slope of unity have been observed; however, much more data are necessary before a conclusion can be drawn as to whether an individual molecule perturbs the crystalline matrix sufficiently enough to alter the
slope or whether the deviations can be attributed to instrumental parameters influencing the precision and accuracy of signal measurements. Present theory assumes the scattering coefficient s, of the entire sample is not a function of solute molecule or its concentration ( 4 ) . A series of ten replicate determinations of 1 pg/ml of 2thiouracil in 10% VjV methanol-water were measured to determine the relative precision of using the rotating sample cell arrangement; the relative standard deviation obtained was 1.47%. This is an improvement of nearly an order of magnitude over the stationary quartz capillary tube system previously used (I); the physical dimensions of the previous capillary tube prevented use of the spinner assembly. The major noise contribution in our system arises from wobble of the capillary tube, caused by a poor fit of the turbine sleeve in the spinner assembly when the tube is spinning. If the capillary tube were held more rigidly in the turbine sleeve, the precision (1.47% in our case) would be reduced even more. The improved precision and limits of detection as well as the greatly facilitated sample handling procedure for phosphorimetric analysis in predominately aqueous solvents should offer additional advantages for use of phosphorimetry for routine analyses in biological and clinical applications.
RECEIVED for review November 12, 1971. Accepted December 21, 1971. Research was carried out as part of a study on the phosphorimetric analysis of drugs in blood and urine, supported by a U.S. Public Health Service Grant (GM-1137309).
Sandwich KBr Disk for Scanning Volatile Pesticides Paul A. Giang Entomology Research Division, Agricultural Research Service, US.Department of Agriculture, Beltsoille, Md. 20705
VOLATILE PESTICIDES invariably evaporate so rapidly that there is not time enough to scan the whole infrared spectrum from 4000 to 200 cm-l with an infrared spectrophotometer. In the laboratory at Beltsville, we have recently found that an excellent spectrum for such pesticides can be obtained by placing a sample of the pesticide in a freshly prepared KBr disk in the barrel of a Wilks Mini-Press (I) and covering this disk with (1) Wilks Scientific Corporation, Norwalk, Conn., Data Sheet No. 16, November 15, 1968. 1340
ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, JUNE 1972
mother KBr disk. This technique allowed us to scan some important volatile chemical pesticides. PROCEDURE
Place a bolt in the barrel of a Wilks Mini-Press (Catalog Number MP-5) and advance five full turns. Deposit 300 mg of KBr on the surface of the bolt inside the barrel, and tap the unit gently to spread the salt uniformly. Insert the second bolt, turn it until it is finger tight, and place the whole press in a bench vise. With a torque wrench, gradually exert 25-
