ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
immunoassay if one couples them to a suitable carrier (24). Thus, our method can be applied to environmental analysis. In clinical analysis, one commonly obtains samples taken from human serum. Serum contains a number of components which exhibit broadband fluorescence on visible excitation. When we injected a serum sample onto the HPLC column, the chromatogram contained strong peaks which overlapped the regions of both labeled insulin and labeled complex. There were no peaks, however, which corresponded to substances of molecular size greater than that of the labeled complex. Thus, a t the present time, fluorescence from interfering components in serum prevents clinical applications of our technique. If one could increase the molecular size of the labeled complex by linking the antibody to a nonfluorescent inert polymer, such as another antibody and separate on a column of larger pore diameter, one could access a region of the chromatogram which is free from fluorescence interference. Other possible routes toward circumventing interference problems include temporal discrimination, enzyme-linked immunoassay, and sandwich immunoassay. These approaches are currently under investigation. ACKNOWLEDGMENT We thank F. D. Howard, Department of Chemistry, Stanford University, for his helpful advice and assistance in carrying out immunochemical reactions. We are also grateful to T. Jupille, Bio-Rad Laboratories, for his counsel on chromatographic methods; G. J. Diebold, Brown University, for discussions on laser fluorimetry; and H. M. McConnell, Department of Chemistry, Stanford University, for use of his laboratory facilities. LITERATURE C I T E D (1) Yalow, R. S. Science 1978, 200, 1236-45. (2) O'Donnell, M. C.; Suffin, S. C. Anal. Chem. 1979, 5 1 , 33A-40A.
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Bradley, A. B.; Zare, R. N. J . Am. Chem. SOC.1976, 98, 620-21. Imasaka, T.; Kadone, H.; Ogawa, T.; Ishibashi, N. Anal. Chem. 177, 49, 667-8. Smith, B. W.; Plankey, F. W.; Omenetto, N.;Hart, L. P.; Winefordner, J. D. Spectrochim. Acta, Part A 1974, 30, 1459-63. Richardson, J. H.; Wallin, B. W.; Johnson, D. C.; Hrubesh. L. W. Anal. Chim. Acta 1976, 86,263-7. Ullman, E. F.; Schwarzberg, M.; Rubenstein, K. E. J . Bid. Cbem. 1876, 251, 4172-8. Smith, D. S. FEBS Len. 1977, 77, 25-7. Spencer, R. D.; Toledo, F. B.; Williams, B. T.; Yoss, N. L. Oh.Chem. Winston-Salem. N.C.) 1973. 79. 838-44. Watson. R A A , Landon, J , Shaw, E J , Smith, D S Clin Chim Acta 1976, 73 51-5 Goklman, M., "Fluorescence Antibody Methods", Academic Press: New York, 1968; pp 97-99. Blakeslee, D.; Baines, M. J . Immunol. Methods 1978, 13, 305-320. Goldman, M. "Fluorescence Antibody Methods", Academic Press: New York, 1968; pp 129-30. Diebold, G. J.; Zare, R. N. Science 1977, 196, 1439-41. Hudson, L.; Hay, F. C. "Practical Immunology". Blackwell Scientific Publications: Oxford, 1976; p 12. We thank E. F. Ullman, Syva Research Institute, for suggesting this explanation. Kronick, M. N.; Little, W. A. J . Immunol. Methods 1975, 8 , 235-40. Skelley, D. S.; Brown, L. P.; Besch, P. K. Clin. Chem ( Winston-Salem, N.C.) 1973, 79, 146-86. Clayton Hopkins, J. A.; Edwards, L.; Herner, A. E.; Van Dreal, P. Clin. Chem. ( Winston-Salem, N.C.), 1977, 23, 403-44. Haugland, R. P. Molecular Probes, Inc:., Plano, Texas, private communication. Kato, K.; Hamaguchi, Y.; Fukui, H.; Ishik.awa, E. J . Biochem. 1975 78, 235-7. Ishikawa E. J . Biochem. 1973, 73, 1319-21. Schuurman, H. J.; de Ligny, C. L. Anal. Chem. 1979, 57, 2-7. Lukens, H. R.; Williams, C. B.;Levison, S. A,; Dandliker, W. B.; Murayama, D.; Baron, R. L. Environ. Sci. Techno/. 1977, 1 1 , 292-7.
RECEIVED for review March 5 , 1979. Accepted June 4, 1979. One of us, S.D.L., acknowledges support from his NIH predoctoral fellowship, Medical Scientist Training Program, Columbia University, College of Physicians and Surgeons. The support of the National Cancer Institute under Grant NIH 2R01 CA23156-02 is gratefully acknowledged.
Determination of Sulfur by Vacuum Ultraviolet Atomic Emission Spectrometry with Hydrogen Sulfide Evolution Paul D. Swaim" and Steve R. Ellebracht Central Laboratory, Dow Chemical U.S.A. Freepori, Texas 7754 1
A method for the determination of sulfur has been developed by which sulfur is reduced to volatile hydrogen sulfide gas and then swept into a dc plasma where it is detected by vacuum ultraviolet atomic emission spectrometry at 180.7 nm. The method makes possible the determination of trace levels of sulfur in samples where the matrix would normally result in spectral and/or chemical interferences. The practical detection limit is 10 ng of sulfur and the linear working range is at least four orders of magnitude. Analysis time is rapld, with less than 2 min being typically required per sample. Analytical procedures are extremely simple, making the technlque ideal for routine applications. Factors which govern the rate of hydrogen sulfide evolution were investigated, as were the effects of possible interferences.
Quick and reliable methods for the determination of trace levels of sulfur in a wide variety of sample matrices have long been pursued by the analytical chemist. As with many nonmetallic elements, sulfur has not proved easily amenable 0003-2700/79/035 1-1605$01.OO/O
to detection of microgram and sub-microgram quantities. This has been further complicated by the fact that techniques for separating and/or concentrating sulfur a t these levels are sometimes cumbersome. The result is that, a t best, methods for traces of sulfur usually are tedious and require considerable operator time and skill. We recently were in need of a method which could be used routinely to determine sulfur in salt samples in the range of 0.5 to 100 ppm. These samples consisted mainly of sodium chloride, potassium chloride, calcium chloride, and magnesium chloride in roughly equal proportions. Sulfur was present primarily as inorganic sulfate although lesser amounts of other inorganic sulfur species may also have been present. Existing titrimetric or colorimetric methods (1-3) were found to be either insufficiently sensitive or excessively time consuming. Turbidimetric and polarographic methods ( 4 , 5) for the determination of sulfate not only required prior conversion of other forms of sulfur but were found t o suffer from lack of specificity and/or reproducibility. Instrumental techniques which have been used to determine sulfur include X-ray fluorescence (6, 7), neutron activation (8),and charged-particle 0 1979 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
Table I. Instrumental Parameters plasma current slit widths argon flow rates plasma nebulizer purge analytical wavelength
7A 25-pm entrance 25-pm exit
2 SCFH 4 SCFH 3 SCFH
180.7 nm
activation analysis (9),but these either are insensitive or would have been impractical. An instrumental technique which has shown great promise for the determination of trace levels of sulfur is vacuum ultraviolet atomic emission spectrometry (10). This technique has exhibited good sensitivity, a wide working range, and is well suited for routine laboratory use. However, it was discovered that direct aspiration of a solution of the salt sample (10% w/v) would not provide adequate sensitivity owing to spectral and/or chemical interferences from the high concentrations of sodium and potassium salts. Efforts to strip the solution of sodium and potassium using a cation-exchange resin failed, largely because of premature saturation of the resin. A technique used routinely in our laboratory for separating sulfur involves reduction with a hydriodic acid reducing solution and distillation of the resulting hydrogen sulfide. Gas evolution techniques have been very successful for the determination of arsenic, selenium, antimony, bismuth, germanium, tellurium, and tin by atomic absorption spectrophotometry (11-131, not only because of the separation they afford from interfering matrices, but also because of the gain in sensitivity resulting from the increase in sample introduction efficiency. By combining a hydrogen sulfide generating apparatus with the vacuum ultraviolet atomic emission spectrometer, we have developed a technique which can be used for the routine determination of trace levels of sulfur in samples for which previously existing methods were inadequate or impractical. EXPERIMENTAL Apparatus. The vacuum ultraviolet atomic emission spectrometer used was essentially the same as that described by Ellebracht et al. ( I O ) with only minor modifications. The purged region from the excitation source to the entrance slit of the monochromator was modified to allow this area to be purged by argon emanating from the monochromator at the entrance slit. Improvements were made in the design of the chamber surrounding the source to eliminate the need of a separate purge for this area. Thus, an inlet of argon to the monochromator provided an adequate purge for the entire system. These modifications resulted not only in a more efficient purge, but also in better plasma stability. The argon purge rate and other instrumental parameters are given in Table I. The hydrogen sulfide generating apparatus, shown in Figure 1, consisted of three sections joined by ground glass joints and held together by retaining springs. A six-turn spiral condenser was used to ensure complete condensation of hot hydriodic acid vapors. A septum support was connected to the 250-mL boiling flask by means of a ‘/,-inch 0.d. glass stem. The stem was not attached perpendicularly to the flask wall, but rather angled toward one side, so that an injected sample would strike the solution rather than the argon inlet tube. The septum support consisted of a ‘/,-inch Swagelok Teflon union and accompanying nuts. One end of the union was attached to the stem using two “0” rings placed between the nut and back ferrule. The other end of the union was ground flat to give a better surface on which the septum could rest. A Microsep F-138 septum (Alltech Associates, Arlington Heights, Ill.) was placed between the union and the nut. This septum is coated on one side with a thin film of Teflon; it was this side which faced the solution. Connections to the emission spectrometer were made by disconnecting the argon line from the nebulizer and reconnecting
1-
I
Figure 1. Hydrogen sulfide generating apparatus
it to the argon inlet of the apparatus. Tygon tubing, as short as possible, was used to connect the argon outlet of the apparatus to the nebulizer of the emission spectrometer. The entire apparatus was secured over an Electrothermal Rotamantle (Electrothermal Engineering Limited, London, England) by means of a ring stand and clamp. Reagents. Stock solutions which were 1000 pg/mL in sulfur were prepared by dissolving the appropriate amounts of the following reagent grade compounds in deionized water: potassium sulfate, sodium sulfite, ammonium persulfate, thiourea, ammonium thiocyanate, and sodium thiosulfate. The sodium sulfite solution was prepared by first thoroughly deaerating the water with nitrogen. Sodium tetrathionate stock solution was prepared by oxidizing a sodium thiosulfate solution with iodine. An aqueous hydrogen sulfide stock solution was prepared by passing hydrogen sulfide gas through deionized water which had previously been deaerated with nitrogen. The sulfide concentration was determined by titration with standard iodine solution. This solution was prepared and standardized immediately before use. Appropriate dilutions of these solutions were made as needed. All other reagents and compounds used were reagent grade quality. Procedure. Preparation of the Reducing Solution. Thirty grams of potassium iodide, 30 mL of hydriodic acid (55-58% by weight), 15 mL of hypophosphorous acid (50-5270 by weight), and a 1.5-inch stirring bar were added to the 250-mL flask of the hydrogen sulfide generating apparatus. After being stirred and heated to reflux under an argon purge for about 20 min, the solution was ready for use. Analysis of High Salt Samples f o r Sulfur. The analysis of high salt samples required no special sample preparation. Analyses were performed by injecting 100 pL of a 20% w/v solution of sample into the reducing solution and comparing the peak height to that obtained from 100 gL of a standard sulfur solution. If several samples were to be analyzed, a standard was run every third injection. RESULTS AND DISCUSSION P a r a m e t e r s Covering the Rate of Hydrogen S u l f i d e Evolution. Composition of the Reducing Solution. Initial trials of this technique were made using a variation of the
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
,
70
80
80
IO0
I10
1 120
T*mp*ratura,
700
800
0 00
1000
I100
I200
Stlrrlng R a t a ,
I
I
I
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'C RPY
Figure 2. Effect of temperature (A) and stirring rate (0)on the rate of hydrogen sulfide evolution
hydriodic acid reducing solution described by Luke (14). This solution consisted of hydriodic acid, hydrochloric acid, and hypophosphorous acid in a 3:2:1 v/v/v ratio. While this solution demonstrated the technique would be feasible, it was not suitable for our purposes because of the slow rate of hydrogen sulfide evolution. This was evidenced by rather ill-defined and very broad peaks, with as long as 10 min needed for 10 Fg of sulfur to be evolved. Better performance was achieved with a solution composed of 30 mL of hydriodic acid and 15 mL of hypophosphorous acid saturated with potassium iodide a t the boiling point of the mixture. The rate of evolution of hydrogen sulfide was increased severalfold in that 10 pg of sulfur were evolved in about 1 min. Moreover, the peaks were well-defined and very sharp in appearance. It is logical that an increase in the iodide concentration should increase the rate of evolution of hydrogen sulfide. Since iodide ion is the active reducing agent (15),an increase in its concentration should aid in driving the reaction toward completion. Even more important may be the fact that the addition of solid potassium iodide (and the removal of HC1) raised the boiling point of the mixture by about 15 "C. Increasing the temperature of the solution should both increase the rate of reduction of the sulfur species and decrease the solubility of H,S in the solution. Temperature, Stirring Rate, and Nebulizer Flow Rate. The effect of temperature on the evolution of hydrogen sulfide is illustrated in Figure 2. The greatest sensitivity in terms of peak height was achieved a t temperatures above 110 "C. Peaks obtained a t temperatures below about 90 "C were broad and ill-defined, indicating a slow rate of evolution. As can be seen from Figure 2, peak height (Le., rate of evolution) varies almost directly with stirring rate up to about 1100 rpm. In practice, stirring rates as high as possible were found to give the best sensitivity and most reproducible results. The rate of hydrogen sulfide evolution was found to be dependent upon the nebulizer flow rate because of the cooling effect the flowing argon stream exerted on the reducing solution: lower flow rates resulted in higher temperatures and thus a higher rate of evolution. However, flow rates much below 4 SCFH resulted in distortion and instability of the plasma, and therefore a decrease in the signal-to-noise ratio.
Table 11. Effect of Form of Sulfur o n Peak Height
a
sulfur species sulfate sulfite thiocyanate persulfate thiosulfate tetrathionate hydrogen sulfide, aqueous thiourea Expressed in millimeters.
peak height ,a
92 96 87 93 90 95 90 0
A nebulizer flow rate of 3.5 to 4 SCFH war found to be the best compromise. Effect of Forms of Sulfur. Since sulfur occurs in a wide variety of chemical forms (both organic and inorganic) and oxidation states, it was necessary to know how the form of sulfur would affect the rate of evolution of hydrogen sulfide. This was done by injecting consecutive 100-pL aliquots of solution which contained 10 pg/mL of sulfur in the following forms: sulfate, sulfite, persulfate, thiosulfate, tetrathionate, thiocyanate, thiourea and aqueous hydrogen sulfide. The data, given in Table 11, show that each form of inorganic sulfur resulted in the same peak height within the limits of long-term reproducibility of the technique. This suggests that the limiting factor for the rate of evolution is the rate a t which hydrogen sulfide can be stripped from the solution. I t is not due to a slow rate of reduction, since aqueous hydrogen sulfide yielded the same peak height as did the other inorganic forms of sulfur. This fact greatly simplified analytical procedures, since it was unnecessary to convert all the various inorganic forms of sulfur to a single state prior to analysis. The data also show that thiourea was not reduced. This is in agreement with Johnson and Nishita who reported that some organic sulfur compounds, such as cystine, cysteine, methionine, taurine, and glutathione do not yield hydrogen sulfide when boiled with a hydriodic acid reducing solution (16). Although other organic sulfur compounds were not tried, it is likely that most would be only partially reduced, if a t all. Linear Range, Limits of Detection, Accuracy, and Repeatability. A well-known virtue of atoniic emission spectrometry when using a high temperature plasma as the
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979 c
Table V. Repeatability sample analpeak ysis heighta 1 70 2
72
3 4 5 6 7 8 9
75 74
10
Figure 3. Typical recorder tracing of (a) 0.02, (b) 0.2, and (c) 0.5 pg sulfur
a
71
74 76 81 79 77
Expressed in millimeters.
standard peak heighta 138
143 148 147 145 150 148 154 153 152
concentration of sulfurb 50.7 50.3 50.7 50.3 49.0 49.3 51.4 52.6 51.6 50.7
- -
X = 50.6 s = 1.06 SI = i2.176 Parts per million.
Table VI. Effects of Possible Interferences solutions, b peak height'
Table 111. Accuracyaib sample no. 1 no. 2 no. 3 titrimetric 93.2 i 0.5 96.7 i 0.3 103.2 + 1.0 present method 95.3 i 0.4 97.3 i. 0.8 105.3 i 1.0 a Results given as mean of triplicate determinations. Results in Darts Der million sulfur. Table IV. Comparison of Techniquesasb X-ray sample fluorescence present method 1 0.12 * 0.01 2 0.22 * 0.01 3 0.36 f 0.01 4 0.32 * 0.01 5 0.40 t 0.01 a Results given as mean of duplicate Expressed as percent SO,'-.
0.13 ir 0.01 0.22 i 0.01 0.36 i 0.00 0.32 i 0.00 0.37 i 0.01 determinations,
excitation source is the wide linear working range for most elements. Peak height was found to be proportional to quantity of sulfur up to about 300 pg. Above about 400 pg, proportionality fails. Figure 3 illustrates the sensitivity of the technique. Based on the quantity of sulfur which would produce a signal equal to twice the standard deviation of the background noise, a detection limit of 0.01 pg of sulfur is obtained. Peak height is therefore proportional to quantity of sulfur over the range of 0.02 to 300 pg, or at least four orders of magnitude. Three salt samples were analyzed by the present method and the titrimetric method for inorganic sulfur proposed by Archer (1). (Although somewhat lengthy, this procedure has been used routinely in our laboratory for a number of years and found to be reliable for quantities of sulfur in the 100-300 pg range.) The results given in Table I11 show good agreement between the methods. Table IV lists the results obtained from the analyses of five saturated brine samples by the present method and by X-ray fluorescence spectrometry. I t can be seen that not only are the results in excellent agreement, but that the present method can also be used for the accurate determination of higher concentrations of sulfur. Repeatability was determined by performing ten consecutive determinations of a 20% w/v solution of salt sample known to contain a small amount of sulfur. Each determination was performed by injecting 100 pL of a 20 pg/mL standard solution, followed by 100 p L of the sample solution.
85 H,O HNO,, 7% 86 H,O,, 3% 84 Pb(NO,),, 2% 88 HgCl,, 2% 89 NaOH, 10% 71 NaOH, 2% 87 48 3" (aq), 2% Concentration of inerferent is on weightivolume basis. All solutions 20 Mg/ml sulfur in the form of sulfate. Expressed in millimeters. ~
The concentration of sulfur in the salt sample was calculated and, from these results, a relative standard deviation was derived. The results are given in Table V. A relative standard deviation of &2.1% shows that the technique has very good short term reproducibility. Further examination of the data reveals that over the period of time required to perform this series of analyses (about 45 min), sensitivity appeared to vary somewhat. This can readily be seen from the values of peak heights for the standards, which varied from a low of 138 mm to a high of 154 mm, representing a change of slightly over 10%. We have observed that the plasma excitation source used in this instrument is not extremely stable over a long period of time, mainly because of aging of the electrodes. However, while stability is not exceptional, changes which occur usually do so gradually. In practice, we have found that if standards are run every third sample, the results will be within 5% relative error of the true value. Interferences. Chemical interferences in the reducing solution would likely occur via two modes of action: (a) the species could react with sulfide and inhibit the evolution of hydrogen sulfide, or (b) the species coud react with the reducing solution and possibly hinder the reduction of sulfur. These possibilities were investigated by spiking a solution of a possible interfering species with a known amount of sulfur and comparing the peak height to that obtained from a standard sulfur solution. The results, given in Table VI, show that strong oxidants did not affect the evolution of hydrogen sulfide, even though reducing agent was consumed. Cations which form acid-insoluble sulfides also did not interfere. However, a high concentration of sodium hydroxide resulted in a reduction in peak height of about 16%. The interference was apparently not due to the consumption of acid, as subsequent injections of standard sulfur solution did not show a similar reduction in peak height. No reason can be advanced for this behavior. An even greater reduction of peak height
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
was observed with ammonia, but this was the result of a thick cloud of ammonium iodide which accompanied the hydrogen sulfide into the plasma. Therefore, the interference was optical in nature rather than chemical. Capacity of the Reducing Solution. The reducing power of the solution will be eventually weakened if repeated injections result in increases in volume and dilution of the mixture. We have observed, however, that significant increases of volume do not occur, probably because of gradual losses which result from incomplete condensation of water and hydriodic acid. Although we have never actually exhausted a solution, we found that as long as samples were nonbasic and relatively free of oxidants, several hundred injections of 100 pL each could be made over a period of about 3 weeks without any apparent loss of quality. Note. The instrumental technology described in this paper is the subject of pending patents and license agreements are being pursued by The Dow Chemical Company.
ACKNOWLEDGMENT The authors express their thanks to C. Vail and C. M. Fairless for their assistance.
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LITERATURE CITED Archer, E. E. Analyst(London), 1956, 8 1 , 181. B o k , P. F. "Colorimetric Determination of Non-Metals"; Interscience: New York, 1958. Sndl, F. D.; Snell, T. S. "colorimetric Methods of Analysis"; Van Nostrand: New York, 1949. Steinbergs, A.; Ilsmaa, 0.; Freneg, J. R.; Barrow, N. J. Anal. Chim. Acta 1962, 2 7 , 158. Mayer, J.; Hluchan, E.; Abel, A. Anal. Chem. 1967, 39, 1480. Bird, R. J.; Taff, R. W. J . Inst. Pet. 1970, 5 6 , 169. Yao, T . C.;Porsche, F. W. Anal. Chem. 1959, 31, 2010. Wayman. C . ti. Anal. Chem. 1964, 36. 665. Clark, P. J.; Neal G. F.; Allen, R. 0. Anal. Chem. 1975, 4 7 , 650. Ellebracht. S.R.; Fairless, C. M.; Manahan, S. E. Anal. Chem. 1978, 50, 1649. Pollock, E . N.; West, S. J. At. Absorpt. News/. 1973, 12, 6. Fernandez, F. J. At. Absorpt. Newsl. 1973, 12, 93. Vijan, P. N.;Chan, C. Y. Anal. Chem. 1976, 48, 1788. Luke, C. L. Ind. Eng. Chem. 1943, 15, 602. Luke, C. L. Anal. Chem. 1949, 21, 1369. Johnson, C. M.; Nishita, H. Anal. Chem. 1952, 24, 736.
RECEIVED for review January 10, 1979. Accepted June 18, 1979. The subject of this work was presented a t the 30th Pittsburg Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 5-9, 1979.
Effects of External Heavy Atoms and Other Factors on the Room-Temperature Phosphorescence and Fluorescence of Tryptophan and Tyrosine Martin L. Meyers and Paul G. Seybold"' Departments of Chemistry and Biological Chemistry, Wright State University, Dayton, Ohio 45435
Luminescence properties of tryptophan, tyrosine, their methyl esters, a tryptophan-tyrosine dipeptide and indole in aqueous solutions and adsorbed on filter paper at room temperature were examined. The room-temperature phosphorescence (RTP) of tryptophan on filter paper is enhanced 455-fold, that of its methyl ester 340-fold, and that of indole 370-fold by addition of 1.0 M sodium iodide. These enhancement factors are apparently the largest ever reported for any compounds. The dipeptide exhibits RTP from its tryptophan residue, and this is also strongly enhanced by added sodium Iodide. The RTPs of adsorbed tyrosine and its methyl ester were not detectable without heavy-atom enhancement, and remained weak even afler addition of sodium iodide. The methyl esters of tryptophan and tyrosine are considerably less fluorescent than are the parent amino acids. Implicatlons for the nonradiative decay mechanisms in the excited states are dlscussed.
T h e luminescence properties of tryptophan and tyrosine have long been of interest, both because these residues can act as intrinsic "fluorescence probes" of protein conformation (1,2) and because of the importance of aromatic amino acids and their metabolites in clinical analyses. Extensive exAuthor to whom correspondence should be addressed. Address during the academic year 1978-79: Institute of Theoretical Physics, University of Stockholm, Vanadisvigen 9, S-11346 Stockholm,Sweden.
aminations of the fluorescence characteristics of the aromatic amino acids and their peptides have been reported (3-9). The usefulness of phosphorescence in identifying tryptophan and other indole derivatives has been recognized for some time (IO,I I ) , and tyrosine phosphorescence has also proved important in some protein studies (12). Interest in the triplet states of these amino acids is further increased by the observations of triplet-singlet energy transfer in proteins from tryptophan donors (13) and of phosphorescence from tryptophan residues of enzymes in room-temperature solutions (14).
The relatively new technique of room-temperature phosphorescence (RTP) depends on the fact that a variety of substituted organic compounds is found to phosphoresce in air at room temperature when spotted and dried on certain surfaces, including filter paper (15-1 7). Tryptophan exhibits R T P (18,19),whereas tyrosine is reported not to exhibit R T P on filter paper (19). In the external heavy-atom effect, environmental atoms of high atomic numbers enhance molecular spin-forbidden transitions via a spin-orbital coupling mechanism (20). This effect has been employed to enhance phosphorescence intensity in both low-temperature (11,21, 22) and room-temperature (18) studies of tryptophan and related compounds. Interpretation of the luminescence variations of aromatic amino acids in solutions and in proteins depends on an understanding of the underlying mechanisms, and some of these mechanisms are still not well established. Differing explanations have been given, e.g., for the factors determining
0003-2700/79/0351-1609$01.00/0 0 1979 American Chemical Society
