68
Anal. Chem. 1983, 55,68-73
Standards for Nanosecond Fluorescence Decay Time Measurements Roger A. Lampert, Leslie A. Chewter, and David Phillips” Davy Faraday Research Laboratory, The Royal Institution, 27 Albemarle Street, London WlX 46.54 United Klngdom
Desmond V. O’Connor Institute for Molecular Sciences, Myodagi, Okazaki 444, Japan
Anthony J. Roberts Unilever Research Laboratories, Port Sunlight, Merseyside, United Klngdom
Stephen R. Meech Department of Chemistry, Wayne State Unlversity, Detroit, Michigan 48202
Wlth a synchronously pumped dye laser as excltation source for fluorescence decay tlme measurements with slngle-photon counting detectlon, a crltlcal reevaluation of literature fluorescence decay parameters for “standard”compounds Is made and new standards are proposed for lifetime measurements. These are 2,5-diphenyloxarole (PPO), 1-cyanonaphthalene, 1-methyllndole, 3-methylindole, 1,bdlmethylIndole, and N,N-dimethyl-1-naphthylamine,In cyclohexane, hexane, or ethanol solution, covering an emlsslon wavelength range of 330 to 440 nm and decay time range of 1.28 to 18.23 ns. Anthracene In solutlon may also be used as a standard If care Is taken with purification and the concentration is known. Qulnlne bisulfate should not be used as a decay time standard. 1-Cyanonaphthalene provides a convenient standard for gas-phase experiments.
Fluorescence decay times represent an additional, relatively easily measured parameter with which to characterize molecular fluorescence. Perhaps the most widely used source of single-exponential lifetime data is Birk’s “Photophysics of Aromatic Molecules” ( I ) ,published in 1970. Without wishing to detract from the reputation of this monumental work, we believe that many of the lifetimes listed therein, although undoubtedly the most accurate available a t the time of publication, have since been shown to be seriously in error. The fact is that relatively crude techniques for lifetime measurements were still in operation a t the time the book was written and the author’s declared intention was merely to classify the reported data. Similarly many workers standardize the performance of SPC equipment against lifetime data given in Berlman’s “Handbook of Fluorescence Spectra of Aromatic Molecules” (2). Since these lifetimes were measured many years ago with a relatively old fashioned pulse sampling oscilloscope technique, it is not surprising that many of them are inaccurate. Any compound with a single exponential decay will serve as a lifetime standard. However, for the sake of convenience the compound should also be easily purifiable and have a known single exponential decay time, independent of excitation and emission wavelength, in an easily purifiable solvent. Among the most commonly used standards are p-bis(2phenylozazolyl)benzene (POPOP), 2,5-diphenyloxazole (PPO), anthracene, and quinine bisulfate. Quoted lifetimes for POP O P in cyclohexane do not show much variation ( 3 , 4 ) but
for the other three compounds the literature values for the decay times contain some serious discrepancies, as shown in Table I (5-27). Thus quinine sulfate in 1 N or 0.1 N H 2 S 0 4 is commonly regarded as having a single exponential decay time of about 19 ns. Recently we have shown that it has, in fact, a double exponential decay t h a t is strongly dependent on temperature and emission wavelength (28). We believe there is a case for a critical new evaluation of decay time data, and in this report we describe a single-photon counting instrument of high sensitivity and time resolution, list a number of parameters, some of them very seldom applied t o this technique, by which least-squares fitting procedures can be judged, and finally propose a set of standard compounds with single-exponential decay times which can serve as calibrants for standard nanosecond measurements. While the decay times of many compounds show only very slight, if any, dependence on excitation and emission wavelength and are relatively insensitive to change in temperature, we believe t h a t the precise conditions under which a decay time is measured should always be specified, as is now common for quantum yield determinations. Moreover it is advisable always t o remove oxygen from organic solvents since the possibility of reversible charge transfer complex formation in the excited state cannot be ruled out.
EXPERIMENTAL SECTION The Instrument. A diagram of the time-resolved fluorescence spectrometer employed for the measurements reported in this paper is shown in Figure 1. Sample excitation in the wavelength range 290-315 nm was achieved with a frequency doubled, cavity dumped, mode-locked synchronously pumped dye laser system. In this a 15-W argon-ion laser (Spectra-Physics Model 171) was mode locked using an acoustooptic device (Spectra-Physics Model 342) providing pulses of around 100 ps fwhm (full width at half maximum intensity), at a repetition rate of 82 MHz, and with an average power of 420 mW. These pulses were used to excite Rhodamine 6G dye in ethylene glycol in a jet stream dye laser (Spectra-Physics Model 375) the normal end mirror of which had been removed and the cavity length extended to match that of the ion laser. Under these conditions the laser was found to mode lock, producing pulses at a repetition rate of 82 MHz. For most photophysical systems, this pulse rate was too fast to allow total system relaxation between excitation events and, consequently, an acoustooptic cavity dumper (Spectra-Physics Model 344) was used to output pulses at a lower rate (single shot-4 MHz). A limited tunability, 570-640 nm, was available by using a multilayer broad band tuning wedge (Spectra-Physics Model 570) incorporated in the dye laser cavity. For wavelengths outside this region
0003-2700/83/0355-0068$01.50/00 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VQL. 55, NO. 1, JANUARY 1983
69
I
Table I. Some Single Euponential Decay Times Measured Since 1970 A , nm
--
compound quinine sulfate
anthracene
concn, M
solvent 0.1 N H,SO, 0.1 N H,S04 0.1 N H,SO, 0.1 N H,S04 3. N H,SO, 1 N H,SO, cyclohexane (degassed)
10-4
10-3 10-4
4 x 10-5 2 x 10-5 2.7 x 10-5 2 x 10-5 2 x 10-5 9.52 X
9-cyanoanthracene
POPOP
PPO
rose bengal
rhodamine B
1-cyanonaphthalene
excitation emission
cyclohexane (undegassed) cyclohexane (undegassed) cyclohexane (undegassed) ethanol (degassed) ethanol (degassed) ethanol (degassed) benzene (degassed) benzene (degassed) cyclohexane (degassed) cyclohexane (degassed) cyclohexane (undegassed) cyclohexane (degassed) cyclohexane cyclohexane (degassed) methanol (undegassed) methanol (undegassed) ethanol (undegassed) ethanol (undegassed) hiexane (degassed) hlexane (degassed)
a Single exponential lifetime not found, moments.
5x
2 x 10-5
5 6 7 8 9 10
6.80 f 0.07 4.79 f 0.05 6.01 f 0.06 5.20 5.2 5.28 5.03 f 0.07 5.22 f 0.04 5.16 5.14 5.42 f 0.04 5.15 i 0.05 3.97
least squares
18
least squares
4
least squares
13
least squares
4 9
19.4 20 19.4 18.8 19.3
365 257 365 365 36 5 340 3 55 340 340 340 365 365
>400 440 >400 400
308
410
room
4.1
355
415
room
3.99
365
405'
25
5.0
355
415
room
5.06
257
400
room
5.67
Fourier
25
3.6
least squares
room
4.00
>405' 415 405' 405' 405+ >400 >400
ref
moments oscilloscope moments log plot fourier transform least squares least squares least squares least squares least squares least squares least squares least squares least squares least squares least squares least squares least squares least squares
room room room room room room 20 room 20 20 25 room room 25 room room 20 20 room
4 70
a
f
i
f
0.03
0.05
0.05
moments
11
12 11 11 11
3 4 14 15 16 11 11
17
19 7
257
440
room
1 2 . 8 f 0.1
least squares
12
8 55
415
room
12.8 f 0.2
least squares
4
340
405+
room
1.13
least squares
3
3 55
415
room
1.10 f 0.02
least squares
4
1.27 1.36
20
370
room room
least squares
310
C
21
room
0.54
least squares
22
room
0.60
least squares
23
moments
24
moments
25
56 8
10-
5 80
room
2.85
10-6
680
room
2.88
2 x 10-5
method of data analysis
257
8X
10-5
rF, ns
T , "C
280
2 x 10-5
325
0.06
25
18.26
least squares
26
25
19.8
least squares
27
Not specified whether solvent is degassed.
alternative laser dyes could be employed. With careful alignment average powers in excess of 50 mW could be obtained with a pulse repetition rate of 4 MHz. The pulses were found to have an autocorrelation width of ca. 6 ps measured using a scanning autocorrelator (Spectra-Physics Model 409). Pulses were also monitored by using fast photodiodes (Hewlett-Packard 4220 in a home-built mount, Spectra-Physics Model 403B). Frequency doubling into the 290-315-nm range was achieved by using temperature and angle tuned ADP crystals (J. Id. Lasers) mounted a t a beam waist formed between two 10-cm focal length lenses. The relatively low pulse peek intensities resulted in a small second harmonic generation efficiency and consequently residual un-
f
Not specified but probably
doubled light was removed with a Corning 7-54 filter. For stability the laser system was mounted on a mechanically isolated optical table (Newport Research Corp.) and operated in a temperature-controlled environment (20 & 1 "C). The sample was contained in a l-cm2 quartz cuvette. Fluorescence was monitored at right angles to the excitation path through a Hilger-Watts 0.33-m D330 monochromator by a fast photomultiplier (Philips XP202OQ) mounted in a homemade voltage divider. The fluorescence resolution was typically 1-2 nm. In order to totally exclude scattered excitation light, it was found necessary to incorporate a Schott WG345 cutoff filter before the monochromator.
ANALYTICAL CHEMISTRY, VOL. 55, NO. 1, JANUARY
70
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1983
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