ANALYTICAL CHEMISTRY, VOL. 51, NO. 12, OCTOBER 1979
in argon which proved unsuccessful in preventing the formation of refractory compounds on the graphite rod.
CONCLUSION The use of halocarbon/argon gas mixture to volatilize refractory compound-forming elements from a graphite rod employed for sample introduction into an ICP has been successfully demonstrated. The halocarbon is decomposed during the heating to vaporization temperature to produce active halide atoms which halogenate the element of interest. This is subsequently vaporized into the plasma as its halide. Zirconium boron, chromium, molybdenum, and tungsten have been successfully vaporized from the graphite rod using an injector gas mixture of 0.1% trifluoromethane in argon and, with the exception of tungsten, rectilinear calibration curves over several orders of magnitude with respect to concentration of the element of interest have been obtained. Good detection limits are observed at the subnanogram level and the precision (RSD 0.06) previously reported ( 1 ) is maintained. The use of 0.1 % trifluoromethane in argon as the chamber/injector gas does not adversely affect elements which do not form refractory compounds and it is therefore proposed that if
1941
multielement investigations were to be carried out, using the vaporization technique, for a number of elements which included those which normally form refractory compounds, an argon gas mixture of the type suggested in this study should be employed to ensure their volatilization.
LITERATURE CITED Gunn, A. M.; Kirkbright, G. F.; Millard, D. L. Analyst(London) 1979, 103, 1066. Renshaw, G. D.; Pounds. C. F.; Pearson, E. F. At. Ahsorpt. News/. 1973, 12, 1955. Dagnall, R. M. I1 CS Cont. Flame Spectry. Zvikov, 1973. Ediger, R. D.; Peterson, G. E.; Kerber, J. D. At. Absorpt. News/. 1974, 13, 61. Alder, J. F.; da Cuhna, M. T. C. In press. Sambueva, A. S.; Shipitsyn, S. A. Zavodsk. Lab. 1965, 31, 1087. Cotton, F. A.; Wilkinson, G. W. "Advanced Inorganic Chemistry, A Comprehensive Text", 2nd ed.;Interscience: New York, London, Sydney, 1966; p 305.
RECEIVED for review April 17,1979. Accepted June 29,1979. We acknowledge the financial support of R.D.S. by the Atomic Weapons Research Establishment, Aldermaston, Berkshire, England.
Fluorescence Quantum Yield Determination by Pulsed Source Single Photon Counting Linda M. Upton and L. J. Cline Love* Department of Chemistry, Seton Hall University, South Orange, New Jersey 07079
A novel method of measuring fluorescence quantum yields by using time correlated single photon counting instrumentation was developed. The principles of single photon counting by using pulsed excitation light sources for quantitative measurements of absorbance and fluorescence intensity are presented. The single photon count rate was found to be proportional to the light flux up to a frequency of about 10% of the pulsed-light-source, 25-kHz repetition rate. Experiment efficiency rates greater than 10 YO produced multiple photon detection and pulse pileup errors. The quantum yields agreed within f0.05 with previously published values and those made by the conventional integration method. The expression for the minimum relative error in the quantum yield due to counting statistics is presented. The actual precision was fO.OO1 and was determined by drift in the lamp intensity.
The fluorescent quantum yield, @f, of a molecule is defined as the fraction of the number of quanta of light absorbed which are emitted by the excited state as fluorescence. Its measurement is a useful determination because it allows a measure of the extent of occurrence of the processes which compete with fluorescence such as internal conversion and intersystem crossing ( I , 2 ) . In addition, if the fluorescent lifetime of the excited state can also be determined, the quantum yield allows a calculation of the natural radiative lifetime of the fluorescent species, r0. This is an intrinsic property of the unperturbed excited singlet state of a molecule ( I ) . Several techniques have been utilized for the measurement of both relative and absolute quantum yields and 0003-2700/79/0351-1941$01 OO/O
these have been reviewed by Demas and Crosby (3). The most widely used technique is that of integrating corrected emission spectra for a measure of the number of quanta fluoresced, with the number absorbed represented by an absorbance value measured on a conventional absorbance instrument. There are several problems associated with most quantum yield measurements (3). A refractive index correction is necessary to take into account solvent properties and differences between standards and unknowns. If the excitation and emission spectra of a compound overlap, there may occur reabsorption of the emitted radiation and, consequently, a low value of calculated quantum yield. Collisional quenching and polarization effects may decrease the actual emitted radiation also. Inner filter effects and detector wavelength response must be accounted for. If a different excitation source is used for the absorbance and fluorescence measurements, the spectral band width can cause errors in the quantum yield values. Much of the work done in the area of' quantum yield measurements has been in trying to solve these problems (4-6). A technique for determining fluorescent quantum yields without directly measuring the sample absorbance has recently been reported (7). This method has the advantages of using the same spectral band-pass for both determinations and greatly reduces the error in absorbance measurements of optically dilute solutions. I t does not correct for overlap in emission and absorption spectra. This laboratory has been involved in the measurement of fluorescent lifetimes, T , by the time-correlated single photon (TCSP) technique. In an effort to calculate natural lifetimes, T,,, where T~ = T / @ ~ ,a method for the determination of fluorescent quantum yields was deemed necessary. A novel 1979 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 12, OCTOBER 1979
technique was developed which utilizes the TCSP instrumentation with a pulsed light source operating at 25 kHz. The method makes use of single photon counting for both the absorbance and the fluorescence intensity measurements and uses the same excitation source and the same detector for each. This way of determining 9ftherefore has the advantage of eliminating band-width differences between the fluorescence and absorbance measurements and, in addition, has the convenience of being able to measure both af and T on the same instrument. Ware (8) and Yguerabide (9) have previously suggested the use of the photon counts produced by the TCSP method for comparing fluorescence intensities, but its use for the determination of relative fluorescent quantum yields has never been reported. The present work described here is the first use of single photon counting (maximum of one photon detected per excitation) using a pulsed light source to measure sample absorbance and fluorescence intensity. The quantum yield is determined according to @f
=
no. of photons fluoresced/no. of photons absorbed (1) by using TCSP instrumentation. Data showing the precision and accuracy of the technique is also presented.
MEASUREMENT PRINCIPLES Photon counting detection of fluorescence and absorbance has been demonstrated (10-12) and has been shown to possess S/N ratio advantages in low light level situations. Its use here for the determination of quantum yields requires different considerations for proper operation. T h e single photon counting mode utilized in these experiments places different constraints on the measurement process due to the fact that a high repetition rate discharge lamp is used for excitation. The use of a narrow pulse width (-2 ns FWHH) excitation light source instead of the conventional continuum light source produces a high flux density of photons (- 107-109 photons per pulse) impinging on the sample. In response to a &type function excitation flash, the sample absorbs light and then begins to decay by various nonradiative or radiative means. Thus the 2-11s excitation pulse is accompanied by multiple radiative decays occurring typically from tenths to hundreds of nanoseconds. The equivalent anode current resulting from one excitation light pulse can be estimated. Suppose a photon flux of lo9 photons per 2 ns pulse excites a sample with a percent transmittance of 99% and a quantum yield of 10%. The subsequent fluorescence is detected by a photomultiplier whose quantum efficiency is 1% . Because of the isotropic nature of the flash lamp and fluorescence light, only a small fraction is collected of each, for example, 1% for the purpose of this estimate, due to solid angle limitations. The calculation is shown in Equation 2.
-
109/(2 X
X
0.01 X 0.1 T @f
X
0.01 e/photon
X
@PMT
(0.01)2=5 x
108
e / s (2)
R/4x This will produce the equivalent flow of - 5 X lo8 electrons per s a t the photocathode. If the P M T has a gain of lo7, typical of the high gain RCA 8850 P M T used in these experiments, the equivalent anode current of one excitation pulse is approximated by Equation 3.
5X
los e / s
X
lo7 X 1.6 X
C/e = 8 X
A
(3)
This is a relatively high peak current and care must be taken to avoid limiting the electronic signal handling equipment. The equivalent count rate produced by the experiment would be -5 X lo8 or 500 MHz. The upper frequency response limit typical of the high gain RCA 8850 can be cal-
culated. These low transit time tubes, typically 1-2 ns, would respond up to 500 MHz to 1 GHz photon count frequencies. This relatively high frequency response is not adequate to accurately follow a 500-MHz experimental photon count rate because the random ejection nature of light produces short-term rates in excess of the average rates. Thus, the frequency response of the photon counting system used for high-intensity, narrow, pulse-light measurements can be limited by any or all parts of the measurement system including the photomultiplier, the amplifiers, and the counter. This high emissive flux density of the fluorescence and percent transmittance would require sophisticated detection devices to correct for pulse pileup (10, 13: 14). I t has been pointed out that the instrumental frequency response should be around 25 times larger than the maximum average pulse rate to keep pile-up error below 1% (10). Fortunately, it is not necessary to detect each photon fluoresced or transmitted to obtain accurate results. Attenuation of the excitation light intensity and/or the light observed by the photomultiplier tube (PMT) allows operation in the single photon counting mode used for fluorescence lifetime measurements. In this mode the beam of photons is diminished so that the probability of detecting more than one photon per excitation pulse is small. The Poisson probability polygons (15)may be used to predict the frequency of occurrence of multiple photon events and the appropriate experimental conditions for minimum pulse pile-up error. This probability may be calculated from Poisson’s equation ,n
P, =
IIL
-
emn!
(4)
where P, is the probability of detecting n photons per excitation pulse, and m is the average number of photons detected per excitation pulse. Normally for these quantum yield measurements an average count rate of 0.01-0.05 photons per excitation pulse is used. Operation in the single photon counting mode is necessary for a linear relationship between photon counts and light flux. This proportional relationship is imperative for accurate percent transmittance and fluorescence intensity values. Figure 1 illustrates the relationship between equivalent average anode photocurrent expressed in millivolts and single photon counts for varying light flux striking the detector. The light was varied by using an adjustable aperature and the analogue current measured simultaneously with the number of photon counts. As can be seen from the plot, the average anode photocurrent is proportional to the light flux at low light levels. Deviations from linearity begin to occur around 2400 counts/s (-3-4 nA), corresponding to a photon collection rate of around 0.10 photon per excitation pulse. This is a 10% experiment efficiency for the flash lamp operating at 25 kHz; Le., only one excitation flash in ten produces an observed fluorescence photon. The deviation from linearity occurs in the nanoampere range of photocurrents shown in Figure 1,in contrast to initial deviations in the microampere range observed by other workers (10). The apparent discrepancy can be understood by considering the duty cycles of the pulsed vs. continuous light excitation. If the excitation pulse is completed in 10 ns and has a repetition rate of 25 kHz, the sample is actually being illuminated only 250 ps per s. Thus, the average current is considerably lower compared with the peak current. However, the peak current determines the onset of pulse pileup and deviations from linearity of photon counts.
EXPERIMENTAL Instrumentation. The instrumentation of the TCSP technique is well documented and will not be described here (16, 17). The options chosen to be used in this laboratory are an excitation
ANALYTICAL CHEMISTRY, VOL. 51, NO. 12, OCTOBER 1979
n
Source
1943
Monochromaloi
El Flgure 3. Sample compartment arrangement for fluorescence intensity measurements 20
40
60
8
Table I. Components Used for Quantum Yield Measurement Ortec Model 9352 nanosecond lamp excitation monochromator Jobin Yvon Model flashlamp
I4 20
sample compartment emission filters photomultiplier electronic modules Source
Monochromalot
single photon counter analogue current amplifier Flgure 2. Sample compartment arrangement for absorbance measurements
monochromator, emission filters, and a counter to monitor the number of photons being detected per second by the photomultiplier tube. It is these photons which are used to provide the number of photons absorbed and the number of photons fluoresced. The number of photons absorbed was measured with the sample compartment set up as shown in Figure 2. The filter used must transmit the excitation wavelength and absorb the emission spectrum. An aluminum plate was used to reflect the transmitted light out to the photomultiplier tube a t right angles to the excitation beam path. The number of photons fluoresced was measured with the sample compartment set up as shown in Figure 3. The filter used must transmit all of the emission spectrum of wavelengths equally for both the reference and the sample. It must also absorb the excitation wavelength used. Commercially available cutoff filters satisfy these requirements. For both of these measurements, a quantum counter solution of 8 g/L rhodamine B in ethylene glycol was used to correct for the errors due to the wavelength-dependent response of the photomultiplier tube. This solution has a fluorescence quantum yield and spectrum independent of excitation wavelength and emits in the 550-650-nm wavelength range. A list of components used and their manufacturers is given in Table I. Reagents. All compounds were obtained from Fisher Scientific or Aldrich Chemical Co. and used as received. Solvents were
v
See Figures 2 and 3 Corning 0-52, 3-72 and Melles Griot U-G-1 RCA 8850 Ortec Model 9352 signal pickoff Ortec Model 436 discriminator Ortec Model 454 timing amplifier Ortec Model 463 constant fraction discriminator Ortec Model 425 delay Heath Model EU 801 counter Keithley Model 427
obtained from Fisher Scientific in spectrograde quality and used as received. No degassing was performed. Procedure. Samples of M concentration or less were prepared. The absorbance of the solution at the excitation wavelength to be used was checked on a Beckman Acta I11 or Spectronic 20 and kept below 0.05 absorbance unit to minimize self-absorption, quenching, and inner filter effects. A solution of M quinine bisulfate in 1 N sulfuric acid was used as the reference compound with a af of 0.546 (6). The number of photons transmitted by 1 N H2S04a t 337 nm was then determined, followed by a determination of the number of photons transmitted by the reference solution of quinine bisulfate in the sample compartment configuration shown in Figure 2. The blank was then repeated. The number of photons transmitted by the sample solvent (blank) was then measured at the excitation wavelength to be used for the sample. This was then followed by the same measurement on the sample solution. Again the blank was repeated. After all samples were done at their respective excitation wavelengths, the reference was repeated. Then, the number of photons absorbed is proportional to the number transmitted by the blank minus the number transmitted by the sample divided by the number transmitted by the blank or fraction absorbed = (no. photons transmitted)b - (no. transmitted), (5) (no. tranSmitted)b The fluorescence set up shown in Figure 3 was then used to measure the number of photons emitted by the reference and
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 12, OCTOBER 1979
Table 11. Comparison of Measured Literature Values
@f
counts for the reference and unknown vary because the solvents are often different, their counts are within an order of magnitude. Because the number of photons absorbed by the reference and sample are kept approximately the same, their errors tend to cancel and the primary uncertainty in the value of +f should be the error in the number of photons fluoresced. The relative error in +f is then given by
and
@f
compound anthracene (ethanol) acridine (ethanol) acridine (0.1 N H,SO,) phenanthrene (ethanol) atabrine (0.1 N HC1) barbital (1N NaOH) atabrine homologue (0.1 N HCl)
single photon count- @ f corr spectrab in$
f
lit.c
0.260
0.27 t 0.05
0.024
0.05
E,, = 0.499
0.5
0.072
0.10 0.03
0.056
0.060
0.002
0.002
0.015
0.020
+0.001based on the uncertainty in numbers of Calculated from integration by photons fluoresced. Uncertainty expressed if reported manual planimeter. in references 18 and 1 9 .
sample solutions when they were excited by their respective excitation wavelengths. Again, they were done in the orderblank, sample, blank. The same emission filter must be used for all samples and the reference in order that they may be compared. Five count readings of 10 s each were taken and averaged. The following calculation was used to give af:
af = [no. photons fluoresced/no. photons absorbed],,k fluoresced/no. photons absorbed],,f
R,T
1-
X
where n is the refractive index of the solvent, unk indicates the sample, and ref indicates the reference quinine bisulfate. The refractive index correction has been found to be necessary by several authors (3, 4).
QUANTUM YIELD RESULTS The fluorescent quantum yields determined by this method are compared with some literature values in Table I1 (18,19). Some of these quantum yields were also determined by the conventional corrected spectra method by using a PerkinElmer MPF-3L spectrofluorometer. This instrument uses a quantum counter and programmed corrections. The fluorescence and absorption spectra (measured on a Beckman Acta 111)were integrated by using a manual planimeter. These results are presented in Table I1 also. The agreement is good within the range of f0.05 found in most interlaboratory comparisons. The ultimate precision in quantum yield measurements by single photon counting is determined by the random fluctuating noise impressed on the signals. The theoretical limit of detection, accuracy, and precision of systems using photomultiplier detection are usually set by the shot noise (20, 21). The expression for this precision can be derived through the standard equations for counting statistics of a difference. The relative error in the quantum yield, Eaf,is given by
(7) where Ef refers to the relative errors in the fluorescence and E A refers to the relative errors in the absorbance of the unknown and reference. As mentioned earlier, experimental conditions were arranged to keep absorbance values
