Analyst, September, 1983, Vol. 108, $9. 1067-1071 1067

Relative Fluorescence Quantum Yields Using a Com puter-controlled Luminescence Spectrometer* Alun T. Rhys Williams and Stephen A. Winfield Perkin-Elmer Ltd., Beaconsfield, Buckinghamshire, H P 9 lQA

and James N. Miller Department of Chemistry, Loughborough University of Technology, Loughborough, Leicestershire, L E 1 1 3T U

Relative fluorescence quantum yields are determined using a computer- controlled luminescence spectrometer. The relative absorbances of the standards and unknowns are measured using the same instrument as for the fluorescence measurements. Relative quantum yields are presented for a wide range of compounds a t room temperature.

Keywords : Relative quantum yields determination ; computer-controlled luminescence spectrometer

The fraction of the number of quanta absorbed by a molecule that are emitted as fluorescence is termed the fluorescence quantum yield. Its determination provides information con- cerning radiationless processes in molecules and, for example, the determination of the potential of fluorophores in assays. Quantum yields are measured either on a relative basis with reference to a standard or by using an absolute method. Both methods have been reviewed by Demas and Crosbyl and by Bridges2

Absolute quantum yields were measured by Weber and Teale3 by using the dipolar scattering of monochromatic light from glycogen solutions as a standard unit of quantum yield. A comparison was then made with the fluorescence from a solution with the same apparent absorbance for the excitation light. Other methods for determining absolute quantum yields include photoacoustic spectroscopy4 and calorimetric rne th~ds .~

The most widely used method of determining quantum yields is by the relative method and the quantum yield of the unknown, Qx, is calculated according to the following equation:

. . EX I R & Q - Q - - _ - Ax * E R * I , ' ni X - R *

where QR is the quantum yield of the standard, A is the absorbance of the solution, E is the corrected emission intensity, I is the relative intensity of the exciting light and n is the average refractive index of the solution. Subscripts R and X refer to the reference and unknown compound, respectively. When a Rhodamine quantum-corrected reference system is used, the relative intensity of the excitation light a t different wavelengths is taken to be unity, which simplifies equation (1) to

The following factors can affect the measurement of relative quantum yields : polarisation; refractive index changes ; re-absorption of the emission ; internal reflection ; variation of optical density with band width ; and calibration errors between ultraviolet absorption and fluorescence spectrometers.

The use of an integrating sphere6 largely overcomes the errors associated with polarisation and refractive index changes. Working at very dilute absorbances or extrapolating to zero absorbance eliminates errors from re-absorption and internal reflection. The errors associ- ated with measuring the absorbance values are based upon the difference in characteristics between an ultraviolet - visible absorption spectrometer and a fluorescence spectrometer.

* Presented at the Pittsburgh Conference and Exposition on Analytical Chemistry and Applied Spectro- scopy, Atlantic City, N J, USA, March, 1983.

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1068 RHYS WILLIAMS et al. : RELATIVE QUANTUM YIELDS USING Analyst, Vol. 108 Unless two identical monochromators are used errors may arise from two sources. Firstly, the effect of spectral band width on the shape of the ultraviolet absorption spectrum, hence absorbance value ; the shape of the fluorescence excitation spectrum, hence relative intensity. For example, the ultraviolet absorption spectrum of pyrene in hexane for two different slit widths is shown in Fig. 1. This illustrates the change in absorbance values and peak ratios, which occur as the spectral band width is changed. Unless the fluorescence spectrometer has an identical monochromator the excitation spectra will also be different.'

0.8

0.6

Q

C 0 D

i Oa4 n a

0.2

n 520 260 300 340 380 220 260 300 340 380

Wavelengthlnm

Fig. 1. Ultraviolet absorption spectra of pyrene in hexane with (a) 2-nm band width and (b) 4-nm band width.

Secondly, wavelength accuracy is particularly important especially where sharp absorbance/excitation bands occur. For pyrene, a 0.5-nm error in the excitation wavelength at 241 nm will result in a 7% intensity error. For a l-nm error the error is 15%.

Several methods have been proposed to overcome these deficiencies, including time- correlated single-photon counting.* Britten et aL9 presented a method where the absorbance of a solution was determined by measuring the fluorescent intensities at two points along the absorbance path. Gains and DawsonlO used absorptivity-related constants derived from the apparent approximately hyperbolic relationship between fluorescence and concentration. In this paper a method is proposed that overcomes the errors associated with measuring absorbances by using the same instrument to measure fluorescence spectra and absorbance values. A mirror placed at the sample focus is used to reflect light into the emission mono- chromator. A 10-mm path length cuvette is placed in the reflected beam and by synchron- ously scanning the excitation and emission monochromators a t the same wavelengths, the instrument is turned effectively into a single-beam absorption spectrometer.

Quinine sulphate is still regarded as the best available, although it has a number of disadvantages such as an emission spectrum that is dependent upon the excitation wavelength and quantum yields, which vary on the type and normality of acid concentration. Velapoldi and Mielenz,ll in a National Bureau of Standards publication, suggest that quinine sulphate in 0.1 M perchloric acid be used in certification measurements. A quantum yield of 0.59 is suggested at an excitation of 347 nm. When measuring quantum yields in organic solvents, 9,lO-diphenylanthracene in cyclohexane has been proposed.12

The choice of primary quantum standard is particularly important.

This is comparable to a value of 0.546 in 1.0 M sulphuric acid.

In this solvent the quantum yield is reported as being unity.

Experimental All fluorescence spectra were measured on a Perkin-Elmer, Model LS-5, luminescence

spectrometer fitted with a red sensitive R928 photomultiplier. Data were recorded using a

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September, 1983 A COMPUTER-CONTROLLED LUMINESCENCE SPECTROMETER 1069 Perkin-Elmer, Model 3600, data station with the PECLS I1 applications software. Absorption spectra were measured on a Perkin-Elmer, Model Lambda 5, spectrometer and quantum yields were measured at room temperature. The Model LS-5 gives quantum- corrected excitation spectra through the use of a Rhodamine 101 quantum-corrected reference detector. Emission spectra were uncorrected and hence a correction graph must be generated. A correction graph from 250 to 630 nm is obtained in the following manner. Over the region 250-410 nm the excitation system is used as a light source of known spectral distribution. Light from the excitation monochromator is reflected into the emission monochromator by a scatterer and the excitation and emission monochromator scanned synchronously at the same wavelength. The excitation band pass is set at 5 nm with the emission band pass set at 20 nm as recommended by Melhuish.13

200 300 400 500 600 Wavelengthlnm

Fig. 2. Emission correction graph for the Model LS-5 fitted with a red sensitive photomultiplier.

In order to overcome errors associated with second-order radiation and the fact that the sharp emission lines of xenon at ca. 470 nm make precise correction difficult, the correction graph from 410 to 630nm is generated by comparing the emission spectrum of quinine sulphate run under identical conditions with that published by Velapoldi and Mielenz .ll Both correction graphs are merged to give the emission correction graph, Fig. 2. The shape of this graph is determined in part by the efficiency of the emission grating and Fig. 3 shows the grating response graph with the grating blazed for maximum efficiency at approximately 450 nm. This graph was generated by dividing the correction graph, generated by scanning both monochromators synchronously at the same wavelength, by that obtained by placing a plane mirror in front of the emission grating and scanning the excitation monochromator. The resultant graph does not include the effect of the photomultiplier response. The latter, also shown in Fig. 3, was obtained by dividing the correction graph by the grating

100

80 %

2 60 2

-$ 40 Q) [L

0

Q)

-

20

200 300 400 500 600 Wavelengthinm

From excitation monochromator

To emission monochromator

10-nm path length cuvette

Plane mirror

Fig. 4. Schematic diagram of the trans- mission cell holder used for the determina- tion of absorbance values.

Fig. 3. (A) Grating response graph and (B) photomultiplier response graph for the Model LS-5.

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1070 RHYS WILLIAMS et al. : RELATIVE QUANTUM YIELDS USING Analyst, VoZ. 108

response graph. All of these procedures were performed on the Model 3600 data station.

The Model LS-5 luminescence spectrometer was used as a single-beam ultraviolet - visible absorption spectrometer by placing a mirror at the sample focus to reflect the excitation light into the emission monochromator. The light needs to be heavily attenuated with a wire-mesh gauze to prevent overloading of the sample photomultiplier. A 10-mm path- length cuvette was placed in the reflected beam (Fig. 4) and by synchronously scanning both monochromators at the same wavelength, a transmission spectrum was obtained. By dividing a sample spectrum by the solvent spectrum the transmission spectrum of the solute was obtained. Fig. 5 compares the absorbance spectrum of pyrene in hexane, as measured on the Model LS-5, with the excita- tion spectrum. Table I compares the ratios of the main peaks as measured on the ultra- violet absorption spectrometer and on the Model LS-5. Excellent agreement between the ultraviolet absorption and fluorescence excitation spectra is observed. In addition, the absorbance value falls midway between the values observed on the ultraviolet spectrometer

The latter was converted to an absorbance spectrum.

run at 2- and 4-nm band widths.

O.* I 0.6 -

8 0 n 5 0.4 n a

0.2 -

Wavelengthlnm

(a) The absorbance spectrum and (b) the excitation spectrum of pyrene in hexane as measured on the Model LS-5.

Fig. 5.

Results and Discussion A series of compounds in a variety of solvents were prepared and the ultraviolet absorption

was measured using the method described under Experimental. The solutions were diluted to give absorbances in the range 0.02-0.35 absorbance unit, i.e., within the expected linear

TABLE I COMPARISON OF PEAK HEIGHTS OF PYRENE IN HEXANE USING AN ULTRAVIOLET

ABSORPTION SPECTROMETER AND LS-5 LUMINESCENCE SPECTROMETER

Lambda 5 ,-A-, Luminescence spectrometer

Band width I A \

Absorption Fluorescence Ratio of peaks 2 nm 4 nm 2.5 nm 2.5 nm

241/273nm .. . * 1.733 1.851 1.786 1.779 2411335nm . . . . 1.585 1.632 1.632 1.634

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September, 1983 A COMPUTER-CONTROLLED LUMINESCENCE SPECTROMETER 107 1

calibration range of fluorescence emission ‘uemws concentration. The solutions were not de-gassed before measurement. The emission spectra were measured using a 2.5-nm band width and corrected using the correction graph in Fig. 2 and the areas under the corrected spectra calculated. The quantum yields were calculated according to equation (2) and the results by this method compared with some literature values, which are given in Table 11. The reproducibility of measuring and calculating the quantum efficiency of the same solution is within *3-5%.

TABLE I1 ROOM TEMPERATURE FLUORESCENCE QUANTUM YIELDS

The following abbreviations are used : 9,10-DPA, 9,lO-diphenylanthracene ; 9-MA, 9-methylanthracene; TPB, tetraphenylbutadiene; QS, quinine sulphate; C, cyclohexane; H, hexane ; MCH, methylcyclohexane ; PA, photoacoustic spectroscopy ; and W, water.

Compound Anthracene .. .. .. Benzene .. .. . . Coronene .. .. .. Fluorene . . . . . . Pyrene .. .. _ .

TPB . . . . . . . . Sodium salicylate . . . . Lucifer Yellow . . . . Fluorescein . . . . . .

9,lO-DPA . . . . . . 9-MA . . . . . . . .

Solvent MCH MCH MCH MCH C C C C 1 M NaOH W 0.1 M NaOH

Excitation1 nm 252 254 303 265 241 262 256 346 302 430 460

Emission rangelnm 360-510 270-360 400-500 280-400 350-550 360-540

360-580 300-500

340-550 460-630 470-620

Literature values Standard I A >

QR QP QP Solvent Ref. DPA (1.00) 0.31 0.33 H 14

15 DPA (1.00) 0.03 0.05 H

DPA (1.00) 0.71 0.54 H 3 DPA (1.00) 0.31 0.32 C 16 QS (0.59) 1.00 1.00 C 12 DPA (1.00) 0.42 - - - DPA (1.00) 0.84 0.86 PA 4 QS (0.59) 0.25 0.25 NaOH 17 QS (0.59) 0.20 0.24 W 18 QS (0.59) 0.82 0.84 0.1 M N ~ O H 5

- - DPA (1.00) 0.18 -

The method proposed for calculating relative fluorescence quantum yield was found to produce results that compare favourably with those previously published. The discrepancy between the observed and literature value for fluorene is probably the result of a relatively low value observed by Weber and Teale3 because Dawson and Windsor15 observed a value of 0.68. The main advantage of this technique is the ability to measure absorbance values on a fluorescence spectrometer thus eliminating errors associated with the use of an absorption spectrometer. The use of a desk-top computer greatly simplifies the experiment and routine quantum yield measurements are easily performed by automating the control of the instru- ment and data calculation.

1. 2.

3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14.

15. 16.

17. 18.

References Demas, J . N., and Crosky, G. A., J . Phys. Chem., 1971, 75, 991. Bridges, J . W., in Miller, J. N., Editor, “Standards in Fluorescence Spectroscopy” Chapman and

Weber, G., and Teale, F. W. J., Trans. Faraday Soc., 1957, 53, 646. Adams, M. J., Highfield, J. G., and Kirkbright, G. F., Anal. Chem., 1980, 52, 1260. Olmsted, J., J . Phys. Chem., 1979, 83, 2581. Ware, W. R., and Rothman, W., Chern. Phys. Lett., 1976, 39, 449. Bendig, J., Kreysig, D., and Schoneich, R., Opt. Spectrosc. USSR, 1980, 49, 29. Upton, L. M., and Cline Love, L. J., Anal. Chem., 1979, 51, 1941. Britten, A., Archer-Hall, J., and Lockwood, G., Analyst, 1978, 103, 928. Gains, N., and Dawson, A. P., Analyst, 1979, 105, 481. Velapoldi, R. A., and Mielenz, K. D., Natl. Bur. Stand. Spec. Publ., 1980, No. 260. Heinrich, G., Schoof, S., and Gasten, H., J . Photochem., 1974/75, 3, 315. Melhuish, W. H., J . Res. Natl. Bur. Stand. Sect. A , 1972, 76, 547. Guilbault, G. G., “Practical Fluorescence, Theory, Methods and Techniques,” Marcel Dekker, New

Dawson, W. R., and Windsor, M. W., J . Phys. Chem., 1968, 72, 3251. Berlman, I. B., “Handbook of Fluorescence Spectra of Aromatic Molecules,’’ Academic Press, New

Inokuchi, H., Harada, Y., and Kondow, T., J . Opt. SOG. Am., 1964, 54, 842. Stewart, W. W., Nature (London), 1981, 242, 17.

Hall, London, 1981, p. 68.

York, 1973.

York, 1965.

Received March 4th, 1983 Accepted March 28th, 1983

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