Illustration of the Principles of Fluorimetry An Apparatus and Experiments Specially Designed for the Teaching Laboratory Stephen W. Wgger; Kenneth P. Ghiggino, Geoffrey A. Meilak, and Bruce verity2 The University of Melbourne, Parkville, 3052, Australia Fluorimetry is a standard technique i n physical and analytical chemistry. However, commercial fluorimeters do not effectively teach the principles of fluorimetry because the student can not dearly see the arrangement of the various components. Commercial instruments are also expensive and usually reserved for research purposes. The Apparatus I n this paper, we describe a n apparatus designed for use in a n undergraduate laboratory. The apparatus has the following advantages from a teaching point of view. It can he constructed cheaply and specifically for the teaching laboratory. It allows the students to become familiar with the components of a fluorimeter and its operation. It is ideal for showing the interfacing of an apparatus with a micmcomputer for experiment control and data collection. It demonstrates the . orincioles . of fluorimetrv to the student.
sample cell
,
optical asis
excktion lens
monochromator
lens
I50 Wxenon arclamp
Optical
axis
1
Figure - 1. Schematic diagram of the fluorimeter As far a s we are aware, this is the first description of such a n apparatus, although the general principles wavelength. (This can he replaced by a bandpass filter to regoverning spectrofluorimeter instrumentation have been duce east.) discussed previously ( I ). lens to focus the excitation beam onto the samole black aluminium housing that contains a temperature-mnThe Experiments trolled cell-holder Although the apparatus can measure fluorescence inten.interference 'kedge" (gratingbwhieh was salvaged from a sities that are quite weak, i t is not intended to be a highdiscarded EEL spedmphotometer-for spectral discemion of the fluorescenc~ resolution instrument. I t is best used for measuring broad, photomultiplier (PM)tube (RCA 1P28 or lP28A). positioned structureless fluorescence bands. Consequently, thGexperat right angles to the excitation,heam imcnts performed with the apparatus should be choscnru"
~ ~ - ~ - ~
I n this nauer we also describe experiments that are suitable for is; with the apparatus. he experiments illustrate the following fundamental principles of fluorimetry. measurement of fluorescence soeetra use of correction fadom calculation of relative fluorescence quantum yield ' collisional quenching the Stern-Volmer equation Design and Setup of the Apparatus A schematic diagram of the fluorimeter and its control system is shown in Figure 1.The components of the fluorimeter are described below.
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150-Wozone-freexenon arc lamp (Osram XBO 150 Wll ofr) with power supply (Cathodeon) high-throughput manoehromator (Bausch and Lamb Model 33-86-01, 1200 groavedmm) for selection of the excitation
'Present address: Depallment of Environmental Management, Victoria Un versify of Technowy,St. Albans Campus, Mcdecnn e Street, St. Albans, 3021, ~ustralia.-'present address: Comalco Research Centre. P.O. Box 316. Thomastown, 3074, Australia.
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The anoaratus is covered with a black cloth durine measuremints. The first four comwnents are alimed on an ootical rail. The position of the &edge is controfied by a steiper motor (Philios Cohase unioolar: steo anele: 1.8")driven bv a stepper-mobr drive doard'(~~'332-698).A slotted o p ~ c a l switch. which com~risesa n IR source and ohotodiode. is fixed above the wedge to act a s a limit sditch. A metal blind with a small window near each end is attached to the wedge. The blind oasses throueh the switch (Fie. 2) so that the switch is activated when either of the Gndows travels through it, that is, when the wedge has reached the limits comof its travel. The stepper-motor/wedge/optical-switch bination is calibrated so that. after initializine the wedee to the lower wavelength limit; any waveleugth-from 3 6 7 G 700 nm can be accessed by executing the appropriate number of steps. For our apparatus, 12.5 steps alter, by 1nm, the wavelength of the emitted light that was selected. The Interface The apparatus is controlled through a n interface with a microcomputer. The general requirements for the interface are given below. Volume 69 Number 8 August 1992
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Flgure 2 Arrangement of the bl~nafor detecl ng me llmlts of trave of the ntelterence weage (a)front view, (0, s oe wew digital input to monitor the status of the optical switch analog input and analog-digital converter (ADC) to digitize the output voltage of the PM tuhe digital output ta set the stepper-motor direction another digital output to initiate the steps The interface unit was designed in our department to operate with a n Apple Macintosh microcomputer. It uses the high-level computer language named Forth. A low-voltage power supply, a stepper motor drive hoard, and a high-voltage Dower s u p ~ l v for the PM tube are contained in a separ&eAbox.A ~ & i & t o s hmicrocomputer sends commands h a the interface unit to the apparatus, using .. - the Macintosh application "Hypercard". The user selects the desired wavelength limits for the spectrum. The stepper motor moves the wedge to the lower waveleneth and Droceeds stenwise to the u w e r wave);made after length h&t. ~ r ~ a d l n ~ o fI'M r h tubcoutput e evwv 10 ritc~s.The rn~rrocomDuterthpn dls~lavsthe duta, stores it in disk files, and plo& the spectrum. This description specifies the interfacing system currently used in our teaching laboratory. However, the apparatus is suitable for interfacing with any computer system. For example, it was used for several years interfaced with a 6809-based microcomputer in our laboratory. Fluorescence Quantum Yield Experiment Fluorescence spectra are taken of two solutions: quinine bisulfate (QBS, Eastman Kodak) in 1 M HzS04, and the commercial optical brightener Leucophor PAF (Sandoz) (2) in water. Both are adiusted to a n absorbance of about 0.5 a t 350 nm and t h e n t h e spectra are measured over the ranee 380400 nm with an excitation waveleneth of 350 nm, which is the wavelength of maximum absorbance for QBS. For Leucophor PAF this wavelength is 343 nm. Reabsorption effects, which can distort fluorescence spectra, are quite small for these two solutes. Typical spectra recorded with the apparatus are shown in Figure 3. The maximum fluorescence intensities occur a t about 450nm for QBS a n d a t about 430 nm for Leucophor PAF. Both values are in good agreement with the literature values of 450 (3a)and 427 n m (2).The QBS spectrum is then normalized by adjusting its intensity scale so that the maximum fluorescence intensity has a value of 1.
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wavelength (nm) Figure 3. Uncorrected spectra of (a) QBS in 1 M H2S04and (b) Leucophor PAF in water. Both solutions were adjusted to have an absorbance of 0.5 at 350 nm. be multiplied to give the corrected normalized intensity
Im.
LA)
=
mzew(w
(1)
Figure 4 shows a plot of .XUvs. wavelength for our apparatus. The corrected spectrum used to calculate dl)was recorded in 0.1 N HzS04 (i.e., 0.05 M HzS04). Its use is justified by our observation that fluorescence spectra of QBS are practically independent of acid concentration. The experimental QBS and Leucophor PAF fluorescence spectra are then corrected using the y(h) values and plotted on one set of axes. Clearly, the QBS spectrum wrrected in this way will he identical to the one given to students for the calculation of .XU. Calculating the ~luoresc&ceQuantum Yield The fluorescence quantum yield qf of a molecule is the ratio of the number of photons emitted q,, to the number absorbed qabs(4a).
*=-
gem
(2)
gabs
The fluorescence quantum yields of two solutions are related by
,,
r p , ~ - (1- 1043(n1)2a1 -%L 21 (I - 1 0 " l ) ( n ~ ) ~ ~
(3)
whereA is the absorbance of the solution at the excitation wavelength; n is the refractive index; a is the area under the corrected fluorescenceemission spectrum; and the subscript numerals refer to the two solutions (5).The factors
The Correction Factor The corrected, normalized fluorescence soectrum of QBS (3a)is suppliedto students. From it, the c&rection fador
Xh) for the apparatus is calculated a t suitable wavelen$zth intervals (e.;.; 10 -1. This accounts for wavelength-dependent factors such as PM-tube response and transmission of the wedge (3b).The correction factor is the number by which the normalized experimental intensity I,, must 676
Journal of Chemical Education
350
400
450 500 550 wavelength (nm)
I
Figure 4. Plot of the correction factorfor our apparatus as a function of wavelength.
4 0
0.01 0.02 0.03 0.04 0.05 0.06
wavelength (nm) Figure 5. Spectra of QBS(solution as in Fig. 3)showing quenching by NaCI. The concentrations of NaCl are (a) 0; (b) 0.00625 M; (c) 0.01875 M; (d) 0.03125 M: and (e)0.05 M.
[NaCII (M) Figure 6. Stern-Volmer plot for the quenching of QBSfluorescenceby NaCI; slope: 72.7 M',intercept: 0.98.
in parentheses take account of the relative amounts of the excitation beam absorbed by each solution. They correspond to the (labsterm in eq 2, and the a terms correspond to the q., term. The refractive index terms cancel in dilute solutio& in the same solvent The fluorescence quantum yield for QBS a t infinite dilution and 25 O C is 0.546 (6).Since this value is not verv sensitive to temperature, we assume that cpf a t 20 'C 2 also equal to 0.546. By combining this value with the areas under the corrected emission spectra and the absorbances of the two solutions a t 350 nm, we can evaluate 91 for Leucophor PAF to be 0.17 0.01 at 20 'C. This agrees well with the literature value of 0.20 at 25 'C (2).
where A is the absorbance of the solution at 350 nm. The Stern-Volmer plot ofI'II vs. [NaCll is a straight line, as shown in Figure 6, which incorporates additional data to that shownin Figure 5. Usingthevalue of 19.02 ns (QBS in 1.0 N H2SOI,425 nm) (8)as z, we can calculate k, from the slope of the line of the best fit in Figure 6.
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Fluorescence Quenchina - ExDeriment . Fluorescence spectra are measured for five solutions of about 10 M QBS in 1 M H2SOdthat mntain NaCl in wncentrations ranging from d t o 0.05 M. These spectra are plotted on one set of axes (Fig. 5). The chloride ion is known to quench the fluorescence of QBS (46), and this process has been mentioned as part of a qualitative fluorescence experiment (7).
The Stern-Volrner Equation Fluorescence quenching can be modelled quantitatively by the Stern-Volmer equation ( 3 ~ ) .
where q$ and cpf are the fluorescence quantum yields in the absence and presence of NaCl respectively; k, is the second-order rate constant for the quenching process; and 7, is the fluorescence lifetime of QBS in the absence of quencher. To simplify analysis of the data, it is assumed that
& P --'pf- 1 where I' and I represent the maximum fluorescence intensities of QBS in the absence and presence of NaCl respectively, corrected for the different amounts of excitation light absorbed by the two solutions. This correction is achieved by dividing the experimental emission intensity by
Comparison with Other Values Such determinations have been carried out manv times with our apparatus. They have consistently given a value of3.6 10.2 x lo9 M-Is-' fork". The same value was obtained A This using a Perkin-Elmer M P $ - ~ ~spectmfluorimeter. value is in reasonable agreement with data in the literature ( 9 , l O ) . It is instructive for students to compare their k, value with the diffusion-limited second-order rate constant, kds (M4s-9 a s calculated from the Debye-Smoluchowski equation (3d).
where R is the gas constant (J m01-~K-'); T is the thermoand q is the viscosity (kgm-Is-') dynamic temperature (K); of the solution at 20 'C. The value of kamiscalculated to be 6.5 x lo9 M-'s-' . his result can be interpreted to mean that, in 1M HzSO4,not every encounter between excited QBS molecules and the quencher results in quenching. Conclusion The a ~ ~ a r a t and u s ex~erimentsdescribed are a cost-efficient &d effective meof teaching experimental technioues and illustratine im~ortant~henomenain fluorimet&to undergraduate &udents. ' Literature Cited 1. Lott.
P E: Hurtubiae, R. J. J Chpm. Edw. 1974,52,.431&320,358364.
2. Smit, KJ.;Chiggina, K P DyosandP&ments
1987,8,8%97. 3. Parker,C.A.Photaiumimanna ofSolutiom; Else%er:Amsterdam, 1968; (a1 p 8 (bl PP 252258 (el pp 12-74ldl pp 7 6 7 8 . NewYork, ~ ~ e n c 194% (a1 4. R i n ~ h ~ , P . F i u o m ~ m ~ n d P h o s p h a m h a h a n h a ; l n p 6 lbl p 328. 5. Rusakoria, R:Teata,k C. J. Phys. Chpm. lW,72,79%796. 6. Melhlush. W H.J. Phvs. Chem. 1961.65.22%235.
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10. Chen, R.F.AmI. Biaehim. 1974,57,593404.
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