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experiments

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Quininew~luorescence Spectra A dry-lab spectral analysis experiment J a m e s E. O'Reilly University of Kentucky Lexington, 40506

Quinine is a strongly fluorescing organic compound that is often used in various analytical chemistry and instrumental analysis courses to illustrate the fundamental principles and analytical applications of molecular fluorescence spectroscopy ( I , 2). In many laboratories, a quantitative fluorescence experiment will usuallv consist in the aeneration of an appropriate calihration c&e and the analysis of a synthetic unknown using a simple filter fluorimeter-primarily because the cost of a scanning spectrofluorimeter is prohibitive for even small, advanced classes. Although students may read or be told about, for example, the general characteristics of the fluorescence spectra of the compound under study, and the relation between these spectra and the selection of proper filters for fluorescence measurements, they usually gain little appreciation of the more subtle aspects of the physicochemical and instrumental aspects of the processes involved hecause of the lack of a complete set of spectra for comparison. Indeed, there is the very real danger that the basic principles involved in the fluorescence method are never truly understood in this sans-snectra annroach. For examole. .. . . a .noint of near-universal misunderstanding, a t least initially, is the difference between a molecular ahsor~tionand a fluorescence excitation spectrum. It is the purpose of this dry-lab experiment to provide a set of quinine spectra, ohtained under carefully controlled conditions, for the student to peruse and interpret. With a proper consideration of the various ways incident electromagnetic radiation can interact with dilute aqueous aui. nine solutions-absorption, subsequent fluorescence, and scatter-the student is able to identify the source of some of the supposedly extraneous peaks in these spectra. Moreover, an interesting instrumental factor should become apparent as the student attempts to assign the various spectral peaks: The fluorimeter used is a grating-dispersion instrument, and second-order spectral peaks are present in the spectra taken a t high sensitivity. The Experiment

As a preliminary reading assignment, the student should read the appropriate chapters concerned with the interaction of electromagnetic radiation with matter, instrumentation for ultraviolet-visible spectroscopy, and molecular absorption and fluorescence spectroscopy in a suitable instrumental analysis text. Introduction It is commonly said that quinine, in dilute acid solution, has two excitation wavelennths-250 and 350 nm-for maximal fluorescence emission at 450 nm. Given this statement, one would then expect to obtain an excitation spec-

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Figure 1 . Specha of quinine in 0.05M H ~ S O I A. . FluOresCence spectrum of 2 ppm quinine, excitation at 250 nm. B. Fluorescence spectrum with excitation at 350 nm. C . Excitation spectrum of 2 ppm quinine, emission monitored at 450 nm. D. Ultraviolet absorption spectrum of 10 ppm quinine: the Y-axis is in mils of absorbance. A.

trum, a plot of fluorescence intensity (at a constant 450 nm) versus the excitine waveleneth. " . consistine of two peaks-one a t 250 and one a t 350 nm. The fluorescence or emission spectra would then consist of a single peak at 450 nm, when using a constant 250- or 350-nm excitation wavelength. The spectra in Figure 1ABC approximate this. However, when one ac&ally obtainithese spectra under carefully controlled conditions, several sup~osedlv"spurious" peaks are also present. These are parthular~; evident in the spectra of very dilute quinine solutions or of the blank (0.05 M HpS04). Basically, this experiment consists in assigning the various peaks in the spectra provided to some basic physical, chemical, or instrumental cause, and in answering some further questions about the fluorescence method. Three points should be noted: First, although it may he tempting to assign one or more peaks to "impurities" or to the sulfuric acid in solution, none of the peaks arise from these. Secondly, the spectra were ohtained using gratings for wavelength dispersion in the excitation and emission monochromators. so the oeculiarities of eratine monochromators will haveto be considered. ~ i n a i l y although , these are supposedly fluorescence spectra, keep in mind the various ways electromagnetic radiation can interact with matter: transmission: absorption. ~ossihlvfollowed hv subsequent fluorescence or phospho;escenc~; Rayleigh, ~ ~ n d a l l , and Raman scatter; and so forth. The Spectra The spectra provided are a)

An excitation and two emission spectra of 2 ppm quinine, a relatively high concentration for fluorescence work; and similar spectra for 0.03 ppm quinine taken at 30X greater instrumental sensitivity. Volume 53, Number 3, March 1976 / 191

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Figwe 2. Excitation and emission spectra of 0.03 ppm quinine in 0.05 M H2S04. A. FluOreScence spectrum with excitation at 250 nm. 8. Fluorescence spectrum w l h excitation at 350 nm. C. Excitation spectrum wlh emis-

Figure 3. Spectra d the background or blank. 0.05 M HsS04. A. ''Fluorescence spectrum" with excitation at 250 nm. B. "Fluorescence spectrum" with excitation at 350 nm. C. "Excitation spectrum" with emission monitored

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at 450 nm.

h) An ultraviolet absorption spectrum of 10 ppm quinine for rompnrmm with the quinine excitation spectrum. C~rcqpondinp,..exrnatinnn and 'emission" spectra of the rdvrnt hlnnk. (,05 M t12S04,rnken at thesamesensitivity ns those far the 0.03 ppm quinine solutions.

1) What are the peaks at 250 nm in emission spectra 2A and 3A due to? What are the peaks at 350 nm in emission spectra 2B and 3 8 due to? 2) What are the very small peaks s t about 275 nm in spectra 2A and 3A due to? What is the small peak at 395 nm in spectrum 3B due to? What effect does this latter peak have on the quinine emission oeak at 450 nm in mectrum 2B. What would the effect he. analvticallv. if the ouinine concentration were even , lower, say 0.01 ppm. 3) What are the peaks a t 500 nm in spectra 2A and 3A due to? The 700-nm peaks in 2B and 3B? 4) Identify the origin of the peaks at 225, 385, and 450 nm in the "excitation spectrum" of the blank, 3C. What effect do these peaks have on the quinine excitation spectrum 2C? 5) Compare the quinine excitation spectrum (1C) with the normal uv absorption spectrum (ID)and comment on the differences in relative peak heights. The table may help. Chen (5)has indicated that the quantum efficiency for quinine fluorescence is very nearly the same, about 0.55, whether the eacitation wavelength is 250 or 350 nm. Furthermore, if the quantum efficiencies are the same at these two wavelengths, why is the fluorescence intensity greater with eacitation at 350 than at 250 nm (1B versus lA)? 6) What effects would these extra peaks have on the analytical sensitivity or detection limit for quinine analysis if you were using (a) a grating-monochromator scanning fluorimeter with a 10-nm bandpass and excitation at 350 nm versus (b) a filter fluorimeter with a mercury vapor Lamp, a primary filter to isolate the 365-nm mercury line, and a short-wavelength cutoff seeondary filter which passes all light above 400 nm? ~~~~~~

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ocompi~edfrom Ref. (31,for a 150-w xenon arc tamp. b compiled from Ref. (4). for a 1P28 pnotomuitiplier t u b e 155 spectral lel~anse).

a t s u 1P28 photomultiplier. T h e slit arrangement in t h e fluorimeter was selected so as t o be symmetrical in both t h e excitation and emission monochromators, a n d corresponded t o a bandpass (full width a t half-height) of approximately 12 n m a s determined from t h e Rayleigh scatter peaks. T h e water used was triply distilled, with the seco n d distillation from alkaline permanganate t o remove t h e fluorescent organic impurities present in both house-distilled a n d deionized water. All solutions were 0.05 M in

H2S04. Ultraviolet a b s o r ~ t i o nsDectra were obtained on a Carv e ~ e 1.00-cm r cells. Model 15 ~ ~ e c t r o ~ h o t o mwith A set of large-scale copies of t h e spectra in Figures 1-3, complete with noise, is available from t h e author on request.

The SDectra

The Interpretation

Fluorescence spectra were obtained on a n Aminco-Bowm a n S ~ e c t r o ~ h o t o f l u o r i m e tequipped er with a 150-W H a novia high-piessure xenon arc l & m d a potted Hamam-

After some consideration, all t h e peaks observed in t h e excitation a n d emission (fluorescence) spectra can be assigned t o one or more of several causes: absorption and

192 / Journal of Chemical Education

subsequent fluorescence by quinine; Rayleigh (and Tyndall) scatter of the exciting wavelength; a water Raman line a t wavelength slightly longer (3380 cm-I) than the exciting line: and several peaks due to the overlapping - - second-order spectra produced by grating monochromators. The spectra of 2 ppm quinine (Fig. 1) show, fairly cleanly, the "desired" e&sion peak at 450 nm and the excitation peaks a t 250 and 350 nm. Figure 1B shows a small scatter peak a t 350 nm from the exciting light. Figure 1C shows a small scatter peak at 450 nm (450-nm light from the excitation monochromator will Rayleigh scatter into the emission monochromator, which is set a t 450 nm, and hence will he detected by the P M tube), and a small peak a t 500 nm due to second-order excitation of quinine fluorescence (setting the excitation monochromator at 500 nm also passes the second-order 250-nm light, which in turn excites quinine fluorescence at 450 nm (6)). The excitation spectrum of 0.03 ppm quinine (Fig. 2C) is \,cry similar in appearance to that of 2 ppm quinine, except that the 450-nm Hayleigh line is relatively murh more pn>nounced, and the actual quinine peaks are somewhat more distorted due to the "extLan peaks which partially overlap. The "excitation spectrum" of the blank (Fig. 3C) clearly shows the 450-nm scatter line and indicates that this peak is not due to the presence of quinine. Figure 3C also clearly shows a second-order scatter peak a t about 225 nm (setting the emission monochromator a t 450 nm will also pass second-order 225-nm scattered light to the detector when the excitation monochromator lets light of this wavelength impinge on the sample). The absence of a 500-nm peak in Figure 3C indicates that this peak is caused hy the quinine in spectrum 2C. Raman Lines

Probably the most subtle aspect of these spectra are the peaks due to the water Raman line. The Stokes Raman hand of water, attributable to the symmetrical 0-H vibration, comes at about 3380 cm-I (6, 7). This band is most clearly visible as the 275-nm peak in Figure 3A, the 390-nm peak in 3B, and the 385-nm peak in 3C. (When the excitation monochromator passes 385-nm light, the resultant Raman band will occur at about 450 nm and therefore be passed by the emission monochromator to the detectorhence a peak a t 385 nm in the excitation spectrum.) While most students are able to assign many of the peaks due to fluorescence, Rayleigh scatter, and second-order grating effects, few are able to assign the Raman scatter peaks. Part of the reason for this is the inherent experimental error in wavelength measurement. The estimated uncertainty in an individual wavelength is about 2-6 nm; thus the resulting uncertainty in the measured Raman band comes to about f300-600 cm-I. For example, eight separate measurements of the Raman band with excitation a t 250 nm resulted in an experimental value of 3630 f 200 cm-I.

Peak Heights

For some classes, the instructor may wish to have the students discuss the relative heights of the various spectral peaks. Toward this end, the lamp-output and photomultiplier-efficiency data in the table are provided. I t should be noted that these data are representative only of the general class of xenon lamp and P M tube used, and not the particular ones used to obtain the spectra herein. For example, the overall light output of a xenon lamp decreases with the age and use of the lamp. and the output at short wavel k g t h s decreases relatively faster than that a t higher waveleneths. So the differences in relative peak heights of the 25cand 350 nm hands in the quininiexcitati& and uvabsorption spectra (1C and ID) cannot be explained totally by simple use of the lamp output data in the table. Summary A set of quinine fluorescence spectra has been provided for students to "dry-lah" an interpretive spectral analysis experiment. The questions in The Spectra section have been written so as to group the various fundamental causes together, as an aid to the student, and with a slant toward the quantitative analytical use of fluorescence. There is no reason why, for example, the questions could not be rewritten for use in a physical chemistry laboratory in an experiment involving fluorescence quenching (2,8). In any event, the instructor is free to tailor questions to better fit the needs of his particular class. The snectral internretation above does not include a discussion of every ohservable peak, but should provide sufficient euidelines for each to be easilv assianed. ~ h k are e a number of standard reference texts on fluorescence available for hackaround reading.(3, 9-12) which also give a good deal of information concerning quinine soectra. The American Instrument Company's fluorescence newAetrer has had short articles on scatter interference 161 and the use of quinine as a tluuromrtric srandsrd (131.

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Literature Cied Willerd, H. H., Memitt. L. L., Jr., and Dean, J. A . "Instrumental M e t h d s of Andysis? 5th Ed., 0. Van Noseand Company, New York. 1974, p 144. 121 O'Reilly, J. E.. J. CHEM. EDUC.,52.610(1975). (3) Udeniriend. S., "Fluorescence Assay in Biology and Medicine: Acsdemie Press, New York, 1962, p 42. (41 "RCA Phofomultiplier Manual? RCA Corporation. Hsrrinon. N.J., 1910. (51 Chen,R.F.,Annl Blochem.. 19,374 (1967). 7. (31. 17 (19731: American Imtrument Com(61 Pamvstar. R A., Fluorescence NOW*. pany, Silver Spring. Md. (71 Ref. i31.p. 110. (81 Eisenhrand, J.. and Raiach. M., 2.A n d Chem., 179,352 11961): 173.406 (19611. 191 Udeniriend, S., "Fluorereenee Assay in Biology end Medicine." Volume IT, Academic Press, New York, 1969. (101 Guilhault. C. C., "Practical Fluoremnco. Theory, Methods, and Techniques,.) MS.. =el Dekker. New York. 1973. (11, Hercules. D. M., (Editor1 Tluoresmnee sad Phosphoreseenm Analysis." Wiley-lnterscience, New York, 1966. (12) White. C. E., and Argauer, R. J., "Fluoreseanee Anslmir A Practical Approach." Marcel Dekker,NewYork, 1970. 1131 Psnnwater, R. A,. and Vsn Deventer, J. H.. (Editors) Fluorescence News, 8, (41, 35 (1974). (1)

Volume 53,Number 3, March 1976 / 193