Illustration of the Principles of Fluorimetry An Apparatus and Experiments Specially Designed for the Teaching Laboratory Stephen W. Bigger,1 Kenneth
P.
Ghiggino, Geoffrey A. Meilak, and Bruce Verity2
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The University of Melbourne, Parkville, 3052, Australia
Fluorimetry is a standard technique in physical and analytical chemistry. However, commercial fluorimeters do not effectively teach the principles of fluorimetry because the student can not clearly see the arrangement of the various components. Commercial instruments are also expensive and usually reserved for research purposes. The Apparatus In this paper, we describe an apparatus designed for use in an undergraduate laboratory. The apparatus has the following advantages from a teaching
point of view.
be 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 microcomputer for experiment control and data collection. It demonstrates the principles of fluorimetry to the stu-
It
can
dent.
Figure 1. Schematic diagram of the fluorimeter.
As far as we are aware, this is the first description of such an apparatus, although the general principles governing spectrofluorimeter instrumentation have been discussed previously (1).
The Experiments Although the apparatus can measure fluorescence intensities that are quite weak, it is not intended to be a highresolution instrument. It is best used for measuring broad, structureless fluorescence bands. Consequently, the experiments performed with the apparatus should be chosen judiciously. In this paper we also describe experiments that are suitable for use with the apparatus. The experiments illustrate the following fundamental principles of fluorimetry. • • • •
•
of fluorescence spectra of correction factors calculation of relative fluorescence quantum yield collisional quenching the Stern-Volmer equation measurement use
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. 150-W ozone-free xenon arc lamp (Osram XBO 150 W/l ofr) with power supply (Cathodeon) •high-throughput monochromator (Bausch and Lomb Model 33-86-07, 1200 grooves/mm) for selection of the excitation •
'Present address: Department of Environmental Management, Victoria University of Technology, St. Albans Campus, McKechnie Street, St. Albans, 3021, Australia. 2Present address: Comalco Research Centre, P.O. Box 316, Thomastown, 3074, Australia.
can be replaced by a bandpass filter to duce cost.) lens to focus the excitation beam onto the sample
wavelength. (This •
re-
•black aluminium housing that contains a temperature-controlled cell-holder • interference “wedge” (grating)—which was salvaged from a •
discarded EEL spectrophotometer—for spectral dispersion of the fluorescence photomultiplier (PM) tube (RCA 1P28 or 1P28A), positioned at right angles to the excitation beam
The apparatus is covered with a black cloth during measurements. The first four components are aligned on an optical rail. The position of the wedge is controlled by a stepper motor (Philips 4-phase unipolar; step angle: 1.8°) driven by a stepper-motor drive board (RS 332-098). A slotted optical switch, which comprises an IR source and photodiode, is fixed above the wedge to act as a limit switch. A metal blind with a small window near each end is attached to the wedge. The blind passes through the switch (Fig. 2) so that the switch is activated when either of the windows travels through it, that is, when the wedge has reached the limits of its travel. The stepper-motor/wedge/optical-switch combination is calibrated so that, after initializing the wedge to the lower wavelength limit, any wavelength from 367 to 700 nm can be accessed by executing the appropriate number of steps. For our apparatus, 12.5 steps alter, by 1 nm, the wavelength of the emitted light that was selected. The Interface The apparatus is controlled through an interface with a microcomputer. The general requirements for the interface are given below. Volume 69
Number 8
August 1992
675
Figure 2. Arrangement of the blind for detecting the limits of travel of the interference wedge: (a) front view; (b) side view.
wavelength (nm) • •
• •
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 tube digital output to set the stepper-motor direction another digital output to initiate the steps
The interface unit was designed in our department to operate with an Apple Macintosh microcomputer. It uses the high-level computer language named Forth. A low-voltage power supply, a stepper motor drive board, and a high-voltage power supply for the PM tube are contained in a separate box. A Macintosh microcomputer sends commands via 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 wavelength and proceeds stepwise to the upper wavelength limit. A reading of the PM tube output is made after every 10 steps. The microcomputer then displays the data, stores it in disk files, and plots 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 H2S04, and the commercial optical brightener Leucophor PAF (Sandoz) (2) in water. Both are adjusted to an absorbance of about 0.5 at 350 nm and then the spectra are measured over the range 380-600 nm with an excitation wavelength 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 at about 450nm for QBS and at about 430 nm for Leucophor PAF. Both values are in good agreement with the literature values of 450 (3a) and 427 nm (2). The QBS spectrum is then normalized by adjusting its intensity scale so that the maximum fluorescence intensity has a value of 1.
Figure 3. Uncorrected spectra of (a) QBS in 1 M H2S04 and (b) Leucophor PAF in water. Bo1h solutions were adjusted to have an absorbance of 0.5 at 350 nm.
be
multiplied to give the corrected normalized intensity (D
^corrA) = lA)4xpA>
Figure 4 shows a plot of y’A) vs. wavelength for our apparatus. The corrected spectrum used to calculate y(X) was recorded in 0.1 N H2S04 (i.e., 0.05 M H2S04). 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(X) values and plotted on one set of axes. Clearly, the QBS spectrum corrected in this way will be identical to the one given to students for the calculation of ?A), Calculating the Fluorescence Quantum Yield
The fluorescence quantum yield cpf of a molecule is the ratio of the number of photons emitted qem to the number absorbed
qabs
(4a). ^em
(2)
