Roberl 1. Duncan, James W. Kirkpatrick, and Roberl E. Near Western llltno~sUn~vers~ty
Macomb, 61455
A Laboratory Experiment in Fluorescence Using the Spectronic 20
The fluorescence process is characterized by high sensitivity and selectivity which makes it an extremely important analytical tool that has become useful and valuable in practically a11 areas of chemistry. We believe the student should he introduced to this technique in the laboratory. ,This paper proposes a simple method by which a fluorometer can be coustructed for use in the laboratory to acquaint the student with the fluorescence technique. This method consists of modifying a Bauscb and Lomb Spectronic 20' to convert it to a filter fluorometer. The equipment required for modification is usually found in the laboratory, thus little or no added expense would he necessary. The modified instrument has been tested in an elementary quantitative analysis laboratory class and proved to be adequate for demonstrating the technique of fluorescence analysis. A discussion of fluorescence and the details of fluorescence techniques may be found in several source^.^ The fluorometer is quite satisfactory for measuring intense fluorescence. Stray light can he easily kept within 5y0 and a typical detection limit of 0.05 ppm for quinine sulfate in 0.1 N sulfuric acid can he obtained. A higher intensity light source would provide even greater sensitivity. The modified Spectronic 20 functions as an uusophisticated filter fluorometer. The exciting wavelength is isolated with primary filters. Secondary filters are used to pass the desired fluorescent light to the detector and to absorb other radiation. The filters can be interchanged for different fluorescing substances. The instrument can be easily converted back to its original function. Given below are the details for modifying the Spectronic 20 and the procedure used for the experiment in the laboratory. Spectronic 20 ALerafiop
The Spectronic 20 is alterid in the following ways: First, a secondary filter (or filters) passing the proper wavelength is inserted between the sample holder and phototube detector. It is important to completely 'Bausch and Lomb, Inc.. 820 Linden Ave., Rochester. N. Y. 14625. G. H., JR., "Quantitative An(a) FRITZ,J. S., AND SCHENK, alytical Chemistry" (2nd ed.), Allyn and Bacon, Inc., Boston, Mass., 1969, pp. 407-13. (b) GWILBAULT, G. G. (Editor), "Fluorescence: Theory, Instrumentiltion and Practice," Marcel D., "FluoresDekker, Inc., New York, 1967. (c) HERCULES, cence," Wiley-Interscience, New York, 1966. (d) WILLARD, H. H., MISRRITT, L. L., JR., AND DEAN,J. A., 'LIn~trumentaI Methods of Analysis" (4th ed.), D. Van Nostrand Company, h e . , New York, 1965, pp. 370-90. Corning Glass Works, Corning, New York 14830.
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Journal of Chemical Education
Figure 1. A view of the bottom of the Spectronic 20 showing the olterotionr to b e mode. Note thot the hinged cover over the phototube and cell area ir open.
enclose the filter so no light passes around the filter and on to the phototube. This can be easily accomplished by building a light-tight bracket out of flexible material such as rubber or cork so the filters can slide in and out. Secondly, the lamp in the instrument is removed to eliminate this source of radiation. These alterations are made underneath the instrument through the accesn sample cuvets are retained. A mercury lamp is placed above the sample compartment which is then irradiated from the top of the instrument with the cap open. See Figure 2. The mercury lamp is set about 5 to 6 in. above the sample holder to provide the maximum intensity, and yet not interfere in removing the sample cuvets. Primary filters can be attached directly to the lamp to isolate the desired wavelength. Filters can be obtained from Corning C ~ m p a n y . ~A description of the experiment and the results obtained are given below.
Figure 2.
The modified instrument as i t appears to the user.
Experimental
A Bausch and Lomb Spectronic 20 was used throughout the experiment. The excitation source was a Coleman mercury lamp model number 14030.' Quinine sulfate in 0.1 N sulfuric acid was used as the fluorescing sample, absorbinga a t 365 w .The two primary filtem used to obtain this wavelength were Corning3 color standard numbers 7-54 and 7-59. Quinine sulfate fluoresces (strong blue) a t approximatelys 460 mp. The Corning 5-57 and Coleman 14218 filters were used to pass this wavelength. A stock solution was prepared by dissolving 1.00 g of quinine sulfate in 0.1 N sulfuric acid, and diluting to 1 l with 0.1 N sulfuric acid. Serial dilutions were made to obtain exactly 0.05, 0.1, 0.2, 0.3, and 0.4 ppm of quinine sulfate in 0.1 N sulfuric acid. The fluorescence of each solution was then measured. The resulting calibration curve is given in Figure 3. Students were directed to adjust the position of thelamp for maximum intensity and then to run all samples, including their unknown sample, without disturbing the lamp position.
mg/ml standard solution into a set of 100-ml volumetric flsuks and dilute to the mark with the 0.1 N HzSO,. (See note 1.) Procedure
Turn on the fluorometer (modified S~ectronic20) and the mer11, alluw the in.lrurnent to warm up. llcc rhk rime 1,) rlcsn and dry rlrr . m p l e c ~ v e t ~.\wid . pu~ting your til.gtr prirtt im the rezr rul,r> a f ~ r they r we d e m . llemirt! the fluorescence intensity of each solution by consecutively placing identioal volumes of each solution in the cuvet and proceeding as follows: Place the blank in the sample holder (align the mark on the test tube with the mark on the fluorometer) of the fluororneter and move the mercurv , lamo until the lieht . shines direc1l.v intu the vuvt.t. Arljurt the rrr,, krn,h unul n rnerer rrudiug of arm i-. obtnlned. Nt.w plarr the mwt conc:enrrrr~da,lut~ort ,rhe 0.001 mg ml r c h r i m i t ) the sample holder and muvc the light source again until a maximum deflection oecun (note 2). Remove this sample, replace it with the blank and once again adjust the zero knob until the instrument reads zero. Measure the fluoraceme of each solution including the dilute stock solution auinine sulfate. Construct a d o t of which contains 0.001 rnelml .. Ru~~re.seer~ce intenairy v t r ; ~c~mccr~trntion.llrawre the mtmsir\- uf a qumine aulfarr .'~nknuwrt"and find it.; c,,ncentrntim from the cdilmrion curve nbcrve. Ckpurt the am~eutmtionin theappropriateunits (note 3). w r y lamp i t , at ~ I t m t 111 ruin
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Notes
1 ) P r e ~ a r all e solutions ahead of time and k e e ~ covered. 110 nor n > w rthe mercury lnmp mrr the euperirnmr 1.i set up. I'l'hls i. verv ioqwrta~tt.) 3) .\lake all your mes.urcment. duriug the one ~ I x a *prriud.
2
Discussion
Preparation of Solutions
The calibration curve which is linear over a wide concentration range indicates that the fluorometer can be used as an analytical instrument in the elementary laboratory. We have shown it to be satisfactory for demonstrating the measuring technique and its usefulness in the many areas of chemistry. The fluorometer constructed is by no means a sophisticated instrument, nor is it intended to be so. It does permit the student to become acquainted with the fluorescence technique which otherwise may have been left out of his background due to limited availability of instrumentation.
In a 1 l volumetric flmk dissolve 0.100 g of quinine sulfate in 0.1 N H9S0, and dilute to the mark with the acid solution. Dilute 10.0 ml (use pipet) of this solution to 1 1in a volumetric flask with the acid solution. The final solution contains 0.001 mg/ml of quinine sulfate. (The 0.1 N &SOt solution can be prepared by
4 Coleman Instruments, Inc., 42 Madison St., Maywood, Ill. 60133. See footnote 2, reference (d).
Figure 3. A working curve showing relative intensity of the quinine sulfate solvtions versus the concentration of quinine rulfote. The intensity reodingr ore the percent transmigrion readings from the Spestronic 20 meter.
Student Direclions
Volume 49, Number 8, August 1972
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