STUDENT-BUILT SPECTROFLUOROMETER JACK M. GOLDSTEIN, WALLACE M. McNABB and J. FRED HAZEL University of Pennsylvania, Philadelphia
T H E rapid acceptawe of spertrofluorometric analysis in such fields as biochemistry and the manufacture of pharmaceuticals suggests that the method is worthy of beiug incorporated iuto t,he customary instrumental analysis course. Commercial iustruments now on the market are patterned on the prot,otype of Bowman, et al. ( I ) , and although these instrumerrts are proving to he extremely useful in research, their present high cost may deter college chemistry departments from purchasing them for pedagogic purposes. A philosophy gaining popularity among educators is that relatively simple, inexpensive, and laboratoryconstructed apparatus can he used advantageously in illustrating principles, since the student's attention is focused on the basic theory and practical methods of operation rather than a mastery of turning the proper dials in the proper sequence ( 2 ) . I n accord with this, a st,udent project was organized for the purpose of constructing a cheap, practical spectrofluoromet,er. Spectrofluorometry can be defined as the measurement of the spectral characteristics of fluorescent or phosphorescent substances by photoelectric means. The luminescence is usually obt,ained using ultraviolet light as the source of excitation. The spectra are due to electronic transitrims within atoms or molecules and bear a close relationship to absorption spectra in the ultraviolet or visible region (3). There are several advantages in the employment of spectrofluorometry in analytical work. For qualitative identification of chemical species, this method may often yield spectra more clearly defined and unique to a single compound than customary spectrophotometry. In quant,it,ative work, the technique is ofteu far more sensitive than ot,her methods, but there are also limitations to its use such as quenching effect,^ and nonadherence to Beer's law (4).
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Sc -Source o f Excitation-UV F Appropriate Filler C - Cell Containin# Flvorracant Liquid
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LI - Cylindrical Condensing Lens S , 8 S* -Slits L. 8 L.-collimating Lensc, PI - Cohtanf Deviation Prim Ph - Photomvtiplier Tube
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- Micrometer
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he used to obtain fluoresceuce spectra of porphyrin compounds. These compounds exhibit maximum fluorewenre when excited wit,h light of approximately 400 millimicrons wave length. A commercial source known as "Mineralite SL 3660" (6) was fouud t.o have high output in this region and was incorporated within t,he instrument. Filter. The purpose of a filter is to narrow the spect,ral range of the exciting source and especially to prevent the t,ransmission of visible light to the cell containing t,he fluoreseent sample. Since in the present work
DESCRIPTION OF THE INSTRUMENT
Figure 1 is a diagram of the optics; Figure 2 is a photograph of the assembled instrument. A discussion of the general 'nd specific aspects of each component follows. The symbols acc,ompanying the topic title pertain to the position of the component in Figure I. Source of Excitation (Sc). I n the selection of the excitiug source, consideration should be given t o the nature of the comuounds which will he studied hv the instrument. There are many ultraviolet sources available ( 5 ) , and for compounds of primary interest there may be one source that will be particularly well-suited. For example, in the present case the instrument was to 'Thin work was supported in part by a grant,-in-aid from the Soconv Mohil Oil Company.
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porphyrin compounds were to be studied, the filter designed for the SL 3660 tube was used. Cell Holder. The cell used with the instrument measured about one centimeter square and ten centimeters long and was one of a pair that came as accessories to a B. & L. "Spectronic 20." The cell holder is pictured in Figure 3. One side of the cell is in close proximity to
the filter (behind which is the source), and, at right angles to this, the fluorescent radiation is observed by the monochromator. Monochromator. (L1,L2, S,, S2,Pr, and D). Of primary importance in the design cf a spectrofluorometer is the choice of an appropriate monochromator. Although there are many articles in the literature describing modifications of the Beckman Model DU spectrophotometer for spectrofluorometric purposes (7), there is little information on the utilization of less costly optical equipment for this purpose. The Beokrnan Model DU is completely equipped with quartz optics in order to fulfill its intended purpose of spectrophotometry in the ultraviolet region. However, much of the fluorescence of interest occurs in the visible region where conventional glass is superior to expensive quartz in spectral dispersion power. For this reason, and also because a quartz spectrophotometer is often fully occupied by its intended function, the instrument to be described uses an old spectroscope as a monochromator. The spectroscope employed was a Hilger of ancient vintage. Itpossesses a constant deviation prism which offers the advantage that the collimating tubes remain stationary during operation. Any unit that can serve as a monochromator could furnish a starting point for the construction of t,his type of instrument. If no such equipment is available, a cheap practical monochromator can be easily constructed. Either an inexpensive grating or an interference wedge can be utilized. Both of these devices offer the convenience of linear dispersion which is not possible in prism instruments. The application of the interference wedge to this type of instrument seems to be particularly appropriate in terms of cost, simplicity, and compactness. The reader is referred to the excellent article by H. W. Safford and D. F. Westneat (8) for details on the construction of such a unit. Slits, if needed, may be obtained commercially or improvised if the budget is limited. A simple but adequate expedient is to scratch a straight line with a razor blade on the hack of a silvered glass. VOLUME 34, NO. 12, DECEMBER, 1957
Detector (Ph). The choice of a suitable detect,ingunit is most critical in this type of instrument. Passage of the fluorescent radiation through a monochromator reduces its intensity manyfold. Thie is especially true if maximum resolution is desired, since small slit widths will then be employed. Very intense U. V. excitation could be employed to increase the signal strength, but this expedient should be avoided because it promotes photochemical decomposition. For this reason, an unusually sensitive detector is needed and this requires the incorporation of a photomultiplier tube. The instrument descrihed incorporates a commercial photomultiplier microphotometer (American Instrument Company, Cat. No. 10-210) in a detachable-manner so that its use is not restricted to this particular application. In all likelihood a homemade detection device would also serve. For example, one can supply high voltage (600-1000 v.) to a photomultiplier tube by means of batteries. The output of the detector tube is then coupled to a meter or recorder via a cathode follower. In this respect it would seem as if one of the popular vacuum tube voltmeters now on the market in kit form (9) would function adequat.ely for this purpose. Furthermore, it would afford the advantage of a direct reading meter. In the present instrument the output of the detector circuit is coupled to a Varian G-10, 10-millivolt recorder. The paper drive of the recorder a180 drives the wave length scroll D of the monochromator, coupling being achieved by means of a string drive. Since the wave length scale will not be linear with the recorder paper (unless a grating or wedge monochromator is used), the following technique is used to assign wave lengths. The input to the recorder is also connected to a microswitch, which, when pressed, shorts the input and registers a pip superimposed on the spectrum. A few such markers on both extremes of the spectral range permit a previously established wave length scale to be copied or pasted on the spectrum. USE OF THE INSTRUMENT
The spectrofluorometer described has been in use for six months, and has given good performance. It is used for qualitative purposes, and its application to qnantitative evaluations has not been tried. Figure 4 shows
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Wave lrnsth (. "1 in Chloroform Figure 4
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the ability of the instrument to differentiate etioporphyrin from protoporphyrin. These two compounds are both degradation products of hemin, the prosthetic group of hemoglobin, and differ only in the fact that etioporphyrin has no carhoxyl groups. Ordinary ahsorption spectrophotometric procedures fail to distinguish between these closely related compounds, and the usual recourse is to make use of paper chromatography. The spectrofluorometer, along with most of the instruments of the department, serves a twofold purpose: for research and for use in the instrumental analysis course. In the latter case, unknowns may be provided which the student must identify by means of the characteristic fluoreecence spectrum. (A list is provided giving the principal fluorescence peaks of a selected group of substances.) Alternately, a more elaborate problem may be assigned, such as letting the student first separate the pigments of leaves by adsorption chromatography (10) and then permitting him to further identify the fractions via their fluorescence spectra. Work has been done using the instrument manually, and although point by point plots are not as convenient as automatic recording, they can be made rapidly and accurately. With this instrument there is always some decomposition of the sample due to the high energy excitation, and also the sample is heated by being near the excitation source. These two problems are minimized by turning on the source only while a run is in progress and by using a rapid scanning rate. In the short wave length region, there is some scattered light, but this appears as sharp mercury emission lines, and instead of being a disadvantage fulfills the useful function of calibration points. The base line is perfectly flat when there is no fluorescent or scattered light emission. Perhaps the greatest asset of the general technique of spectrofluorometry is that recording instruments can be built to cost a fraction of the price of recording spectrophotometers. This is largely due to the fact that this "single beam" instrument examines light emi~sion rather than absorbed radiation and therefore does not involve compensation for solvent effects. (A solvent can usually be utilized which is nonfluorescent.) Also,
no complex electronic circuitry is needed to provide for a flat base line as in the case of absorption spectrophotometry. Simple d.-c. amplification of the output of a photomultiplier tube (1P28 is recommended) has given satisfactory results. Although a major disadvantage of this instrument is that it can only be used to examine a select group of compounds, the incorporation of an atomizer burner accessory is expected to extend its function to that of a recording flame photometer. In concluding this article the authors would like to emphasize that the primary purpose of reporting this student project is not to permit duplication in detail of the instrument described, since i t is highly unlikely that identical starting materials would he available t o other groups. The principal objective of this articleis to encourage the construction of equipment by students themselves preferably by the employment of unitized components which are flexible enough to permit the assembly of a variety of instruments. Each component such as a monochromator or a photomultiplier photometer could have been constructed originally as a student project, or purchased. In this way, otherwise nnobtainable equipment may he provided, new instruments may be developed, and, most important, it is felt that the students themselves will acquire a sound practical background. LITERATURE CITED (1) BOWMAN, R. L.: P. A. C A U L ~ E LS. D ,UDENPRIEND, Science, 122, 32 (1955). (2) LAITINEN, H. A., J. CHEM.EDUC.,33, 422 (1956). (3) PRINOSHEIM, P.. "Fluorescence and Phosphorescence," Interscience Publishers, Ino., New York, 1949, pp. 1-9. (4) DELAHAY, P., "Instrumental Analysis," The Maemillan Co., New York, 1957, pp. 24144. (5) KOLLER,L. R., "Ultraviolet Radiation," John Wiley & Sons, Ine., New York, 1952, pp. 32-76, (6) . . Ultra Violet Products Ino., 145 Pasadena. Ave., South Pasadens, California. iii See for examole MCANALLY. J. S.. A n d . Chem.. 26. 1526 1954). PFORD. H. W.. AND D . F. WESTNEAT. J. CHEM.EDUC.. (8) SAI 30, 343 (1953): (9) Heath Company, Benton Harbor, Mieh., Heathkit Model ~
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(10) I)INIELS, F., J. H. MATHEWG, J. W. WILLIAMS, P. BENDER.
G. W. MURPHY, R. A. ALBERTY, "Experimental Physical Chemistry," 4th ed., MeGraw-Hill Book Co., Inc., Yew York, 1949, pp. 262-66.
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