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Energy Mode = \ Single Beam \ Dynode Voltage = 700 v \ λΕχ = 448 nm V Ex Slit = 1 0 n m Em Slit = 1 0 n m — 100 μg/^ 0 0 ml Estrogen · — - - Reagent Blank 460
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Figure 6. Fluorescence emission spec trum of 100 μς/ΙΟΟ ml concentration of estrogen Standard, —; emission spectrum of blank,
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Figure 7. Double-beam fluorescence emission spectrum of estrogen control vs. reagent blank
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C O R C O
Double-Beam Mode DV = 700 V λΕχ = 448 nm Ex Slit = 10 nm Em Slit = 10 nm
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Reference = Reagent Blank Double-Beam Mode DV = 700 V λΕχ = 4 4 8 nm Ex Slit = 1 0 n m Em Slit = 1 0 n m
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Figure 8. Double-beam fluorescence emission spectrum of reagent blank vs. reagent blank for estrogen analysis
1106 A · ANALYTICAL CHEMISTRY, VOL. 4 8 , NO. 13, NOVEMBER
1976
analyzed by fluorescence, are an ex ample of this. T h e spectra in Figure 6 are of the estrogen standard whose emission peak is at about 560 nm and the reagent blank used in this proce dure. Observe t h a t the blank has com pletely distorted the spectrum of es trogen. Figure 7 shows a double-beam comparison of the estrogen sample and reagent blank reference. Figure 8 shows a similar comparison of the blank vs. the same blank. The doublebeam technique completely cancels out the blank and permits a lower level of estrogen to be detected rela tive to single-beam measurements. T h e use of double-beam fluores cence provides an extended linear range for some analyses. T r y p t o p h a n is one example where the linear range can be extended approximately one hundredfold from 10" 6 to 10~ 8 Μ by eliminating the solvent interference. Detecting tryptophan at these low concentrations is essential in research presently being done on cataracts, in t h a t there appears to be a correlation between the concentration of trypto phan and the degree of opalescence causing the cataract (6). Double-beam fluorescence is currently being used to determine the tryptophan content of soluble lens proteins. After the sam ples are hydrolyzed, the proteins are then diluted with urea buffer, and an initial fluorescence double-beam mea surement is recorded. A series of fluo rescence readings is taken with each additional aliquot of tryptophan. Fig ure 9 is a spectrum of such a sample with 2.1 nmol of tryptophan added to the original solution in 0.3-nmol ,\liquots showing the tryptophan emis sion peaking at 353 nm when excited at 280 nm. T h e buffer fluorescence at 368 nm is eliminated in the doublebeam measurement. Figure 10 is a plot of the data leading to t h a t shown in the previous figure by seven successive additions of tryptophan to the original hydrolyzed protein solution. Values were taken at the emission maximum when excited at 280 nm. T h e trypto p h a n content in the original solution can be determined by extrapolation to zero intensity. T h e double-beam measurement in this case completely canceled an inter fering background fluorescence per mitting greater sensitivity than pre viously measured using single-beam techniques (7). Summary We have endeavored in this paper to describe the relatively new tech nique of double-beam fluorescence spectrophotometry and its advan tages, disadvantages, and instrumen tation. We have shown applications where the technique provides greater
