Parts of this paper were presented at the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pennsylvania on March 4, 1960.
J. M. Price, M. Kaihara, and H. K. Howerton, "Influence of Scattering on Fluorescence Spectra of Dilute Solutions Obtained with the Aminco–Bowman Spectrophotofluorometer," Appl. Opt. 1, 521-533 (1962)
Various factors affecting the intensity of scattered radiation appearing in fluorescence and excitation spectra of molecules in dilute solution obtained with the Aminco–Bowman Spectrophotofluorometer have been investigated. The spectra can be modified and in some cases improved by the addition of colored glass filters, interference filters, and polarizing elements to the optical system. The polarizing elements permit the measurement of fluorescence polarization, and results are given for the polarization of fluorescence of some xanthydrol derivatives in glycerol at room temperature. The effects of strong sample absorption and highly intense scattered radiation on observed fluorescence spectra and quantitative assays are shown.
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Concentrations were 3.6 × 10−6M for rhodamine B, 3.4 × 10−6M for rhodamine 6G, 4.5 × 10−6M for eosin, and 5.0 × 10−6M for fluorescein. An XBO-162 lamp, 10 × 10 mm cell, and 1P21 photomultiplier tube were used. The slit arrangement was 1—4 mm, 2—polarizer with EA, 3—1 mm, 4—1 mm, 5—polarizer with EF and BF alternately, 6—4 mm, and 7—1 mm.
Reported by Weber10 at 18°C with concentrations of 3.63 × 10−6M for rhodamine B, 3.4 × 10−6M for rhodamine 6G, 2.6 × 10−6M for eosin, and 4.94 × 10−6M for fluorescein.
Table IV
Depolarization Factors of Scattering from Water, Ethyl Alcohol, Chloroform, and Cyclohexanea
Substance
Depolarization factor, ρn
Rayleigh and Tyndall
Raman
Water
0.053
0.23
Ethyl alcohol
0.060
0.23
Chloroform
0.18
0.28
Cyclohexane
0.044
0.31
The measurements were made at 296°K using a 10 × 10 mm cell, XBO-162 lamp, and a 1P21 photomultiplier tube. The excitation wavelength was set at 345 mμ except the chloroform where it was set at 300 mμ. The slit arrangement was 1—none, 2—1 mm, 3—2 mm, 4—2 mm, 5—polarizer with EF and BF alternately, 6—1 mm, and 7—1 mm. The accuracy is no better than 25%.
Table V
Stray Light (%) from Excitation Monochromator with Slit Arrangement No. 2 and XBO-162 Lamp
Excitation wavelength (mμ)
Wavelength (mμ)
300
350
400
450
500
550
600
650
210
0.15
1.0
0.3
0.2
1.5
1.0
1.3
0
220
0.07
0.3
0.07
0.09
0.4
0.15
0.05
0
230
0
0.08
0.02
—
0.1
0.03
0
0
240
0.02
0.03
0.02
0.02
0.01
0.01
0.01
0
250
0.05
0.03
0.02
0.02
0.01
0.01
0.01
0
260
0.08
0.03
0.02
0.008
0.02
0.009
0
0
270
0.08
0.02
0.01
0.01
0.01
—
0.02
0.008
280
0.1
0.05
0.01
0.02
0.007
—
0.03
0.05
290
—
0.03
0.007
0.007
0.005
0.007
—
0
350
0
—
0.02
0.01
0.005
0.01
0
0
450
0
0
0.02
—
0.02
0
0
0
Table VI
Relative Intensity of Scattered Radiation from Ludox in Distilled Water
Concentrations were 3.6 × 10−6M for rhodamine B, 3.4 × 10−6M for rhodamine 6G, 4.5 × 10−6M for eosin, and 5.0 × 10−6M for fluorescein. An XBO-162 lamp, 10 × 10 mm cell, and 1P21 photomultiplier tube were used. The slit arrangement was 1—4 mm, 2—polarizer with EA, 3—1 mm, 4—1 mm, 5—polarizer with EF and BF alternately, 6—4 mm, and 7—1 mm.
Reported by Weber10 at 18°C with concentrations of 3.63 × 10−6M for rhodamine B, 3.4 × 10−6M for rhodamine 6G, 2.6 × 10−6M for eosin, and 4.94 × 10−6M for fluorescein.
Table IV
Depolarization Factors of Scattering from Water, Ethyl Alcohol, Chloroform, and Cyclohexanea
Substance
Depolarization factor, ρn
Rayleigh and Tyndall
Raman
Water
0.053
0.23
Ethyl alcohol
0.060
0.23
Chloroform
0.18
0.28
Cyclohexane
0.044
0.31
The measurements were made at 296°K using a 10 × 10 mm cell, XBO-162 lamp, and a 1P21 photomultiplier tube. The excitation wavelength was set at 345 mμ except the chloroform where it was set at 300 mμ. The slit arrangement was 1—none, 2—1 mm, 3—2 mm, 4—2 mm, 5—polarizer with EF and BF alternately, 6—1 mm, and 7—1 mm. The accuracy is no better than 25%.
Table V
Stray Light (%) from Excitation Monochromator with Slit Arrangement No. 2 and XBO-162 Lamp
Excitation wavelength (mμ)
Wavelength (mμ)
300
350
400
450
500
550
600
650
210
0.15
1.0
0.3
0.2
1.5
1.0
1.3
0
220
0.07
0.3
0.07
0.09
0.4
0.15
0.05
0
230
0
0.08
0.02
—
0.1
0.03
0
0
240
0.02
0.03
0.02
0.02
0.01
0.01
0.01
0
250
0.05
0.03
0.02
0.02
0.01
0.01
0.01
0
260
0.08
0.03
0.02
0.008
0.02
0.009
0
0
270
0.08
0.02
0.01
0.01
0.01
—
0.02
0.008
280
0.1
0.05
0.01
0.02
0.007
—
0.03
0.05
290
—
0.03
0.007
0.007
0.005
0.007
—
0
350
0
—
0.02
0.01
0.005
0.01
0
0
450
0
0
0.02
—
0.02
0
0
0
Table VI
Relative Intensity of Scattered Radiation from Ludox in Distilled Water