Abstract

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.

© 1962 Optical Society of America

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References

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  1. H. K. Howerton, ISA J. 6, 50 (1959).
  2. H. K. Howerton (to American Instrument Co., Inc.), U.S. Patent2,971,429 (Feb.14, 1961).
  3. R. L. Bowman, P. A. Caulfield, S. Udenfriend, Science 122, 32 (1955).
    [CrossRef] [PubMed]
  4. D. E. Duggan, R. L. Bowman, B. B. Brodie, S. Udenfriend, Arch. Biochem. Biophys. 68, 1 (1957).
    [CrossRef] [PubMed]
  5. K. Satoh, J. M. Price, J. Biol. Chem. 230, 781 (1958).
    [PubMed]
  6. C. A. Parker, Analyst 84, 446 (1959).
    [CrossRef]
  7. S. F. Velick, J. Biol. Chem. 233, 1455 (1958).
  8. S. F. Velick, “Spectra and Structure in Enzyme Complexes of Pyridine and Flavin Nucleotides,” in Light and Life edited by W. D. McElroy, H. Bentley Glass (Johns Hopkins Press, Baltimore, Md., 1961).
  9. D. H. Rank, Anal. Chem. 19, 766 (1947).
    [CrossRef]
  10. G. Weber, J. Opt. Soc. Am. 46, 962 (1956).
    [CrossRef]
  11. D. H. Rank, R. J. Pfister, H. H. Grimm, J. Opt. Soc. Am. 33, 31 (1943).
    [CrossRef]
  12. K. Yagi, Z. Yoshida, T. Tabata, Fluorescence (Nankodo, Tokyo, Japan, 1958), Chap. 6, p. 87. (Translated by M. Kaihara.)
  13. R. E. Kemp, Rev. Sci. Instr. 26, 1120 (1955).
    [CrossRef]
  14. R. R. Brown, J. M. Price, J. Biol. Chem. 219, 985 (1956).
    [PubMed]
  15. J. M. Price, L. W. Dodge, J. Biol. Chem. 223, 699 (1956).
    [PubMed]
  16. G. Herzberg, Molecular Spectra and Molecular Structure, II. Infrared and Raman Spectra of Polyatomic Molecules (Van Nostrand, New York, 1959).
  17. C. E. White, M. Ho, E. Q. Weimer, Anal. Chem. 32, 438 (1960).
    [CrossRef]
  18. E. J. Bowen, F. Wokes, Fluorescence of Solutions (Longmans, Green, New York, 1953), p. 20.

1960

C. E. White, M. Ho, E. Q. Weimer, Anal. Chem. 32, 438 (1960).
[CrossRef]

1959

H. K. Howerton, ISA J. 6, 50 (1959).

C. A. Parker, Analyst 84, 446 (1959).
[CrossRef]

1958

S. F. Velick, J. Biol. Chem. 233, 1455 (1958).

K. Satoh, J. M. Price, J. Biol. Chem. 230, 781 (1958).
[PubMed]

1957

D. E. Duggan, R. L. Bowman, B. B. Brodie, S. Udenfriend, Arch. Biochem. Biophys. 68, 1 (1957).
[CrossRef] [PubMed]

1956

G. Weber, J. Opt. Soc. Am. 46, 962 (1956).
[CrossRef]

R. R. Brown, J. M. Price, J. Biol. Chem. 219, 985 (1956).
[PubMed]

J. M. Price, L. W. Dodge, J. Biol. Chem. 223, 699 (1956).
[PubMed]

1955

R. E. Kemp, Rev. Sci. Instr. 26, 1120 (1955).
[CrossRef]

R. L. Bowman, P. A. Caulfield, S. Udenfriend, Science 122, 32 (1955).
[CrossRef] [PubMed]

1947

D. H. Rank, Anal. Chem. 19, 766 (1947).
[CrossRef]

1943

Bowen, E. J.

E. J. Bowen, F. Wokes, Fluorescence of Solutions (Longmans, Green, New York, 1953), p. 20.

Bowman, R. L.

D. E. Duggan, R. L. Bowman, B. B. Brodie, S. Udenfriend, Arch. Biochem. Biophys. 68, 1 (1957).
[CrossRef] [PubMed]

R. L. Bowman, P. A. Caulfield, S. Udenfriend, Science 122, 32 (1955).
[CrossRef] [PubMed]

Brodie, B. B.

D. E. Duggan, R. L. Bowman, B. B. Brodie, S. Udenfriend, Arch. Biochem. Biophys. 68, 1 (1957).
[CrossRef] [PubMed]

Brown, R. R.

R. R. Brown, J. M. Price, J. Biol. Chem. 219, 985 (1956).
[PubMed]

Caulfield, P. A.

R. L. Bowman, P. A. Caulfield, S. Udenfriend, Science 122, 32 (1955).
[CrossRef] [PubMed]

Dodge, L. W.

J. M. Price, L. W. Dodge, J. Biol. Chem. 223, 699 (1956).
[PubMed]

Duggan, D. E.

D. E. Duggan, R. L. Bowman, B. B. Brodie, S. Udenfriend, Arch. Biochem. Biophys. 68, 1 (1957).
[CrossRef] [PubMed]

Grimm, H. H.

Herzberg, G.

G. Herzberg, Molecular Spectra and Molecular Structure, II. Infrared and Raman Spectra of Polyatomic Molecules (Van Nostrand, New York, 1959).

Ho, M.

C. E. White, M. Ho, E. Q. Weimer, Anal. Chem. 32, 438 (1960).
[CrossRef]

Howerton, H. K.

H. K. Howerton, ISA J. 6, 50 (1959).

H. K. Howerton (to American Instrument Co., Inc.), U.S. Patent2,971,429 (Feb.14, 1961).

Kemp, R. E.

R. E. Kemp, Rev. Sci. Instr. 26, 1120 (1955).
[CrossRef]

Parker, C. A.

C. A. Parker, Analyst 84, 446 (1959).
[CrossRef]

Pfister, R. J.

Price, J. M.

K. Satoh, J. M. Price, J. Biol. Chem. 230, 781 (1958).
[PubMed]

R. R. Brown, J. M. Price, J. Biol. Chem. 219, 985 (1956).
[PubMed]

J. M. Price, L. W. Dodge, J. Biol. Chem. 223, 699 (1956).
[PubMed]

Rank, D. H.

Satoh, K.

K. Satoh, J. M. Price, J. Biol. Chem. 230, 781 (1958).
[PubMed]

Tabata, T.

K. Yagi, Z. Yoshida, T. Tabata, Fluorescence (Nankodo, Tokyo, Japan, 1958), Chap. 6, p. 87. (Translated by M. Kaihara.)

Udenfriend, S.

D. E. Duggan, R. L. Bowman, B. B. Brodie, S. Udenfriend, Arch. Biochem. Biophys. 68, 1 (1957).
[CrossRef] [PubMed]

R. L. Bowman, P. A. Caulfield, S. Udenfriend, Science 122, 32 (1955).
[CrossRef] [PubMed]

Velick, S. F.

S. F. Velick, J. Biol. Chem. 233, 1455 (1958).

S. F. Velick, “Spectra and Structure in Enzyme Complexes of Pyridine and Flavin Nucleotides,” in Light and Life edited by W. D. McElroy, H. Bentley Glass (Johns Hopkins Press, Baltimore, Md., 1961).

Weber, G.

Weimer, E. Q.

C. E. White, M. Ho, E. Q. Weimer, Anal. Chem. 32, 438 (1960).
[CrossRef]

White, C. E.

C. E. White, M. Ho, E. Q. Weimer, Anal. Chem. 32, 438 (1960).
[CrossRef]

Wokes, F.

E. J. Bowen, F. Wokes, Fluorescence of Solutions (Longmans, Green, New York, 1953), p. 20.

Yagi, K.

K. Yagi, Z. Yoshida, T. Tabata, Fluorescence (Nankodo, Tokyo, Japan, 1958), Chap. 6, p. 87. (Translated by M. Kaihara.)

Yoshida, Z.

K. Yagi, Z. Yoshida, T. Tabata, Fluorescence (Nankodo, Tokyo, Japan, 1958), Chap. 6, p. 87. (Translated by M. Kaihara.)

Anal. Chem.

D. H. Rank, Anal. Chem. 19, 766 (1947).
[CrossRef]

C. E. White, M. Ho, E. Q. Weimer, Anal. Chem. 32, 438 (1960).
[CrossRef]

Analyst

C. A. Parker, Analyst 84, 446 (1959).
[CrossRef]

Arch. Biochem. Biophys.

D. E. Duggan, R. L. Bowman, B. B. Brodie, S. Udenfriend, Arch. Biochem. Biophys. 68, 1 (1957).
[CrossRef] [PubMed]

ISA J.

H. K. Howerton, ISA J. 6, 50 (1959).

J. Biol. Chem.

K. Satoh, J. M. Price, J. Biol. Chem. 230, 781 (1958).
[PubMed]

S. F. Velick, J. Biol. Chem. 233, 1455 (1958).

R. R. Brown, J. M. Price, J. Biol. Chem. 219, 985 (1956).
[PubMed]

J. M. Price, L. W. Dodge, J. Biol. Chem. 223, 699 (1956).
[PubMed]

J. Opt. Soc. Am.

Rev. Sci. Instr.

R. E. Kemp, Rev. Sci. Instr. 26, 1120 (1955).
[CrossRef]

Science

R. L. Bowman, P. A. Caulfield, S. Udenfriend, Science 122, 32 (1955).
[CrossRef] [PubMed]

Other

S. F. Velick, “Spectra and Structure in Enzyme Complexes of Pyridine and Flavin Nucleotides,” in Light and Life edited by W. D. McElroy, H. Bentley Glass (Johns Hopkins Press, Baltimore, Md., 1961).

G. Herzberg, Molecular Spectra and Molecular Structure, II. Infrared and Raman Spectra of Polyatomic Molecules (Van Nostrand, New York, 1959).

E. J. Bowen, F. Wokes, Fluorescence of Solutions (Longmans, Green, New York, 1953), p. 20.

K. Yagi, Z. Yoshida, T. Tabata, Fluorescence (Nankodo, Tokyo, Japan, 1958), Chap. 6, p. 87. (Translated by M. Kaihara.)

H. K. Howerton (to American Instrument Co., Inc.), U.S. Patent2,971,429 (Feb.14, 1961).

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Figures (13)

Fig. 1
Fig. 1

Optical diagram.

Fig. 2
Fig. 2

Location of polarizers in cell compartment.

Fig. 3
Fig. 3

Polarizer assembly.

Fig. 4
Fig. 4

Location of filters in cell compartment.

Fig. 5
Fig. 5

Camera arrangement for photographing spectra on the oscillograph. The camera is an inexpensive single lens reflex type and is attached to the oscillograph with a light-tight cardboard tube. The camera support is attached to the oscillograph with hinges to facilitate rapid positioning of the unit when a photograph record of the observed fluorescence spectrum is desired.

Fig. 6
Fig. 6

Fluorescence spectra of quinine sulfate (0.0375 μg/ml) in 0.1 N sulfuric acid at room temperature. The 1P21 photomultiplier tube was operated with a 975 volt battery and slit arrangement No. 4 (Table I) was used. To obtain the curve with the excitation monochromator set at 310 mμ the oscillograph Y axis was set at 1.0 dc and the multiplier at 4. For the fluorescence spectrum with the excitation monochromator set at 620 mμ the oscillograph Y axis was set at 0.1 dc and the multiplier at 1.0. It should be noted that the curves have identical characteristics with peaks representing from left to right, Rayleigh and Tyndall scattering, Raman scattering, fluorescence, and scattered radiation in the second order of diffraction.

Fig. 7
Fig. 7

Scattering from distilled water —1, ethyl alcohol —2, chloroform —3, and cyclohexane —4 at room temperature. The peaks marked (C) are due to Rayleigh and Tyndall scattering and those marked (I) are due to Raman scattering. A 1P21 photomultiplier tube and an XBO-162 lamp were used with the excitation wavelength set at 310 mμ. Slit arrangement was 1—4 mm, 2—1 mm, 3—2 mm, 4—2 mm, 5—1 mm, 6—4 mm, and 7—1 mm. The meter multiplier and sensitivity settings were respectively 0.003 and 22 for water, 0.01 and 43 for ethyl alcohol, 0.01 and 19 for chloroform, and 0.01 and 43 for cyclohexane.

Fig. 8
Fig. 8

Rayleigh and Tyndall scattering and Raman scattering from water (upper curve), deuterium oxide (middle curve), and an equal mixture of the two (lower curve) at room temperature. The excitation wavelength was 410 mμ (24,390 cm−1). The Raman scattering due to the Stokes Raman band of water in polymeric association was expected at 24,390 cm−1–3300 cm−1, or 21,090 cm−1, and the observed values were 21,322 cm−1 (upper curve) and 21,276 cm−1 (lower curve). For deuterium oxide, the Raman scattering for the Stokes Raman frequency was expected at 24,390 cm−1–2300 cm−1, or 22,090 cm−1, and the observed values were 22,173 cm−1 (middle curve) and 22,075 cm−1 (lower curve). As has been emphasized in the text, the exact frequency of the Stokes Raman band cannot be accurately measured under these conditions, but the values observed may make it possible to identify quickly extraneous peaks in fluorescence spectra as those likely to be this type of scattering and not additional fluorescence peaks. These records were obtained using the XBO-162 Xenon lamp and the 1P21 photomultiplier tube with a 975 volt battery. Slit arrangement No. 3 (Table I) was used with the oscillograph Y-axis set at 0.1 dc and the multiplier set at 1.0.

Fig. 9
Fig. 9

The effects of certain filters on the fluorescence spectra of quinine sulfate (0.0375 μg/ml) in 0.1 N sulfuric acid and xanthurenic acid (0.165 μg/ml) in 9.5 N sodium hydroxide. Each of the six records has one curve which was photographed with the filter and one curve which was recorded without the filter. When the scattering is small (quinine sulfate) it is readily removed by any of the filters used. When the scattering is very large (xanthurenic acid) it is not possible to remove it adequately with the polarizers, but the Corning filter No. 3-72 is very satisfactory. The relatively low transmission of the interference filters renders their use unsatisfactory. These records were made with the 1P21 photomultiplier tube with a 975 volt battery. The oscillograph Y axis was set at 0.1 dc and the multiplier was set at 6 for quinine sulfate and at 10 for xanthurenic acid. Slit arrangement No. 3 (Table I) was used for quinine sulfate and No. 4 was used for xanthurenic acid.

Fig. 10
Fig. 10

Excitation spectrum (peaks at 263, 395, and 500 mμ) and emission spectrum (peaks at 320 and 395 mμ) of fused quartz at room temperature. The records were made with the XBO-162 lamp and the 1P21 photomultiplier tube with a 975 volt battery. Slit arrangement No. 3 was used with the oscillograph Y-axis set at 0.1 and the multiplier at 4.2. The excitation wavelength was set at 250 mμ for the fluorescence spectrum and the fluorescence wavelength was set at 395 mμ for the excitation spectrum.

Fig. 11
Fig. 11

Work curve and fluorescence spectra of quinine sulfate in 0.1 N sulfuric acid at room temperature. The concentration of quinine sulfate for each spectrum is indicated by the vertical arrows. The work curves and fluorescence spectra were obtained under three conditions: (1) with no filters; (2) with a Corning 3-74 glass filter in position “F” (see Fig. 4); and (3) with the polarizing filters set with the electric vector vertical at position A and the electric vector horizontal at position F (see Fig. 2). The three horizontal arrows at the left of the work curves indicate the relative intensity of the blanks for the three curves. The fluorescence spectra reveal that both Raman and Rayleigh or Tyndall scattering were present at the lowest concentrations of quinine sulfate. Raman scattering should be suspected as the cause of the second peak of these fluorescence spectra because it occurs at 25,300 cm−1 (395 mμ) which is shifted 3300 cm−1 from the excitation (28,600 cm−1 or 350 mμ), and thus corresponds to the strong Stokes Raman band for —OH stretch of intermolecular hydrogen bonds in water in polymeric association. The removal of this peak by the polarizing filters is further evidence that this peak represents Raman scattering. The work curve was obtained with the excitation wavelength set at 350 mμ and the fluorescence wavelength set at 450 mμ The meter multiplier settings were as shown below the fluorescence spectra. Other instrumental parameters were: lamp, XBO-162; photomultiplier tube, 1P21; slit arrangement No. 3; sensitivity, minimum; oscillograph Y axis 1 dc; oscillograph multiplier 1.0 except for a concentration of 10 μg/ml where it was set at 2 and for a concentration of 100 μg/ml where it was set at 4. (See Figs. 6, 8, 9, 10, for λ and mμ numbering.)

Fig. 12
Fig. 12

Work curve and fluorescence spectra of kynurenic acid in 14.4 N sulfuric acid at room temperature. The concentration of kynurenic acid for each spectrum is indicated by the vertical arrows. The work curves and fluorescence spectra were obtained under three conditions: (1) with no filter; (2) with a Corning 0-51 glass filter in position F; and (3) with the polarizing filters set with the electric vector vertical at position A and the electric vector horizontal at position F. The horizontal arrows at the left of the work curves indicate the relative intensity of the blanks for the three curves. The excitation wavelength was set at 340 mμ and the fluorescence wavelength was set at 435 mμ for the work curve. The meter multiplier settings were as shown below the fluorescence spectra. Other instrumental parameters were: lamp, XBO-162; photomultiplier tube, 1P21; slit arrangement No. 3; sensitivity, minimum; oscillograph Y axis 1 dc; oscillograph multiplier 1.0. (See Figs. 6, 8, 9, and 10 for λ and mμ numbering.)

Fig. 13
Fig. 13

Work curve and fluorescence spectra of xanthurenic acid in 9.5 N sodium hydroxide at room temperature. The concentration of xanthurenic acid for each spectrum is indicated by the vertical arrows. The work curves and fluorescence spectra were obtained under three conditions: (1) with no filter; (2) with a Corning 3-71 glass filter in position F; and (3) with the polarizing filters set with the electric vector vertical at position A and the electric vector horizontal at position F. The horizontal arrows at the left of the work curves indicate the relative intensity of the blanks for the three curves. The excitation wavelength was set at 370 mμ, and the fluorescence wavelength was set at 530 mμ for the work curve. The meter multiplier setting were as shown below the fluorescence spectra. Other instrumental parameters were: lamp, XBO-162; photomultiplier tube, 1P21; slit arrangement No. 4; sensitivity, minimum; oscillograph Y axis 1 dc; oscillograph multiplier 1.0 (See Figs. 6, 8, 9, 10 for λ, mμ numbering.)

Tables (7)

Tables Icon

Table I Slit Arrangements

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Table II Position and Placement of Polarizers and Filters

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Table III Polarization of Fluorescence of some Xanthydro Derivatives in Glycerol at 296°Ka

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Table IV Depolarization Factors of Scattering from Water, Ethyl Alcohol, Chloroform, and Cyclohexanea

Tables Icon

Table V Stray Light (%) from Excitation Monochromator with Slit Arrangement No. 2 and XBO-162 Lamp

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Table VI Relative Intensity of Scattered Radiation from Ludox in Distilled Water

Tables Icon

Table VII Identification of Peaks of Scattered Radiation Appearing in Spectra of Figs. 11, 12, and 13

Equations (6)

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p = I I I + I .
T = S 0 P 0 [ unpolarized ]
I I = R E T R B
p = R E T R B R E + T R B
ρ n = I I = T R B R E
ρ l = ρ n 2 ρ n = T R B 2 R E T R B

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