Absorbance-Corrected Synchronous Fluorescence with a Fiber-Optic-Based Fluorometer

Abstract

Synchronous fluorescence spectroscopy is frequently used to resolve multiple components in mixtures without separation. However, uncompensated inner-filter effects can lead to unexpected and/or large quantitative errors. An instrument and a method that correct for primary and secondary inner-filter effects in synchronous fluorescence spectroscopy have been designed and characterized. A bifurcated fiber-optic-based diode array fluorometer that simultaneously measures front-surface fluorescence and absorbance was constructed. A transfer function that mathematically describes the coupling efficiency between the excitation and emission legs of the fiber optics is defined and characterized. Once characterized, such a fluorometer is capable of calculating what the fluorescence measurement would be in the absence of inner-filter effects. Several experimental procedures for determining the instrument's transfer function are compared and discussed. Applications of the various procedures to experimental fluorescence inner-filter corrections to problematic samples are demonstrated.

Keywords

Fluorescence Synchronous fluorescence Absorption-corrected fluorescence

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References

Vo-Dinh

, Anal. Chem. 50, 396 (1978).

Santana

Hernandez

and Bernal

, and Martin-Lazaro

, Analyst (London) 118, 917 (1993).

Abbot

D. W.

Moody

R. L.

and Mann

R. M

, and Vo-Dinh

, J. Am. Ind. Hyg. Assoc. 47, 379 (1986).

Nithipatikom

and McGown

L. B.

, Appl. Spectrosc. 41, 395 (1987).

Inman

E. L.

Jr. , and Winefordner

J. D.

, Anal. Chem. 54, 2018 (1982).

Lloyd

J. B. F.

, Analyst (London) 105, 97 (1980).

Spino

L. A.

and Armstrong

D. W

, and Vo-Dinh

, J. Chromatogr. 409, 147 (1987).

Kerkhoff

J. M.

and Winefordner

J. D.

, Anal. Chim. Acta 175, 257 (1985).

Kato

and Yokoyama

, and Sanada

, Fuel 59, 845 (1980).

10.

Lloyd

J. B. F.

, Anal. Chem. 52, 189 (1980).

11.

Siano

S. A.

, J. Quant. Spectrosc. Radiat. Transfer 47, 55 (1992).

12.

Plaza

Dao

N. Q.

Jouan

and Fevrier

, and Saisse

, Appl. Opt. 25, 3448 (1986).

13.

Vo-Dinh

, Appl. Spectrosc. 36, 576 (1982).

14.

Lloyd

J. B. F.

, Analyst 99, 729 (1974).

15.

Bowman

L. E.

and Crouch

S. R.

, Appl. Spectrosc. 46, 925 (1992).

16.

Latz

H. W.

and Ullman

A. H

, and Winefordner

J. D.

, Anal. Chem. 50, 2148 (1978).

17.

Tietz

N. W.

, Fundamentals of Clinical Chemistry (W. B. Saunders, Philadelphia, 1976).

18.

Kubista

Sjoback

and Eriksson

, and Albinsson

, Analyst (London) 119, 417 (1994).

19.

Puchalski

M. M.

and Morra

M. J

, and Von Wandruszka

, Fresenius' J. Anal. Chem. 340, 341 (1991).

20.

Yappert

C. M.

and Ingle

J. D.

Jr. , Appl. Spectrosc. 43, 759 (1989).

21.

Wiechelman

, Am. Lab. 18, 49 (1986).

22.

Lutz

H. P.

and Luisi

P. L.

, Helv. Chim. Acta 66, 1929 (1983).

23.

Ratzlaff

E. H.

and Harfmann

R. G

, and Crouch

S. R.

, Anal. Chem. 56, 342 (1984).

24.

Smith

R. M.

and Jackson

K. W

, and Aldous

K. M.

, Anal. Chem. 49, 2051 (1977).

25.

Dye

J. L.

and Nicely

V. A.

, J. Chem. Ed. 48, 443 (1971).

26.

Ingle

J. D.

Jr. , and Crouch

S. R.

, Spectrochemical Analysis (Prentice Hall, Engelwood Cliffs, New Jersey, 1988).

27.

Product Brochure MR-3 (Highlight Fiber Optics, Caldwell, Idaho, 1986).

28.

Zhu

Z. Y.

and Yappert

M. C.

, Appl. Opt. 46, 919 (1992).

29.

Komives

and Schultz

J. S.

, Talanta 39, 429 (1992).

30.

Zhu

Z. Y.

and Yappert

M. C.

, Appl. Opt. 46, 912 (1992).

31.

Deaton

Ph.D. Dissertation, University of California at Davis, Davis (1984).

32.

Miller

J. N.

, Standards in Fluorescence Spectrometry (Chapman and Hall, London/New York, 1981).