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The phenomenon of Fluorescence has two main areas of application in practical chemistry and those are mostly involved with analysis. Fluorescence can permit the measurement of a substance that uniquely has fluorescent properties when mixed with a large number of other compounds that do not fluoresce. In addition because Fluorescence can provide a relatively strong signal against a very low noise background, the technique can be easily used to detect substances at the 10-8- 10-9g/ml concentration level. In a10 μl sample cell this would be equivalent to a mass of 10-10 - 10-11 g (i.e.,.10-100 pg). Such sensitivities are readily obtainable and are nowhere near the lower limit of measurement.
Unfortunately relatively few substances fluoresce and so a number of reagents have been developed to provide fluorescent derivatives that can improve the sensitivity of the analysis. Such reagents are also employed in liquid chromatography to improve levels of detection. Examples of some effective fluorescent reagents are as follows.
One of the most popular fluorescent reagents is 5-dimethyl aminonaphthalene-1-sulphonyl chloride (dansyl chloride, DNS-chloride or DNS-Cl). dansyl chloride reacts with phenols and primary and secondary amines under slightly basic conditions to form a fluorescent suphonate ester or suphonamide. The quantum efficiency of dansyl derivatives is high; whereas the reagent itself does not fluoresce. Unfortunately, the hydrolysis product, dansyl ic acid, is strongly fluorescent and causes interference with water-soluble derivatives. The derivatives, however can often be removed by a subsequent chromatographic process. The detection limits of the dansyl derivatives are often in the low nanogram range (ca>1 x10-9 g/ml) and the excitation and emission maxima can vary between 350-370 nm for excitation and 490-540 nm for emission. This reagent has been used successfully in the analysis of amino acids, alkaloids , barbiturates and pesticides .
4-Chloro-7-nitrobenz-2,1,3-oxadiazole (NBD chloride) reacts with aliphatic primary and secondary amines to form highly fluorescent derivatives. Aromatic amines, phenols and thiols yield weakly or non-fluorescent derivatives; consequently, the reagent is specific for aliphatic amines. The reaction is carried out under basic conditions and the products are extractable from aqueous mixtures by solvents such as benzene or ethyl acetate. The Fluorescence can be significantly reduced by the presence of water and so the solution should be dry. Detection limits are in the fraction of a nanogram range ( 2-5x 10-10g/ml). The advantage of this reagent over dansyl chloride is that both the reagent and its hydrolysis products are not fluorescent.The excitation and emission wavelengths are also higher (480 nm excitation and 530 nm emission). NBD chloride derivatives have been used for the analysis of amino acids, amphetamines , alkaloids and nitrosamines.
Fluorescamine (4-phenylspiro(furan-2-(3H),1'-phthalan)3,3'-dione) is also a commonly used Fluorescence reagent. It reacts almost instantly and selectively with primary amines, while the excess of the reagent is hydrolyzed to a non-fluorescent product. The reagent itself is non-fluorescent. The reaction is carried out in aqueous acetone at a pH of about 8-9. The excitation and emission wavelengths are 390 nm and 475 nm respectively. Two disadvantages of the reagent are its cost and unfortunately the products are less stable, cannot be stored and should be used for analysis immediately after formation. Fluorescamine has been employed in the analysis of polyamines , catecholamines and amino acids.
A less costly alternative to Fluorescamine is o-phthaldehyde (OPT), the derivatives of which are more stable and consequently can be stored overnight if necessary. It is used in a similar manner to fluorescamine the detection limits being about 0.1 ng (ca 4 x 10-10g/ml). OPT has been used in the analysis of dopamine, catecholamines and histamines. Other Fluorescence Reagents that are sometimes used include 4-bromoethyl-7-methoxycoumarin, diphenylindene , sulphonyl chloride, dansyl -hydrazine and a number of fluorescent isocyanates .
For further information on derivatizing reagents the reader is strongly recommended to refer to the Handbook of Derivatives for chromatography edited by Blau and Halket .
About the Author
RAYMOND PETER WILLIAM SCOTT was born on June 20 1924 in Erith, Kent, UK. He studied at the University of London, obtaining his B.Sc. degree in 1946 and his D.Sc. degree in 1960. After spending more than a decade at Benzole Producers, Ltd. Where he became head of the Physical Chemistry Laboratory, he moved to Unilever Research Laboratories as Manager of their Physical Chemistry department. In 1969 he became Director of Physical Chemistry at Hoffmann-La Roche, Nutley, NJ, U.S.A. and subsequently accepted the position of Director of the Applied Research Department at the Perkin-Elmer Corporation, Norwalk, CT, U.S.A.
In 1986 he became an independent consultant and was appointed Visiting Professor at Georgetown
University, Washington, DC, U.S.A. and at Berkbeck College of the University of London; in 1986 he retired but continues to write technical books dealing with various aspects of physical chemistry and physical chemical techniques. Dr. Scott has authored or co-authored over 200 peer reviewed scientific papers and authored, co-authored or edited over thirty books on various aspects of physical and analytical chemistry. Dr. Scott was a founding member of the British chromatography Society and received the American Chemical society Award in chromatography (1977), the M. S. Tswett chromatography Medal (1978), the Tswett chromatography Medal U.S.S.R., (1979), the A. J. P. Martin chromatography Award (1982) and the Royal Society of Chemistry Award in Analysis and Instrumentation (1988).
Dr. Scott’s activities in gas chromatography started at the inception of the technique, inventing the Heat of Combustion Detector (the precursor of the Flame Ionization Detector), pioneered work on high sensitivity detectors, high efficiency columns and presented fundamental treatments of the relationship between the theory and practice of the technique. He established the viability of the moving bed continuous preparative gas chromatography, examined both theoretically and experimentally those factors that controlled dispersion in packed beds and helped establish the gas chromatograph as a process monitoring instrument. Dr. Scott took and active part in the renaissance of liquid chromatography, was involved in the development of high performance liquid chromatography and invented the wire transport detector. He invented the liquid chromatography mass spectrometry transport interface, introduced micro-bore liquid chromatography columns and used them to provide columns of 750,000 theoretical plates and liquid chromatography separations in less than a second. Dr. Scott has always been a “hands-on” scientist with a remarkable record of accomplishments in chromatography ranging from hardware design to the development of fundamental theory. He has never shied away from questioning “conventional wisdom” and his original approach to problems has often produced significant breakthroughs.