Organic Chemistry Applications of Fluorescence Spectroscopy*

Organic Chemistry Applications of Fluorescence Spectroscopy*

Organic Chemistry Applications of Fluorescence Spectroscopy Stephen G Schulman, Qiao Qing Di, and John Juchum, University of Florida, Gainesville, FL,...

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Organic Chemistry Applications of Fluorescence Spectroscopy Stephen G Schulman, Qiao Qing Di, and John Juchum, University of Florida, Gainesville, FL, USA & 1999 Elsevier Ltd. All rights reserved. This article is reproduced from the previous edition, volume 3, pp 1718–1725, & 1999, Elsevier Ltd.

Symbols c E h kex kf m

velocity of light energy Planck’s constant excitation wavelength fluorescent wavelength frequency

Introduction The fluorescence of organic molecules consists of the emission of light by molecules, which have previously absorbed visible or ultraviolet radiation. The measurement of fluorescence often permits very low analyte detection limits (1015–104 mol dm3) and is widely employed in quantitative analysis, especially as a detection and quantitation method in liquid chromatography. Most applications are derived from the relationship between analyte concentration and fluorescence intensity and are therefore similar in concept to other spectrochemical methods of analysis. However, as well as spectral intensity, other features of fluorescence spectral bands of organic molecules, such as position in the electromagnetic spectrum, emission lifetime and excitation spectrum are exquisitely sensitive to the molecular environment and to molecular structure and, therefore, are also analytically useful, especially for probing the environment of the fluorophore. This article will deal with the nature of organic molecular fluorescence, its dependence on molecular structure, reactivity and interactions with the environment and its utility in the trace analysis of organic compounds.

There is also an efficient radiationless pathway for the demotion of the excited molecule from higher to lower electronically excited states called internal conversion. In aliphatic molecules which have a high degree of vibrational freedom, vibrational relaxation and internal conversion may return the excited molecule to the ground electronic state radiationlessly within 1012 s after excitation, in which case fluorescence is not observed. However, in aromatic molecules, the degree of vibrational freedom is restricted. In this case, the excited molecule may, radiationlessly, arrive in the lowest vibrational level of the lowest electronically excited singlet state. Subsequently (after 1011–107 s), it may return to the ground electronic state by emitting nearultraviolet or visible fluorescence. With very few exceptions, fluorescence always originates from the lowest excited singlet state. This means that only one fluorescence band may be observed from a given molecule, even though it will usually have several absorption bands. Therefore, the observation of several fluorescence bands in a solution of pure sample suggests the occurrence of a chemical reaction in either the ground or excited state, resulting in two or more fluorescent species. Alternatively, the purity of the sample must be questioned. Because the fluorescence spectrum of any one organic compound can demonstrate only one fluorescence band, a band which is usually broad and lacking features, the fluorescence spectrum does not reveal the detailed information about molecular structure that NMR, IR or mass spectrometry does. Nevertheless, fluorescence spectra can give information about the molecular environment that is unobtainable by other methods.

Chemical Structural Effects on Fluorescence The Origin of Molecular Fluorescence The electronic excitation of molecules occurs as the result of the absorption of near-ultraviolet or visible light. Subsequent to excitation, the loss of excess vibrational energy, known as vibrational relaxation, takes place in approximately 1012 s the excess energy being lost by inelastic collisions with solvent molecules.

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Most fluorescence spectra arise from functionally substituted aromatic molecules. Consequently, the compounds of interest in this article are those derived from organic compounds that possess aromatic rings, such as benzene, naphthalene or anthracene, or their heteroaromatic analogues pyridine, quinoline, acridine, etc. The fluorescence spectra of these substances may often be understood in terms of the electronic interactions

Organic Chemistry Applications of Fluorescence Spectroscopy

between the simple aromatic structures and their substituents.

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Chemical Structure and Position of the Fluorescence The energies of the ground and excited states of fluorescing molecules are affected by molecular structure. This is reflected in the positions of the fluorescence maxima in the spectrum.

Chemical Structure and Fluorescence Intensity

E ¼ hn ¼ hc=l

½1

The intensity of fluorescence observable from a given molecular species depends on the probability of light absorption and the probability of fluorescence. The molar absorptivity is a macroscopic manifestation of the probability of light absorption by molecules in the optical path. For most aromatic molecules, the p, p* absorption bands lying in the near-ultraviolet and visible regions of the spectrum have molar absorptivities of 1  103– 1  105 dm3 mol1 cm1 so that the appropriate choice of the transition to excite can influence the intensity of fluorescence by about two orders of magnitude. If the absorbance of the potentially fluorescing species, at the wavelength of excitation, is below 0.02, the intensity of fluorescence is proportional to the molar absorptivity at that nominal wavelength. At high absorbances, the sample is not equally illuminated along the optical path, giving rise to ‘self-shadowing’ effects. More important is the quantum yield or efficiency of fluorescence, which may affect the intensity of fluorescence over about four orders of magnitude and may determine whether fluorescence is at all observable. The quantum yield of fluorescence is dependent on the rates of processes competing with fluorescence for the deactivation of the lowest excited singlet state. Aromatic molecules that contain lengthy aliphatic side chains generally tend to fluoresce less intensely than those without the side chains because of greater opportunity for vibrational deactivation (the ‘loose-bolt’ effect). In unsubstituted aromatic molecules, the rigidity of the aromatic ring results in lower probabilities of vibrational deactivation and hence higher quantum yields. The fluorescence of organic molecules is quenched (diminished in intensity) by heavy atom substituents such as –As(OH)2, Br and I and by certain other groups such as –CHO, –NO2 and nitrogen in six-membered heterocyclic rings (e.g. quinoline). These substituents cause mixing of the spin and orbital motions of the valence electrons. Spin–orbital coupling obscures the distinct identities of the singlet and triplet states and thereby enhances the probability or rate of singlet-triplet intersystem crossing. This process favours population of the lowest triplet state at the expense of the lowest excited singlet state and thus decreases the fluorescence quantum yield. Consequently, aromatic arsenites, nitro compounds, bromo and iodo derivatives, aldehydes, ketones and N-heterocyclics tend to fluoresce very weakly or not at all.

According to eqn [1], where E is the energy, n, the frequency and l, the wavelength of fluorescence and h and c are, respectively, Planck’s constant and the velocity of light, the greater the separation between the ground and excited states the greater will be the frequency and the shorter will be the wavelength of fluorescence. This separation depends on the energy difference between the highest occupied and the lowest unoccupied molecular orbitals and the repulsion energy between the electronic configurations corresponding to the ground and excited states. In aromatic hydrocarbons, the greater the degree of linear annulation the closer together will be the highest occupied and lowest unoccupied orbitals. Consequently, benzene fluoresces at shorter wavelengths than naphthalene which fluoresces at shorter wavelengths than anthracene. Phenanthrene, which is angularly annulated, emits at wavelengths between those of naphthalene and anthracene. In functionally substituted aromatic molecules, the substituents with lone electron pairs, e.g. –NH2, –OH, will have highest occupied orbitals, higher in energy than those of the unsubstituted hydrocarbons while the substituents with vacant p* orbitals, e.g. –CHO, –CO2H, will have p* orbitals lower in energy than those of the unsubstituted hydrocarbons. This means that in substituted aromatic molecules, fluorescence will be at wavelengths longer than in the unsubstituted molecules. This is so regardless of whether the substituents are electron donating or electron withdrawing.

Influence of the Chemical and Physical Environment on Fluorescence Spectra The Solvent The solvents in which fluorescence spectra are observed play a role secondary only to molecular structure in determining the spectral positions and intensities with which fluorescence bands occur and occasionally determine whether fluorescence is observed. The electronic transition accompanying excitation entails a change in electronic charge distribution. If the excited state is more polar than the ground state, a more polar solvent will stabilize the excited state more than the ground state and cause the fluorescence to shift to longer wavelengths relative to that observed in a less polar

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Organic Chemistry Applications of Fluorescence Spectroscopy

solvent. If, however, the ground state is more polar than the excited state, which is rarely the case, the fluorescence will tend to shift to shorter wavelengths upon going to a more polar solvent. Hydrogen bonding in the lowest excited singlet states occasionally results in a decrease in fluorescence quantum yield upon going from hydrocarbon to hydrogen bonding solvents. Many arylamines and phenolic compounds demonstrate this behaviour which is due to internal conversion enhanced by coupling of the vibrations of the molecule of interest to those of the solvent. Molecules having the lowest excited singlet states of the n, p* type rarely fluoresce in hydrocarbon solvents because the n, p* singlet state is efficiently deactivated by intersystem crossing. However, in polar, hydrogen bonding solvents, such as ethanol or water, these molecules often become fluorescent. This results from the stabilization of the lowest singlet p, p* state relative to the lowest n, p* state by hydrogen-bonded interaction. If the interaction is sufficiently strong, the lowest p, p* state drops below the n, p* state in the strongly solvated molecule, becoming the lowest excited singlet state and permitting intense fluorescence. Quinoline and 1-naphthaldehyde, for example, do not fluoresce in cyclohexane but do so in water. The Influence of pH The spectral shifts accompanying protonation or dissociation of basic or acidic functional groups depend on whether the functional group undergoing protonation or dissociation is directly coupled to the aromatic system and whether it gains or loses electronic charge upon going from the ground to the excited state. The higher the positive charge on electron-attracting groups and the higher the negative charge on electron-donating groups, the lower, in general, will be the energy of fluorescence. Thus, the protonation of electron-withdrawing groups, such as carboxyl, carbonyl and pyridyl nitrogen, causes shifts of fluorescence spectra to longer wavelengths while the protonation of electron-donating groups, such as the amino group, produces spectral shifts to shorter wavelengths. The protolytic dissociation of electron-donating groups, such as hydroxyl, sulfhydryl or pyrrolic nitrogen, produces spectral shifts to longer wavelengths while the dissociation of electron-withdrawing groups, such as carboxyls, shifts the fluorescence spectra to shorter wavelengths. In some molecules, the presence of nonbonded electrons obviates the occurrence of fluorescence. However, protonation of the functional group possessing the nonbonded electron pair raises the n, p* lowest excited singlet state above the lowest excited singlet p, p* state and thereby allows fluorescence to occur. Benzophenone, for example, does not fluoresce as the neutral molecule

but does so, moderately intensely, as the cation in concentrated sulfuric acid. An interesting aspect of acid–base reactivity of fluorescent molecules is derived from the occurrence of protonation, and dissociation, during the lifetime of the lowest excited singlet state and is occasionally observed in the pH dependence of the fluorescence spectrum. The lifetimes of molecules in the lowest excited singlet state are typically 1010–107 s. Typical rates of proton transfer reactions are r1010 s1. Consequently, excited state proton transfer may be much slower, much faster or competitive with radiative deactivation of the excited molecules. If excited state proton transfer is much slower than fluorescence, the relative fluorescence intensity will vary with pH exactly the same way as does the absorbance, reflecting only the ground-state acid– base equilibrium. If excited state proton transfer is much faster than fluorescence, the fluorescence intensity will vary with pH in a way that reflects the acid–base equilibrium in the lowest excited singlet state. Equilibrium in the excited state is a rare phenomenon and will not be dealt with further here. If the rate of proton transfer, in the excited state, is comparable to the rates of photophysical deactivation of excited acid and conjugate base, the variations of the fluorescence intensities of acid and conjugate base, with pH, will be governed by the kinetics of the excited-state proton transfer reactions and the fluorescence of acid and conjugate base will be observed over a wide pH range. The Influence of High Solute Concentrations Several types of excited-state solute–solute interaction are common at high solute concentrations. The aggregation of excited solute molecules with unexcited molecules of the same type may result in a new excited molecule called an excimer, which either may not luminesce or may luminesce at lower frequency than the monomeric excited molecule. Because excimer formation takes place in the excited state, it is sometimes demonstrable as a shifting of the fluorescence spectrum. However, after fluorescence, the deactivated polymer, which is unstable in the ground state, rapidly decomposes. Hence, the absorption spectrum does not reflect the presence of the excited-state complexes. Occasionally, excited-state complex formation may occur between two different solute molecules. The term ‘exciplex’ has been coined to describe a heteropolymeric excited-state complex. Excimer and exciplex formation are usually observed only in fluid solution because diffusion of the excited species is necessary to form the excited complexes. One concentration effect that is observed in molecules in fluid or rigid media is resonance energy transfer. Energy transfer entails the excitation of a molecule which, during the lifetime of the excited state, transmits its excitation

Organic Chemistry Applications of Fluorescence Spectroscopy

energy to a nearby molecule. The probability of resonance energy transfer decreases as the inverse sixth power of the distance between donor and acceptor and can occur between molecules which are separated by up to 100 nm. Because the mean distance between molecules decreases with increasing concentration, energy transfer is favoured by increasing the concentration of the acceptor. For energy transfer to occur between two dissimilar molecules, the fluorescence spectrum of the energy donor must overlap the absorption spectrum of the energy acceptor. Fluorescence may be diminished in intensity or eliminated due to the deactivation of the lowest excited singlet state of the analyte by interaction with other species in solution. This is called quenching of fluorescence. Mechanisms of quenching appear to entail internal conversion, intersystem crossing, electron transfer and photodissociation as modes of deactivation of the excited fluorescer–quencher complexes. Quenching processes may be divided into two broad categories. In dynamic or diffusional quenching, interaction between the quencher and the potentially fluorescent molecule takes place during the lifetime of the excited state. As a result, the efficiency of dynamic quenching is governed by the rate constant of the quenching reaction, which is usually typical of that for a diffusion-controlled reaction, the lifetime of the excited state of the potential fluorescer and the concentration of the quenching species. Interaction between quencher and fluorophore results in the formation of a transient excited complex which is non-fluorescent and may be deactivated by any of the usual radiationless modes of deactivation of excited singlet states. Because interaction occurs only after excitation of the potentially fluorescing molecule, the presence of the quenching species has no effect on the absorption spectrum of the fluorophore. Many aromatic molecules, for example, quinolines and acridines, are dynamically quenched by halide ions such as Cl, Br and I. Static quenching is characterized by complexation in the ground state between the quenching species and the molecule which, when excited alone, should fluoresce. The complex is generally not fluorescent and, as a result, the ground-state reaction diminishes the intensity of fluorescence of the potentially fluorescent species. The quenching of the fluorescence of o-phenanthroline by complexation with iron(II) is an example of static quenching.

Applications Native Fluorescence of Organic Compounds Numerous organic compounds are intrinsically fluorescent and so may be assayed directly, eliminating any need of derivatization or labelling. This section lists some

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examples of types of compounds which possess native fluorescence and some factors which exert influence on that fluorescence. The assay of organic compounds by fluorescence spectroscopy is covered in greater detail by Guilbault and that of organic natural products by Wolfbeis. Simple fluorescence spectra of 2000 compounds have been published by Sadtler Research Laboratories. Three of the twenty common amino acids exhibit fluorescence. These are phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp). Phenylalanine, as the name implies, consists of alanine with one methyl hydrogen substituted by a phenyl group It is weakly fluorescent at lf ¼ 282 nm (lex ¼ 260 nm) and cannot be detected in the presence of Tyr or Trp. Tyrosine is phenolic and fluoresces (lex ¼ 275 nm; lf ¼ 303 nm) with much greater intensity than Phe just as phenol fluoresces far more intensely than benzene. The phenolic group, which ionizes at pH above 10, introduces the need to control pH in the assay. The phenolate form of Tyr fluoresces with far less intensity than the non-ionized form and redshifts to 345 nm. Tryptophan has a quantum yield very close to that of Tyr but absorbs far better and so fluoresces (lex ¼ 287 nm; lf ¼ 348 nm) more intensely. The nitrogen-containing indole moiety in Trp becomes protonated at low pH, thus decreasing its fluorescence. The fluorescence of Trp is also quenched at high pH and so Trp fluoresces best over a range in pH from 4 to 9. The fluorescence properties of these amino acids as mentioned apply only to the free amino acids in solution. Such properties can change significantly when they become part of a protein. The indole moiety seen in Trp is a common structure in nature and is seen in abundance in alkaloids. Indole in water fluoresces at lf ¼ 352 nm (lex ¼ 275 nm). Many simple natural derivatives of indole fluoresce in the 330–350 nm range and can be maximally exited at 270–290 nm. The ‘indole alkaloid’ is a major class of alkaloids, which include a number of well-known drugs, legal as well as illicit. The ergot alkaloids are in this class. The famous hallucinogen lysergic acid diethylamide (LSD) at pH 7 fluoresces at 365 nm with an excitation maximum at 325 nm. Bromolysergic acid diethylamide is not hallucinogenic. It is brominated at the 2 position on the indole moiety. It fluoresces (lex ¼ 315 nm; lf ¼ 460 nm) at pH 1 with far less intensity than LSD. Other classes of alkaloids, which exhibit fluorescence, include the quinoline and isoquinoline alkaloids. Quinoline is weakly fluorescent. The anti-malarial drug quinine includes a methoxy substituent on the 6 position of the quinoline moiety and fluoresces very intensely. Quinine, in sulfuric acid solution, is often used as a standard in fluorescence spectroscopy for determining a quantum yield. Its fluorescent properties are sensitive to pH. At pH 2 it has an excitation maximum of 347 nm with fluorescence at 448 nm. At pH 7 the peaks shift to

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Organic Chemistry Applications of Fluorescence Spectroscopy

absorb at 331 nm and emit at 382 nm. In hydrochloric acid solution, absorption is unaffected but fluorescence intensity is quenched greatly by the halide anions. Isoquinoline alkaloids can be subdivided into two groups. Those alkaloids which preserve the isoquinoline moiety and those which have a reduced ring structure, the tetrahydroisoquinolines. Papaverine, a smooth muscle relaxant, possesses the isoquinoline moiety. In chloroform, papaverine fluoresces at lf ¼ 347 nm (lex ¼ 315 nm). The addition of some trichloroacetic acid to this protonates the isoquinoline moiety and causes a shift in absorption and fluorescence peaks (lex ¼ 415 nm; lf ¼ 452 nm). Berberine is an isoquinoline alkaloid which has a quaternary nitrogen with hydroxide as the standard counterion. In ethanol it has excitation maxima of 432 and 352 nm and fluoresces at 548 nm. A change of solvent to DMF causes a shift of the excitation maximum to 380 nm and emission to 510 nm. Berberine deposited on a TLC plate shows excitation maxima of 433 and 353 nm and fluoresces at 510 nm. 13-Methoxyberberine in ethanol has one excitation peak, at 433 nm, and its emission peak shifts to 562 nm. Tetrahydroisoquinoline alkaloids have a benzenoid fluorophore rather than isoquinoline and so fluoresce at shorter wavelengths. The opiate alkaloids are of this class of alkaloids. Morphine and codeine differ only at the 3 position. Morphine has a hydroxy substituent, making it phenolic, and codeine has a methoxy substituent. In water at pH 1 both compounds fluoresce at 350 nm. Both are excited at 285 nm. Codeine has an additional excitation peak at 245 nm. They can be assayed in admixture because morphine loses its fluorescence under basic conditions by way of phenolate formation but codeine retains its fluorescence at high pH. Caffeine is a member of the xanthine alkaloids. Caffeine in water fluoresces at lf ¼ 303 nm (lex ¼ 270 nm). Caffeine is 1,3,7-trimethylxanthine. Xanthine in water at pH 1 fluoresces at lf ¼ 435 nm (lex ¼ 275 nm). The fluorophore of caffeine and xanthine is that of purine (lex ¼ 300 nm; lf ¼ 360 nm at pH 13). Purine is, likewise, the fluorophore associated with the purine bases adenine and guanine. Adenine at pH 1 fluoresces at lf ¼ 380 nm (lex ¼ 265 nm). Adenosine and its various phosphates all fluoresce at lf ¼ 390 nm (lex ¼ 272 nm) in 5 M sulphuric acid. Guanine at pH 1 fluoresces at lf ¼ 350 nm (lex ¼ 275 nm), and at pH 11 at lf ¼ 360 nm (lex ¼ 275 nm). Guanosine and GMP (guanosine 50 -phosphate) at pH 1 fluoresce at lf ¼ 390 nm (lex ¼ 285 nm). The other bases associated with DNA and RNA are the pyrimidines: cytosine, thymine and uracil. They also exhibit fluorescence. Cytosine in water fluoresces at lf ¼ 313 nm (lex ¼ 267 nm) but CMP (cytidine 50 -phosphate) in water fluoresces at lf ¼ 330 nm (lex ¼ 248 nm). Thymine in water at pH 7 fluoresces at lf ¼ 320 nm

(lex ¼ 265 nm) but TMP (thymine 50 -phosphate) fluoresces at lf ¼ 330 nm (lex ¼ 248 nm). Uracil in water at pH 7 fluoresces at lf ¼ 309 nm (lex ¼ 258 nm) but UMP fluoresces at lf ¼ 320 nm (lex ¼ 248 nm). The quantum yields of all the bases, nucleosides and nucleotides are extremely low, so extraordinary conditions must be applied to the fluorescence analysis of DNA, RNA and their component parts. Examples of compounds possessing native fluorescence listed so far have had a benzene ring or a nitrogencontaining heterocycle as the fluorophore. There is also a great number of oxygen-containing heterocyclic compounds that fluoresce. Coumarins and flavonoids are the two largest classes of oxygen heterocycles. Coumarins fluoresce more intensely under basic conditions where flavones fluoresce weakly. In 30% sulfuric acid solution, flavones fluoresce intensely and coumarins do not. Coumarin is not normally fluorescent, but hydroxy substitution in any position except 8 leads to intense fluorescence at room temperature. In methanol, 3-hydroxycoumarin fluoresces at lf ¼ 372 nm (lex ¼ 316 nm), 4hydroxycoumarin fluoresces at lf ¼ 357 nm (lex ¼ 300 nm), 6-hydroxycoumarin fluoresces at lf ¼ 431 nm (lex ¼ 341 nm) and 7-hydroxycoumarin fluoresces at lf ¼ 392 nm (lex ¼ 333 nm). Other oxygen-containing heterocycles are also of interest. Cannabinols in ethanol fluoresce at lf ¼ 318 nm (lex ¼ 280 nm). Tocopherols (vitamin E) in ethanol fluoresce at lf ¼ 340 nm (lex ¼ 295 nm). Other vitamins and coenzymes exhibit fluorescence. Riboflavin (vitamin B2) is a three-ring heterocycle which in water at pH 7 fluoresces at lf ¼ 565 nm (lex ¼ 370 or 440 nm). The various forms of vitamin B6 all have native fluorescence: pyridoxine (lex ¼ 340 nm; lf ¼ 400 nm), pyridoxal (lex ¼ 330 nm; lf ¼ 385 nm) and pyridoxamine (lex ¼ 335 nm; lf ¼ 400 nm). Vitamin B12, as cyanocobalamin, has a porphyrin moiety (cf. haem and chlorophyll) and fluoresces at lf ¼ 305 nm (lex ¼ 275 nm). All D vitamins fluoresce when treated with strong acid but that is due to a degradation product. Vitamin D2 (calciferol) has been reported to exhibit native fluorescence in ethanol at lf ¼ 420 nm (lex ¼ 348 nm). Calciferol’s fluorescence is due to a rigid conjugated triene rather than an aromatic ring. Vitamin A (retinol) also has no aromatic ring but rather has an extended conjugation of p-bonds which leads to fluorescence. Retinol in ethanol fluoresces at lf ¼ 470 nm (lex ¼ 325 nm) and in pentane–hexane it fluoresces at lf ¼ 513 nm (lex ¼ 321 nm).

Fluorescent Derivatization Not all substances are fluorescent. Nonfluorescent substances may be analysed by indirect methods of which there are several.

Organic Chemistry Applications of Fluorescence Spectroscopy

(1) Some organic compounds, themselves nonfluorescent, can be converted into fluorescent compounds by a chemical reaction with another organic compound which is itself also nonfluorescent. o-Phthalaldehyde (OPA), itself nonfluorescent, is one of the most widely used reagents for the assay of amines, amino acids, peptides, amino carbohydrates, etc. It reacts with the primary amino group, in the presence of a thiol (usually 2-sulphanylethanol), to give strongly fluorescent condensation products. The fluorescence is measured at B455 nm with the excitation wavelength at B340 nm. The assay can be conducted in the nanomole range.

For example, this method has been employed for analysis of carbamate pesticides in surface water after the pesticides are hydrolysed in strong base to yield methyl amine and phenols. New reagents such as naphthalene-2,3-dicarboxaldehyde (NDA), 1-phenylnaphthalene-2,3-dicarboxaldehyde (fNDA), and anthracene-2,3-dicarboxaldehyde (ADA) have been synthesized as improved OPA/thiol type reagents. While similar in many respects, there are important differences in these isoindole products. For example, the products formed with NDA, fNDA or ADA are considerably more stable than the corresponding OPA derivatives and possess substantially higher fluorescence quantum efficiencies and minimal interference compared with the latter. (2) Some nonfluorescent organic compounds can react with fluorescent dyes to give fluorescent products which usually show altered fluorescence properties with regard to the free dye. 4-(Aminosulfonyl)-7-(1-piperazinyl)-2,1,3-benzoxadiazole (ABD-PZ), maximum wavelength 565 nm with excitation at 413 nm, has been synthesized as a fluorescent reagent for determination of carboxylic acids. It reacts with carboxylic acids in the presence of diethyl phosphorocyanidate (DEPC) to produce fluorescent adducts with fluorescence at longer wavelengths. For example, the maximum wavelength of fluorescence of arachidic acid labelled with ABD-PZ is 580 nm with excitation at 440 nm. This method has been applied to a reversed-phase HPLC column for fatty acid mixture analysis. The detection limits for eight fatty acids are in the 10–50 fmol range.

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When the piperazinyl group in ABD-PZ is substituted by a chiral group such as the 3-aminopyrrolidinyl group, it becomes a chiral derivatization reagent (D-ABD-APy or L-ABD-APy). This chiral derivatization reagent reacts with carboxylic acid enantiomers to form diastereomers for fluorescence detection. The diastereomers derived from antiinflammatory drugs and N-acetylamino acids are efficiently resolved by a reversed-phase column after they react with D-ABD-APy. The detection limit, for example, of ABD-APy-naproxen on HPLC chromatograms is 30 fmol. (3) Fluorometric enzyme assay involves a reaction catalysed by an enzyme, in which the product must show different fluorescence properties compared with those of the substrate. One example is the fluorometric peroxygenase assay for lipid hydroperoxides in meats and fish. In the reaction, catalysed by pea peroxygenase, the lipid hydroperoxide is reduced to an equimolar amount of alcohol during hydroxylation of the substrate, 1methylindole, which shows no fluorescent product in the absence of the peroxygenase. The maximum wavelengths of excitation and emission for the hydroxylated product, 3-hydroxy-1-methylindole, appear at 410 and 485 nm, respectively, in n-butanol. The detectabilities of hydroperoxides are in the range of 25–150 nmol and a-tocopherol, an antioxidant at levels equivalent to those in meats and fish, did not affect the peroxygenase reaction. The method enables determination of total lipid hydroperoxides in sample homogenates without extracting total lipids from meats and fish. (4) Fluoroimmunoassay involves attaching a fluorescentlabelled antibody to its specific antigen or vice versa, making use of a complexation reaction between the antigen and the antibody, for fluorescence detection at nanogram and lower levels. The specific affinity reactions may include the following: enzyme–substrate, hormone–receptor, neurotransmitter–receptor, pharmacological agent–receptor, etc. Such fluoroimmunoassays are divided into two categories – heterogeneous assays, which involve physical separation of the assay mixture before detection, and homogeneous assays, in which no separation steps are required. The most common fluorescent labels employed for fluoroimmunoassay are fluorescein isothiocyanate (FITC), which emits apple-green fluorescence (520 nm) when excited by ultraviolet or, preferably, by blue light

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(494 nm), and tetramethylrhodamine isothiocyanate (TRITC), which emits orange fluorescence (575 nm) when excited by ultraviolet or, preferably, by green light (550 nm). One example is the fluoroimmunoassay for the routine detection of buprenorphine in urine samples of persons suspected of Temgesics abuse. Buprenorphine antibody is labelled with pseudobuprenorphine, the dimer of buprenorphine. In this case, pseudobuprenorphine has a higher affinity for the antibody than that of FITC–norbuprenorphine. Pseudobuprenorphine shows an intense blue fluorescence with maximum at 435 nm when excited at 326 nm. The minimum detectable dose of buprenorphine by the fluoroimmunoassay is calculated to be 20 ng ml1. See also: Biochemical Applications of Fluorescence Spectroscopy, Fluorescence Microscopy, Applications, Fluorescent Molecular Probes, Fluorescence Polarization and Anisotropy, Inorganic Condensed Matter, Applications of Luminescence Spectroscopy, X-Ray Fluorescence Spectrometers, X-Ray Fluorescence Spectroscopy, Applications.

Further Reading Baeyens WRG, de Keukeleire D, and Korkidis K (ed.) (1991) Luminescence Techniques in Chemical and Biochemical Analysis. New York: Marcel Dekker.

Brand L and Johnson ML (ed.) (1997) Fluorescence Spectroscopy. San Diego: Academic Press. Eastwood D and Love LJC (ed.) (1988) Progress in Analytical Luminescence. Philadelphia: ASTM. Goldberg MC (ed.) (1989) Luminescence Applications in Biological, Chemical, Environmental, and Hydrological Sciences. Washington, DC: American Chemical Society. Guilbault GG (ed.) (1990) Assay of organic compounds. In: Practical Fluorescence, 2nd edn., pp. 231--366. New York: Marcel Dekker. Lakowicz JR (ed.) (1991) Topics in Fluorescence Spectroscopy, vol. 1–5. New York: Plenum Press. Lumb MD (ed.) (1978) Luminescence Spectroscopy. London and New York: Academic Press. Mason WT (ed.) (1993) Fluorescent and Luminescent Probes for Biological Activity: A Practical Guide to Technology for Quantitative Real-time Analysis. London, San Diego: Academic Press. Rendell D (1987) Fluorescence and Phosphorescence Spectroscopy. Published on behalf of ACOL, London. Chichester and New York: Wiley. 1987. Sadtler Research Laboratories (1974–1976) Fluorescence Spectra, Chapter 3, vol. 1–8. Philadelphia. Schulman SG (1977) Fluorescence and Phosphorescence Spectroscopy: Physicochemical Principles and Practice. New York: Pergamon Press. Schulman SG (ed.) (1985–1988) Molecular Luminescence Spectroscopy: Methods and Applications, Part I–II. New York: Wiley. Soper SA, Warner IM, and McGown LB (1998) Molecular fluorescence, phosphorescence and chemiluminescence. Analytical Chemistry 70: 477R--494R. Winefordner JD, Schulman SG, and O’Haver TC (1972) Luminescence Spectrometry in Analytical Chemistry. New York: Wiley-Interscience. Wolfbeis OS (1985) The fluorescence of organic natural products. In: Schulman SG (ed.) Molecular Luminescence Spectroscopy: Methods and Applications, Part I, Chapter 3, pp. 167--370. New York: Wiley.