Analytical applications of o-hydroxymercuribenzoic acid, dithiofluorescein and mercurated fluorescein

The application of the reagents mentioned in the title to titrimetric, photometric and fluorimetric analysis of several compounds of sulphur, nitrogen and mercury, including procedures for selective determinations, detection, location, identification and estimation in trace quantities, is reviewed.     

Correction for quenching in fluorimetric determinations using steady state fluorescence

Quenching corrections in fluorimetric estimations are arrived at using time resolved fluorometry, where the reduction in the lifetimes of the fluorophore in the presence of quenchers is a measure of the correction. A novel procedure for the correction of quenching in fluorimetric determinations using steady state fluorescence spectroscopy is described here. The method is based on the variation in the slope of the fluorescence versus concentration plots, as the quencher concentration is changed. As a test case, the procedure for the determination of uranium has been demonstrated in the presence of a number of metal ion quenchers.     

QSPR studies on the photoinduced-fluorescence behaviour of pharmaceuticals and pesticides

Fluorimetric analysis is still a growing line of research in the determination of a wide range of organic compounds, including pharmaceuticals and pesticides, which makes necessary the development of new strategies aimed at improving the performance of fluorescence determinations as well as the sensitivity and, especially, the selectivity of the newly developed analytical methods. In this paper are presented applications of a useful and growing tool suitable for fostering and improving research in the analytical field. Experimental screening, molecular connectivity and discriminant analysis are applied to organic compounds to predict their fluorescent behaviour after their photodegradation by UV irradiation in a continuous flow manifold (multicommutation flow assembly). The screening was based on online fluorimetric measurement and comprised pre-selected compounds with different molecular structures (pharmaceuticals and some pesticides with known ‘native’ fluorescent behaviour) to study their changes in fluorescent behaviour after UV irradiation. Theoretical predictions agree with the results from the experimental screening and could be used to develop selective analytical methods, as well as helping to reduce the need for expensive, time-consuming and trial-and-error screening procedures.     

Three-dimensional high-performance liquid chromatography based on time-resolved laser fluorimetry

Microcolumn high-performance liquid chromatography is used for trace determinations of polynuclear aromatic hydrocarbons. The detector is a laser fluorimetric system with subnanosecond time resolution. The detection limit is 210 fg for perylene. In chromatography, the fluorescence decay curves are measured successively as the sample elutes by a microcomputer-controlled data-processing system. A three-dimensional chromatogram, in which retention times and delay times are displayed, is constructed after sample elution. A real sample extracted from airborne particulates is measured, and perylene, benzo(a)pyrene, and benzo(ghi)perylene are shown to be present.     

Kinetic fluorimetric determination of histidine,histamine and their mixtures

Kinetic fluorimetric methods for the determination of histidine and histamine, both separately and in mixtures, are described; the methods are based on the accelerating effect of these compounds on the oxidation of 1,1,3-tricyano-2-amino-1-propene by hydrogen peroxide in the presence of copper. A differential rate method is used for resolution of mixtures of these compounds (10^?5 M level), in which synergic effects are taken into account. The relative standard deviations for histidine and histamine are 1.4% and 2.1%, respectively, for separate determinations but 4.0% and 3.4%, respectively, for determinations of mixtures. Histamine/histidine ratios between 1:1 and 16:1 are satisfactorily resolved.     

A fluorimetric study of pindolol and its complexes with cyclodextrins

Spectrofluorimetric characteristics of pindolol have been investigated with the aim of using this technique for analytical determinations. Other monosubstituted indole derivatives, 4-methoxy and 5-methoxyindole, have been also studied for comparative purposes. Corrected excitation and emission wavelengths in different solvents are reported and the effect of solvent on the Stokes shifts of these compounds has been analysed using the Lippert equation. In addition, the Stokes shift of pindolol has been determined in dioxan-water solvent mixtures and the presence of specific solvent effects is discussed. The fluorescence of pindolol is pH dependent, the quantum yields determined in water are lower than those in other solvents. With respect to the sensitivity, it has been found that the detection limits in aqueous solutions are improved in the presence of beta and methyl-beta-cyclodextrin. Finally, a fluorimetric analysis of the interaction between pindolol and different cyclodextrins has been carried out in order to determine the apparent stability constants of the complexes and the thermodynamic parameters associated to complexation.     

New colorimetric and fluorimetric methods for the determination of 1,4-cyclohexanedione

1,4-Cyclohexanedione has been determined by reaction with o-phthalaldehyde in sulfuric acid. The determinations depend on the formation of a dicationic salt of pentacenequinone. A sensitive and highly selective fluorimetric method is recommended, but the reaction can also be used spectrophotometrically. Many organic compounds, as well as the isomers of 1,4-cyclohexanedione, give negative results. A spot test is described which can be used for quick preliminary investigations of complex mixtures for I,4-cyclohexanedione.     

Nonextractive spectrofluorimetric determination of aluminium in real, environmental and biological samples using chromotropic acid

An ultra-sensitive and highly selective nonextractive fluorimetric method is presented for the rapid determination of aluminium at nano-trace levels using chromotropic acid as a fluorimetric reagent [lambda(ex) = 360 nm and lambda(em) = 390 nm] in the pH range of 4.1-4.7. The fluorescence intensity of the metal chelate (2:3 complex) reaches a constant value within 1/2 hr and remains unchanged for over 48 hr. The fluorescence intensity aluminium concentration calibration curve is collinear between 1 and 300 ng/ml of Al. A constant fluorescence intensity is obtained over a wide range (1:50-1:1500) of Al:reagent molar concentrations. Large excesses of over 60 cations, anions and complexing agents (like tartrate, oxalate, phosphate, thio-urea, SCN(-), etc.) do not interfere in the Al determination. The developed method was successfully used in assaying aluminium in several standard reference materials (Al-bronze, brass, stainless steel) as well as in some environmental and biological samples. The method is very precise and accurate (S.D = +/-0.001 on 10 ng/ml; 11 determinations).     

Simultaneous determination of metals in two-component mixtures with 5-sulfo-8-quinolinol by using phase-resolved fluorimetry

Determinations of metals in two-component mixtures are described in which 5-sulfo-8-quinolinol is used to produce fluorescent chelates of the metals. The metal chelates have broad, overlapping fluorescence spectra but the fluorescence lifetimes (τ) of the chelates are sufficiently different to permit phase-resolved fluorimetric determinations in which the fluorescence contributions of the two chelates can be resolved. Average determination errors ranged from ?4.1% to 2.1% for each metal chelate in mixtures of zinc and cadmium (Δτ = 1.0 ns), gallium and indium (Δτ = 1.7 ns) and aluminum and gallium (Δτ = 5.3 ns). Limits of detection and determination for each individual metal chelate were found to be three times and five times greater, respectively, for the phase-resolved measurements relative to steady-state measurements under the same experimental conditions. Effects of pH on the fluorescence lifetimes and intesities of the metal chelates were studied.     

Theoretical prediction of the native fluorescence of pharmaceuticals

At present, to search fluorescent compounds or to increase the native fluorescence is an active research line specially and not only with analytical purposes. On some analytical areas and from the early times of applications of fluorescence (mid-fifties) the fluorimeter was defined as the suitable detector for determination of pharmaceuticals and subsequently, this detection mode has been widely applied. Therefore, it is mandatory to develop new strategies to discover or to enhance in a simple way the native fluorescence of organic compounds to increase the number of analytes to be determined by direct fluorescence.In the present paper are studied further applications of a new tool suitable to increase the research in analytical field. Calculations on molecular connectivity and discriminant analysis are applied to a certain number of pharmaceuticals (and some pesticides) on which fluorescence behaviour was observed in an experimental screening or obtained from scientific literature. The screening tests were based on the on-line fluorimetric measurement by using a continuous-flow assembly. The screening comprised pre-selected compounds with different molecular structures. The theoretical predictions agree with the empirical results from the screening test.     

Determination of uranium in silicate rocks using laser induced fluorescence

A rapid method for fluorimetric estimation of uranium in silicate rocks is described. The fluorescence of uranyl complex is induced by laser beam in the ultraviolet region provided by nitrogen laser tube. The emission spectrum is quite intense and relatively persistent. For direct estimation of uranium in geological silicate materials without prior extraction, the interference of certain cations and anions that might be present in silicate rocks on uranium determination was studied. The limit of detection is 0.5 ppb.     

Fluorimetric method for the determination of uranium in natural waters

A method is described for the fluorimetric determination of uranium in natural waters. The limit of detection is 0.3 ppM. Ion-exchange is used to preconcentrate the uranium by a factor of 22 and separate it from quenching ions in the sample. The fluorescence is measured in a medium that is 1.35M in both sulphuric and phosphoric acids. The uranyl ions are excited by radiation of wavelength 280 nm and bandwidth 40 nm. The emitted signal is scanned from 470 to 510 nm. After spiking of the sample solution with a small volume of standard uranium solution, the fluorescence signal is scanned again and the uranium content of the sample calculated from the two readings. The coefficient of variation is 8.5% for determinations of U in a synthetic water sample having a uranium content of 1.9 ppM. Destruction of organic matter in the eluates gives 0.1 ppM detection limit.     

Fluorimetric determination of monosubstituted guanidino compounds with benzoin—dimethylformamide reagent

A sensitive fluorimetric method for the determination of monosubstituted guanidino compounds is based on their reaction in potassium hydroxide solution with benzoin in the presence of dimethylformamide. The fluorescence produced is stabilized by β-mercaptoethanol and shows excitation and emission maxima around 325 and 435 nm, respectively. The method is simple, selective for monosubstituted guanidino compounds, including polypeptides with one or two arginyl residues, and sensitive; compounds can be determined at concentrations as low as 0.08—0.22 nmol ml^-1.     

Study of phenolic compounds as natural antioxidants by a fluorescence method

Much work has been carried out in recent years on the beneficial effect of phenolic compounds as natural antioxidants which help to neutralize free radicals. In fact, researchers have focused their attention on the pathological role of free radicals in a variety of diseases, among which the most important are atherosclerosis and cancer. Thus, among the components of the so-called 'Mediterranean Diet', phenolic compounds have received increased attention as epidemiological studies have shown that consumption of foods and beverages rich in phenolics is correlated with reduced incidence of heart disease. In this study, four phenolic compounds: (1) 3,4,5-trihydroxybenzoic acid (gallic acid); (2) trans 3,4',5-trihydroxystilbene (trans-resveratrol); (3) 3,3',4',5,7-pentahydroxyflavone (quercetin) and its glycoside (4) 3,3',4',5,7-pentahydroxyflavone-3-rutinoside (Rutin) have been subjected to antioxidant study by a fluorimetric assay. In this method, the rate of peroxidation induced by 2,2'-azobis (2-methylpropionamidine) dihydrochloride was monitored through the loss of fluorescence of the protein B-phycoerythrin (B-PE). Under appropriate conditions, the loss of B-PE fluorescence in the presence of reactive species is an index of oxidative damage of the protein. The inhibition of the action of reactive species by phenolic compounds, reflected in the protection against the loss of B-PE fluorescence in the fluorimetric assay, is a measure of its antioxidant capacity against the reactive species. The antioxidant effects of phenolic compounds have been investigated at different concentrations to relate activity to structural effects. It has been observed that the fluorescence decay due to peroxyl radical attack on B-PE decreases exponentially with time. As a reference compound for antioxidant capacity we used 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylicacid (trolox), a water soluble tocopherol analogue. This compound reacts rapidly with peroxyl radicals, and, until the trolox is consumed, no loss in phycoerythrin fluorescence is observed. A linear correlation of the net protection value with the concentration of trolox was demonstrated. The phenolic compounds studied react with peroxyl radicals in a similar way to trolox. Quercetin and rutin were shown to have strong antioxidant activities. The results obtained here are in agreement with previous studies confirming that quercetin is the most antioxidant of the four polyphenolics.     

Kinetics and equilibria of proton exchange in the lowest excited singlet states of 8-methoxyquinoline and 8-methoxyquinaldine: analysis of some complex fluorimetric titration curves

The determination of the rate constants for proton exchange in the lowest excited singlet states of 8-methoxyquinoline and 8-methoxyquinaldine from the pH dependences of their fluorescence spectra is complicated by overlap of the spectra of the conjugate acids and bases of both compounds and by the enhancement of 8-methoxyquinoline fluorescence at high pH by a process apparently unrelated to proton exchange. The fluorimetric titration data are analyzed in ways which compensate for these difficulties and the kinetic parameters of excited-state proton exchange determined. Qualitative relationships between the parameters of excited-state proton exchange, electronic structure and steric factors are considered.     

A simple method for the determination of benzo[a]pyrene and benzo[k]fluoranthene in a mixture

Atmospheric particulate matter is routinely analysed for the concentration of the carcinogen benzo[a]pyrene (BaP). Benzo[k]fluoranthene (BkF) has identical spectral characteristics and interferes in the fluorimetric estimation. A simple method for the determination of BaP in the presence of BkF is described. Based on quenching of the fluorescence of BaP by the addition of small amounts of nitromethane, the method can be used for accurate estimation for wide concentration ratios of BaP to BkF ranging from 0.1 to 10.0.     

交替最小化协变矩阵误差算法与三维荧光分析法相结合同时分辨利尿类药品

氨苯喋啶、呋塞米和阿米洛利是3种常用的利尿药,被广泛用来治疗多种疾病。近来这3种药物被用作兴奋剂提高运动员在比赛中的成绩,因此,国际奥林匹克委员会(IOC)将其列为违禁药品[1]。因此对这3种物质进行同时分辨和测定具有极其重要的现实意义。然而这3种物质具有相似的理化性质     

Direct determination of nalidixic acid in urine by matrix isopotential synchronous fluorescence spectrometry

A new method for the determination of nalidixic acid in urine is proposed for concentrations between 25 and 1000 ng ml(-1) by means of matrix isopotential synchronous fluorescence spectrometry. This new technique is useful for the determination of compounds in samples with unknown background fluorescence, such as nalidixic acid in urine, without the need for tedious preseparation. The method was performed in ethanol/water medium (80% v/v), at an apparent pH of 2.9 provided by adding sodium monochloracetate/monochloroacetic acid buffer solution. The method was successfully applied to the determination of nalidixic acid in urine. Better sensitivity and reproducibility are achieved in these matrices than with the fluorimetric methods described in the literature.     

Improved trihydroxyindole method for the simultaneous stopped-flow spectrofluorimetric determination of epinephrine and norepinephrine in urine

A modification of the trihydroxyindole method for the simultaneous fluorimetric determination of epinephrine and norepinephrine in urine is reported. The overall reaction (oxidation and tautomerization) develops in a single step and the lutins formed are monitored by the stopped-flow technique. The determinations are based on the initial rate and fluorescence amplitude of the kinetic curves. An extension of the proportional equations method is used for the simultaneous resolution of the two catecholamines. The linear range of the proposed method is 0.1–460 ng ml^?1 for epinephrine and 0.2–480 ng ml^?1 for norepinephrine and the analytical recoveries are about 95% and 91%, respectively. The proposed method offers simplicity, improved sensitivity and a low, steady reagent blank signal.