APPLICATION OF TIME-RESOLVED FLUOROMETRY TO THE RESOLUTION OF PORPHYRIN-PHOTOPRODUCT MIXTURES

Abstract —The quantitation of porphyrin and its photoproducts in biological samples is important in diverse applications including, but not limited to, tumor phototherapy. Differences in fluorescence properties, e.g., emission spectra and lifetimes, of the various components can be utilized to resolve the individual contributions. In this report time-resolved measurements using multifrequency phase and modulation rluorometry are employed to study mixtures of protoporphyrin IX and photoprotopor-phyrin. Individual lifetimes of 14.85 ns and 5.95 ns were determined for dioxane solutions of protoporphyrin IX and photoprotoporphyrin, respectively. These lifetime values were utilized to resolve the individual emission spectrum of each component in the mixture using the phase-resolved Huorometry technique.     

Homogeneous immunoassay of phenobarbital by phase-resolved fluorescence spectroscopy

A new technique for determination of phenobarbital by homogeneous fluoroimmunoassay is described. Simultaneous determination of free and antibody-bound fluorescein-labelled phenobarbital is accomplished by making phase-resolved fluorescence measurements, in which selectivity is provided by the difference between the fluorescence lifetimes of the two species, as well as by the difference in fluorescence intensities. The technique requires no specialized reagents other than those inherent in immunoassay (antibody, antigen and labelled antigen). Problems due to non-specific binding of labelled antigen to sample matrix components can be alleviated by addition of a relative excess of albumin.     

High-performance liquid chromatography of metal chelates: Environmental and industrial trace metal control

The methodological aspects of the use of HPLC in determination of metals in the form of their chelates are discussed. Rational schemes for the analysis of complex environmental and industrial samples are presented, including choice of chelating reagent, chelate preparation, metal concentration, separation method and detection method. Examples of the application of HPLC of metal chelates to environmental, production and quality control are presented.     

Metal Ion Chromatography with Fluorescence Detection

Abstract

Nonpolar, agglomerated anion exchanger, and surface-sulfonated cation exchanger stationary phases have been used in conjunction with 8-hydroxyquinoline-5-sulfonic acid (HQS) in the eluent or as a postcolumn reagent for the separation and detection of a number of metals that form fluorescent HQS complexes. Several metals, notably those classified as transition metals, form nonfluorescent HQS chelates and quenches the fluorescence of other metal-HQS metal chelates. Such transition metals have been detected by introducing the fluorescent Al-HQS chelate postcolumn. Cation exchange stationary phases are the most useful for chromatographic applications involving HQS and are able to provide a variety of useful separations by tailoring elution conditions. Although not sensitive to Ba, the approach may be particularly good for the determination of the other alkaline earth metals. Fluorescence quenching resulting from Fe and Ni leaching from stainless steel chromatographic systems present a problem for trace analysis and accentuate the need for nonmetallic hardware. Subpicomole detection limits are attainable for Cd, Mg and Zn.     

Simultaneous determination of traces of iron, cobalt, nickel, copper, mercury and lead in water by energy-dispersive x-ray fluorescence spectrometry after preconcentration as their piperazino-1,4-bis(dithiocarbamate) complexes

A bidentate chelating agent has been proposed to preconcentrate seven metal ions dissolved in an aqueous sample for their simultaneous determination using energy-dispersive x-ray fluorescence spectrometry. The metal ions are precipitated as their polymeric piperazino-1,4-bis(dithiocarbamate) chelates, which are then collected by vacuum filtration on a Millipore membrane filter for direct examination by x-ray fluorescence analysis. Iron, cobalt, nickel, copper and zinc are determined by means of their K x-rays and mercury and lead by means of their L x-rays. A detection limit in the μg1⁻¹ range can be achieved for all metals tested in 250-ml water samples with a counting time of 600 s. Effective precipitation of all metals occurs at pH 6–7. The recoveries of eight analyses of the metals in a multielement standard using the proposed method ranged from 97 to 105% and the precision ranged from 2.3 to 3.1%. High concentrations of calcium and magnesium do not interfere with the method. The method is simple, sensitive and accurate, and has been used for the simultaneous determination of the seven metals under study in environmental samples and synthetic mixtures.     

Fluorescence polarization immunoassays for metal ions

Antibodies raised against a given metal ion complex of a polyaminopolycarboxylate chelating agent can display specificity for the immunizing chelate and, when used in conjunction with a fluorophore-labeled analog of that chelate, can form the basis for highly sensitive and specific methods for detecting that metal ion by competitive inhibition fluorescence polarization immunoassay (FPIA). Chelate complexes of ethylenediamine-N,N,N',N'- tetraacetic acid (EDTA) and of a hetrocyclic ring-substituted derivative of diethylenetriamine-N, N', N"-triacetic acid (DTTA) have been used to configure such assays for the heavy metal ions lead(II) and cadmium(II) respectively. Limits of detection for the 1:1 metal chelates under ideal conditions are 20 ppt for lead(II) and below 100 ppt for cadmium(II). Standard curves for 0 - 100 nM cadmium (II) chelate can be constructed in the presence of fixed 250 nM concentrations of the corresponding, potentially cross-reactive chelates of zinc(II), copper(II) and mercury(II). Cross-reactivity of the lead (II) FPIA with 15 non-target metals is below 0.2% in all cases except for mercury(II) (0.37%). These characteristics have allowed the development of FPIA methods for the quantitative analysis of lead in a variety of samples relevant to environmental monitoring, including soil, dust, solid wastes and drinking water. Although applied thus far to heavy metals that are of concern as toxic contaminants in the environment, anti-chelate FPIA methods are also in principle applicable to a wide variety of other metal ions, including precious metals and various transition and main group elements used or monitored in a range of industrial applications. As conventional methods for trace metal analysis based on atomic spectroscopy are relatively slow, expensive and cumbersome, anti-chelate FPIA methods have the potential to supplant many existing techniques and in so doing extend the use of immunoassay technology beyond the biomedical, veterinary and agricultural spheres in which it has historically found use.     

A spectrophotometric study of o,o'-dihydroxyazobenzene and its chelates with aluminum,gallium and indium

The composition of the chelates of aluminum, gallium and indium with o,o'-dihydroxyazobenzene has been established in acidic alcoholic -water mixtures to be pH dependent by means of spcctrophotometric measurements. Only chelates with a 1 : 1 and 2. : 1 ratio of o,o'-dlihydroxyazobenzene to metal are formed. The first acid dissociation constant of o,o'-dlihydroxyazobenzene and the formulas and stability constants for -the chelates were determined, The stability of the chelates increased in tle order indium, aluminum, gallium.     

Simultaneous Determination of Heavy Metals in Bovine Liver and Oyster Tissue by Solvent Extraction-Reversed Phase High Performance Liquid Chromatography

Abstract

High performance liquid chromatographie method for the simultaneous determination of ppm levels of cadmium, nickel, lead, zinc, cobalt, copper and bismuth in biological samples has been developed. Each 250 mg of Bovine Liver(NBS 1577) or Oyster Tissue(NBS 1566) was ashed in a muffle furnace over night at 500 to 55O°C. Then the ash was treated with 1.5 ml of 2 N hydrochloric acid; and the solution was put into a separatory funnel. The dissolved heavy metals were extracted into chloroform as hexamethylenedithiocarbamato chelates. The metal chelates were separated on a reversed phase column(5 μm, ODS, 4.6 × 15O mm), and determined by measuring the peak height of each metal chelate. Cd, Ni, Pb, Zn and Cu were determined accurately over the concentration range of 0.5–850 ppm with standard deviation ca. 7%.     

Phase-resolved fluorimetric determination of two albumin-bound fluorescein species

A new approach is described for phase-resolved fluorescence spectroscopy, for use in resolving mixtures of two components with very similar fluorescence spectra and life-times. Results are given for application of the technique to solutions containing fluorescein physically bound to albumin and fluorescein isothiocyanate covalently bound to albumin. Because the two fluorescein species have essentially identical fluorescence spectra and a phase-angle difference of only 2°, the conventional phase-resolved method in which measurements are made at the two phase angles at which the fluorescence contribution from one or the other of the components is zero will not resolve the components. Solutions containing 25–50 nM of each component were successfully resolved by making measurements at two other phase angles and solving the pair of simultaneous equations that is generated. Accuracy is best (average relative error, 3%) using detector phase angles corresponding to a 45° shift from the phase angles of the components. Relative standard deviations of ±16% are obtained at these phase angles. Solutions containing 5–50 nM fluorescein and 50–500 nM fluorescein isothiocyanate conjugated to albumin could also be resolved, with an average relative error of 16% and ±2.4 r.s.d. The method could be used for simultaneous determination of a fluorophore in two different microenvironments, as in protein-ligand binding studies and in homogeneous immunoassay.     

3,5,7,4'-Tetrahydroxyflavone (kaempferol) as a chromogenic reagent for gallium and indium

3,5,7,4'-Tetrahydroxyflavone (kaempferol) forms yellow chelates with indium and gallium. Based on this reaction, a sensitive spectrophotometric method for the determination of gallium and indium alone and in presence of other metals and alloys has been developed. Beer's law is obeyed up to 2.8 and 1.44 ppm for indium and gallium respectively.     

Fluorescence and liquid room-temperature phosphorescence of the aluminium-ferron chelate in vesicles

In order to evaluate the analytical potential of vesicles for enhancing the room-temperature luminescence of metal chelates, the photoluminescence emission of the aluminium-ferron complex in vesicular solutions was examined. The presence of vesicles of didodecyldimethylammonium bromide produced a six-fold enhancement in the fluorescence intensity of the metal chelate. Vesicles also proved to be efficient stabilizers to obtain analytically useful liquid room-temperature phosphorescence for aluminium. Optimum experimental conditions and luminescence characteristics of the aluminium complex in the presence of vesicles are described and compared with those obtained for the same chelate in micellar media. As a result, a vesicle-enhanced fluorimetric procedure is proposed which has been successfully applied to the determination of low levels of aluminium in milk powders.     

Structure, bonding, and fluorescence of bivalent metal chelates of o,o'-dihydroxyazobenzene

The structure and bonding of bivalent metal chelates of o,o'-dihydroxyazobenzene (DHAB) have been studied by PMR and absorption spectroscopic methods. These studies indicate that fluorescence, which is observed only for the MgDHAB chelate, may arise from a greater degree of ionic character in the metal-oxygen bonds of this compound than in the other metal(II) chelates examined. Possible structures of these chelate compounds are proposed and discussed.     

Liquid adsorption chromatography of chelates : The effect of structure on the thin-layer chromatographic behaviour of chelates

The thin-layer chromatographic behaviour of chelates belonging to different classes is discussed. The major adsorption interactions of chelates are hydrogen bonding between the ligand donor atoms and the surface hydroxyl groups and reactions between the metal atom and the electron-donor active centres of the sorbent. Predominance of either of these general mechanisms depends on the chelate structure and particularly on the coordination saturation of the chelate. Coordination-saturated chelates are retained because of hydrogen bonding, while the metal atom does not participate directly but can influence sorption by affecting the electron density distribution in the chelating ring. Atomic electronegativity is used as a measure of the electron-acceptor ability of the metal. Electronegative atoms located outside the functional group of the chelate can participate in the adsorption either directly or by affecting the proton-acceptor ability of the donor atoms as a result of induction and steric effects. The relationship between chelate retention factors and the parameters characterizing the electron and spatial structure of ligands can be described quantitatively by an equation of the type log [(/R_f)?1] = A + Σσ. In the case of coordination-unsaturated chelates, adsorption interactions with participation of the metal atom predominate, either by ion exchange (with ligand replacement) or by a donor-acceptor mechanism (with introduction of the adsorption centre into the coordination sphere without decomposition). In general, the adsorbability of chelates is directly related to the proton-acceptor ability of donor atoms and the acceptor ability of the metal atom. Classification of chelates by their adsorption interactions is proposed. Recommendations are given for selecting the optimal chelating reagent for the separation of metals by liquid-adsorption chromatography.     

Structure and behavior of organic analytical reagents : Heats and entropies of formation of several divalent metal chelates of 2- and 4-methyl-8-hydroxyquinoline1

The Calvin-Bjerrum titration technique for the determination of chelate formation constants has been applied to the Cu(II), Ni(II), Co(II), Zn(II) and Mn(II) chelates of 2- and 4-methyl-8-hydroxyquinoline. Measurements were made at several temperatures in order to evaluate ΔH and ΔS values of chelation. The results obtained were interpreted in terms of steric hindrance of the 2-methyl group. In all cases the heats of formation of the chelates of 2-methyl-8-hydroxyquinoline were remarkably morepositive than those for the corresponding chelates of 4-methyl-8-hydroxyquinoline. This large difference in the strengths of the metal-chelate bonds is apparently due to the hindrance of the methyl groups which prevent the close grouping of the two reagent molecules around the metal in chelates of 2-methyl-8-hydroxyquinoline. The lower bond strength in chelates of 2-methyl-8-hydroxy-quinoline is partially compensated by a relatively larger entropy of formation. This is attributed to decreased solvent chelate interaction caused by the shielding of the polar O, N, and metal atoms by the 2-methyl groups. The determination of chelate fortmation constants of 2-phenyl-8-hydroxyqumoline has been carried out to further extend our study of steric effects in metal chelates.     

Fiber-Optic time-resolved fluorimetry for immunoassays

Rare-earth metal chelates used as fluorescent labels for immunoassays possess extremely long fluorescence lifetimes and permit the elective use of time-resolved detection. This is very important in fiber-optic fluorimetry, a technique that ordinarily exhibits large signal backgrounds from back-scattered radiation. With time-resolved detection to reject the back-scattered radiation, the limit of detection for Eu 2-naphthoyltrifluoroacetonate is 10(-12)M, nearly three orders of magnitude lower than for the fiber-optic measurement of the most common fluorescent label, fluorescein isothiocyanate. Commercially available reagents labeled with an europium chelate are used to demonstrate the potential utility of time-resolved fluorimetry in fiber-optic immunoassays. Rabbit immunoglobulin G (IgG) is covalently bonded to the distal end of quartz optical fibers prior to exposure to anti-rabbit IgG labelled with europium chelate. The limit of detection for the assay is approximately 0.1 mug/ml.     

Systematic atomic number effects in complexes exhibiting ligand luminescence: Part 2. Salicylidene-o-aminophenolates

The room-temperature fluorescence properties of Group IIIA metal complexes with salicylidene-o-aminophenol (SoAP) in aqueous 20% methanol are reported. The data include absorption, emission and excitation maxima, quantum yields and luminescence lifetimes. A systematic heavy atom effect influences the fluorescence decay to an extent sufficient for simultaneous kinetic analysis of ternary metal ion mixtures, based on decayrate measurements.     

Metal ion capillary zone electrophoresis with direct UV detection: determination of transition metals using an 8-hydroxyquinoline- 5-sulphonic acid chelating system

The application of capillary zone electrophoresis to the separation and determination of metal ions after the precolumn formation of negatively charged chelates is described. Multi-component mixtures of transition metal complexes with 8-hydroxyquinoline-5-sulphonic acid (HQS) were separated in about 10 min in a fused-silica capillary column with a borate buffer of pH 9.2 at an applied voltage of 15 kV followed by direct UV detection. The capillary pretreatment with an electroosmotic flow modifier, namely a tetraalkylammonium salt, is necessary to achieve resonable migration times of these metal complexes. Incorporating the chelating reagent in the electrophoretic buffer markedly improves the detectability of relatively unstable chelates, such as those of Co(II), Zn(II) and Cd(II), and allows the separation of metal ions that form unstable HQS chelates, such as Mn(II) and alkaline earth metals. The effects of electrophoretic buffer parameters affecting the complexation reaction and migration behaviour are discussed. Linearity of calibration graphs is observed for about three orders of magnitude with sub-ppm detection limits. The applicability of the method to the analysis of real samples is demonstrated.     

Vapor-Liquid Equilibrators To Aid In Making Measurements To Determine Solvent Adducts On Metal Chelates

Abstract

Two vapor-liquid equilibrators to saturate water with organic solvent without forming two liquid phases were developed, thus permitting a more accurate determination of organic adducts to metal chelates. An equation was developed to show that vapor pressure measurements could be used as an alternative method for determining the number of organic adducts on a metal chelate in the aqueous phase.     

Mass spectrometry of metal chelates-I Detection on the picogram scale

A method has been developed for the detection and estimation of metals in the form of chelates in amounts down to 10(-12) g. The chelate is evaporated into the source of a mass spectrometer and a continuous record of the ion current at a significant mass number is made. The integrated ion current is directly proportional to the concentration of metal chelate.     

COMPLEXES OF GALLIUM(III) WITH HYDROXYAROMATIC LIGANDS

Abstract

The equilibria between gallium(III) ion and selected hydroxyaromatic and dihydroxyaromatic ligands at 25°C, μ=0.100 M (KNO₃) have been determined. Potentiometric measurements on 1:1, 2:1, and 3:1 molar ratios of ligand to Ga(III) have been made as a function of degree of neutralization over the entire accessible ?log [H⁺] scale. Calculations were carried out so as to take account of competing hydrolytic reactions, and formation constants of gallium(III) with chromotropic acid, 8-hydroxyquinoline-5-sulfonic acid, 5-sulfosalicylic acid, and 1,2-dihydroxy-benzene-3,5-disulfonic acid were obtained. Stable hydroxo chelates do not form under the reaction conditions employed. The protonation constants of the ligands and the formation constants of the gallium chelates are discussed and compared with previously published work on these gallium chelates and on chelates of “analogous” metal ions such as those of Fe(III) and A1(III).