Fluorimetry specificity

Hirschfeld, T., Deaton, T, Remote Fiber Optic Fluorimetry Specific Analyte Optrodes , invited paper, Pittsburg Conference Atlantic City, NJ, Match 1982. 

As a consequence of the optimization of performance, different performing instruments were first designed for one specific task. However, the best instruments from the latest generation correspond to universal instruments that make it possible to run several types of experiments such as fluorimetry, luminometry, and densitometry for instance. This has interesting consequences for users who work in different domains of luminescence for research and development purposes and do not have to buy a whole range of instruments. 

It must be kept in mind, however, that CE data resemble a macroscopic effect of complexation and, like ultrafiltration or size exclusion chromatography CE, do not give information on specific binding sites. Despite the existence of a well-established theoretical basis, it is not possible to get reliable complexation constants, for practical reasons, if too many complexation processes are involved at the same time. Often the combination with spectroscopic investigations, especially with NMR and fluorimetry, may provide more details. 

HPLC units have been interfaced with a wide range of detection techniques (e.g. spectrophotometry, fluorimetry, refractive index measurement, voltammetry and conductance) but most of them only provide elution rate information. As with other forms of chromatography, for component identification, the retention parameters have to be compared with the behaviour of known chemical species. For organo-metallic species element-specific detectors (such as spectrometers which measure atomic absorption, atomic emission and atomic fluorescence) have proved quite useful. The state-of-the-art HPLC detection system is an inductively coupled plasma/MS unit. HPLC applications (in speciation studies) include determination of metal alkyls and aryls in oils, separation of soluble species of higher molecular weight, and separation of As111, Asv, mono-, di- and trimethyl arsonic acids. There are also procedures for separating mixtures of oxyanions of N, S or P. 

GC-MS has found wide application in studies of monoamines in both animal models and in human neuropharmacology [452]. Interest has centred on the use of selected ion monitoring in the determination of trace amounts of the amines, their metabolites and related substances with a possible function as neurotransmitters. The SIM approach complements established assay methods such as gas chromatography with electron capture detection (ECD), fluorimetry or enzymic assay. A check on specificity is afforded and in many cases enhancement in sensitivity and precision of measurement can be obtained. Method development, principally relating to estimation of central amine turnover, is noted in this Section and an outline of work on human depression serves to illustrate the potential of GC-MS to the study of CNS dysfunction. 

Analytical applications have been found for all parts of the electromagnetic spectrum ranging from microwaves through visible radiation to gamma (y) rays (Table 1). The emission and absorption of electromagnetic radiation are specific to atomic and molecular processes and provide the basis for sensitive and rapid methods of analysis. There are two general analytical approaches. In one, the sample is the source of the radiation in the other, there is an external source and the absorption or scattering of radiation by the sample is measured. Emission from the sample may be spontaneous, as in radioactive decay, or stimulated by thermal or other means, as in flame photometry and fluorimetry. Both approaches can be used to provide qualitative and quantitative information about the atoms present in, or the molecular structure of, the sample. 

The presence of two monochromators, and the fact that not all molecules with a chromophore fluoresce, means that fluorimetry is more specific than ordinary ultraviolet spectroscopy. This allows drugs that fluoresce to be assayed in the presence of other compounds that would interfere in an ultraviolet assay. 

More recently, confocal fluorimetry itself has been impressively extended. In particular, the implementation of multi-photon excitation opened the potential to excite different fluorescent labels by a single laser line [47]. This considerably simplified the optical setup of confocal instruments. For example, Heinze et al. [48] described a setup for two-photon excitation confocal fluorimetry where three molecular species were quantified simultaneously using a single laser. When included in screening systems, these spectroscopic advancements enable the quantification of enzymatic reaction rates on several substrates in parallel or, when applied for peptide or protein ligands, the simultaneous measurement of binding affinities on different target receptors. In this way, biopharmaceuticals can be selected on the basis of their specificity and selectivity. As a consequence, undesired side activities can be controlled very early in the hit identification process. 

Electrocapillary methods, described in Sections 13.2 and 13.3, are very useful in the determination of relative surface excesses of specifically adsorbed species on mercury. As discussed in Section 13.4, such methods are less straightforward with solid electrodes. For electroactive species and products of electrode reactions, the faradaic response can frequently be used to determine the amount of adsorbed species (Section 14.3). Nonelectro-chemical methods can also be applied to both electroactive and electroinactive species. For example, the change in concentration of an adsorbable solution species after immersion of a large-area electrode and application of different potentials can be monitored by a sensitive analytical technique (e.g., spectrophotometry, fluorimetry, chemiluminescence) that can provide a direct measurement of the amount of substance that has left the bulk solution upon adsorption (7, 44). Radioactive tracers can be employed to determine the change in adsorbate concentration in solution (45). Radioactivity measurements can also be applied to electrodes removed from the solution, with suitable corrections applied for bulk solution still wetting the electrode (45). A general problem with such direct methods is the sensitivity and precision required for accurate determinations, since the bulk concentration changes caused by adsorption are usually rather small (see Problem 13.7). 

The applications of automatic continuous segmented analysers can aiso be classified according to the type of detection system involved. Thus, 70-75 of all the methodologies described on this topic used molecular UV absorption spectroscopy (spectrophotometry, photometry), followed by ISE potentiometry (10-15 ) and, much less often, nephelometry, fluorimetry, etc. The applications described below were mostly developed with the aid of Technicon technology and are classified according to this criterion —other applications to specific problems related to laboratory processes are described in the corresponding chapters. 

Nash et al. (2000) (Methodologies for determination of antimony in terrestrial environmental samples). The review by Tolg (1987) (Extreme trace analysis of the elements -the state of the art today and tomorrow) is an insightful review by an experienced trace analyst concentrating on atomic spec-trometric methods including AAS, OES, XRE, MS with many variants of excitation. A table is provided comparing the capability of determinative methods listing the method, the specific technique, limit of determination, matrix effects, multielement determination, and speciation analysis. Methods compared include AAS, ZAAS, OES-DCP, OES-ICP, OES-MIP, OES-HC, EANES, AES, XRS, MS, NAA, voltammetry, LAS and fluorimetry. 

Except for this key target, the methodological task has been put, namely, to demonstrate the unique capabilities of the nonlinear laser fluorimetry method (which is not, so far, well known in a wide cirde of optidans) on the specific object. 

It was realized early on that the fluorescence exhibited by certain substances was a valuable property for their analysis because it was measurable at lower concentrations than optical absorbance, because it was linear with concentration over a wide concentration range, and because its relatively high specificity permitted determination even in the presence of other substances. Numerous analytical methods for the determination of compounds with native fluorescence have therefore been developed. Many methods were also developed where a compound of interest was converted to a fluorescent product by a specially devised chemical reaction to enable it to be determined by the sensitive technique of fluorimetry. Our main concern is with the application of reagents designed to make fluorescent derivatives of compounds of interest, in order to permit their determination or detection by fluorimetric methods allied to chromatographic (or electrophoretic) separation processes. 

The measurement of low concentrations of compounds by fluorimetry is normally the main purpose of fluorescence labelling of compounds which have no native properties that allow sensitive detection. Where there is fluorescence quenching by known or unknown components of the sample solutions or a lack of specificity of the fluorescence measurements, the application of alternative methods for the determination of fluorescent derivatives may be necessary. In such cases fluorescence may be used to monitor the chromatographic separation even though alternative quantitation methods are applied. Radioactivity measurement and mass spectrometry are frequently used. If a reagent is selected for a certain analytical purpose, besides its reactivity and the fluorescence and chromatographic characteristics of its derivatives, its accessibility to radioactive labelling and its suitability for qualitative and quantitative mass spectrometry should therefore also be considered. 

Radioactive Dns-Cl. [N-Meffiy/- C]Dns-CI (specific radioactivity 10—30 Q mol ) and [G- H]E)ns-Q (specific radioactivity 3—10Ci mmol ) are avciilable from several commercial sources. The labelled reagents can be applied just like unlabelled Dns-Cl. Their application allows the substitution of fluorimetry by the more convenient and automated liquid scintillation counting. The fluorescence... 

For quantitative derivative formation Dns-Cl has to be employed in large excess, so that even micro techniques for the preparation of the derivatives are expensive. Users of labelled Dns-Cl are thus frequently tempted to use too little reagent. More importantly, the specificity of the method is not increased, as compared with fluorimetry, by the use of a labelled reagent. Nevertheless, both I C] and [ H]Dns-Cl have been used for the identification or estimation of amino acids and amines [93, 94]. 

Molecular techniques based on ultraviolet (UV)-visible spectrophotometry and fluorimetry have been also proposed for direct determination of a specific element form. Colored species, such as those of Cr or Mn, can be directly determined spectrophotometri-cally. However, most of such species will require the use of a selective color-forming reagent, lacking the sensitivity, and often the selectivity, required for... 

---