Fluorometry limitations

Riboflavin can be assayed by chemical, en2ymatic, and microbiological methods. The most commonly used chemical method is fluorometry, which involves the measurement of intense yeUow-green fluorescence with a maximum at 565 nm in neutral aqueous solutions. The fluorometric deterrninations of flavins can be carried out by measuring the intensity of either the natural fluorescence of flavins or the fluorescence of lumiflavin formed by the irradiation of flavin in alkaline solution (68). The later development of a laser—fluorescence technique has extended the limits of detection for riboflavin by two orders of magnitude (69,70). 

The types of compounds that can be analyzed by fluorometry are rather limited. Benzene ring systems, such as the vitamins riboflavin (Figure 8.13) and thiamine, are especially highly fluorescent compounds and are analyzed in foods and pharmaceutical preparations by fluorometry. Metals can be analyzed by fluorometry if they are able to form complex ions by reaction with a ligand having a benzene ring system. 

Fluorometry and absorption spectrophotometry are competing techniques in the sense that both analyze for molecular species and complex ions. Each offers its own advantages and disadvantages. As stated above, the number of chemical species that exhibit fluorescence is very limited. However, for those species that do fluoresce, the fluorescence is generally very intense. Thus we can say that while absorption spectrophotometry is much more universally applicable, fluorometry suffers less from interferences and... 

Although AS and AES can be detected at a low UV wavelength, sensitivity is lacking and a more suitable detection was achieved using indirect photometric detection, post-column colour formation reactions, or a pre-column derivatisation, suppressed conductivity detection and refractive index detection [1,42,43]. A comparison of detection limits for the determination of these anionic surfactants shows that photometric and conductivity detectors are better (picomole or nanogram range) than refractive index or fluorometry detectors by about a factor of 1000 [40],... 

Information concerning the tertiary structure of the proteins has been obtained from fluorometry, proton magnetic resonance spectroscopy, limited proteolysis, and X-ray analysis of protein crystals. 

Thus, by the technique of in situ fluorometry on tic it is possible to analyze the parent compound and all of the common degradation products simultaneously. The detection limit is 0.01 yg per spot with an average standard deviation of 7.25% for 0.4 yg spots. 

It has long been recognized that both the diffuse spectra and quenching problems can be alleviated by performing the fluorescence measurement in a low-temperature solid matrix, rather than in a fluid solution. The most common low-temperature matrices used in molecular fluorometric analysis are frozen liquid solutions the analytical characteristics of frozen-solution luminescence spectrometry have been discussed extensively in the literature (2-10). Obviously, MI represents an alternative technique to use of frozen liquid solutions for low-temperature fluorometric analysis. There are two principal advantages of MI over frozen-solution fluorometry. First, in MI, any material which has an appreciable vapor pressure at room temperature can be used as a matrix one is not limited by the... 

Comeal organ culture combined with objectively quantifiable assays for comeal epithelial barrier disruption reduces the high variability associated to the subjectively scored Draize Test. The FITC-Dextran retention has been studied as a quantitative evaluation of the comeal epithelial barrier (Lopez et al. 1991) following chemical exposure of bcnzal konium chloride (BAC), Polyquad, and Thimerosal. Sodium dodecyl sulfate (SDS) has also been tested for disruption of the tight junctions via FITC-Dextran retention assay. However, as an objective outcome measure for ocular toxicity, the scoring system is not yet quantitatively comparable for assessment of ocular irritancy to multiple test products. This limitation is similar to surface biotinylation assays. As fluorometry is utilized more extensively in varied laboratories with numerous test chemicals a standardized scoring system can be elicited similar to the familiar Draize Test. 

The decay times and detection limits of some lanthanides measured with time resolved fluorometry using different enhancement systems are given in Table 12.33. 

While luminescence in vapor-deposited matrices accordingly should be a powerful technique for detection and quantitation of subnanogram quantities of PAH in complex samples, it suffers from two major limitations. First, it is obviously limited to the detection of molecules which fluoresce or phosphoresce, and a number of important constituents of liquid fuels (especially nitrogen heterocyclics) luminesce weakly, if at all. Second, the identification of a specific sample constituent by fluorescence (or phosphorescence) spectrometry is strictly an exercise in empirical peak matching of the unknown spectrum against standard fluorescence spectra of pure compounds in a hbrary. It is virtually impossible to assign a structure to an unknown species a priori from its fluorescence spectrum qualitative analysis by fluorometry depends upon the availabihty of a standard spectrum of every possible sample constituent of interest. Inasmuch as this latter condition cannot be satisfied (particularly in view of the paucity of standard samples of many important PAH), it is apparent that fluorescence spectrometry can seldom, if ever, provide a complete characterization of the polycyclic aromatic content of a complex sample. 

A laser system that delivers pulses in the picosecond range with a repetition rate of a few MHz can be considered as an intrinsically modulated source. The harmonic content of the pulse train - which depends on the width of the pulses - extends to several gigahertz. The limitation is due to the detector. For high frequency measurements, it is absolutely necessary to use microchannel plate photomultipliers (that have a much faster response than usual photomultipliers). The highest available frequencies are then about 2 GHz. As for pulse fluorometry, Ti sapphire lasers are most suitable for phase fluorometry, and decay times as short as 10-20 ps can be measured. 

The first automatic immunoanalyzers used fluorescent labels. These provided automation and short turn-around times, but due to a fairly high nonspecific background the sensitivity of assays was not improved over that obtained by RIA. Introduction of time-resolved fluorescence facilitated the development of assays with substantially reduced detection limit [1], here called improved sensitivity. This was based on reduction of the background, which facilitated the use of a large excess of label in sandwich-type immunometric assays and a much extended measuring range [3-5]. Other nomadioactive reporter molecules, e.g., luminescent labels or enzymes combined with luminescent or fluorescent substrates, can provide similar sensitivity of assays [2]. However, time-resolved fluorometry is unique in facilitating development of fully automated in-house assays, and it has therefore become widely used in research laboratories. 

Solid pellet fluorometry (or fluorimetry) is one of the classic older methods that were widely used to determine the uranium content in urine (Centanni et al. 1956). The urine sample is added to solid NaF or NaF/LiF that is fused by heating so that water and volatile organic and inorganic compounds are evaporated. The sample is then excited by UV radiation at 320-370 nm and the fluorescence at 530-570 nm is measured (perpendicular to the incident beam). The sensitivity is about 30 pg L for a 0.1 mL sample, but after preconcentration by ion exchange detection limits of 0.1 0.1 pg L for a 10 mL have been reported (Dupzyk and Dupzyk 1979). Even this improved MDL is insufficiently sensitive for monitoring unexposed populations where the expected concentration of uranium in urine is below 0.02 pg L (20 ng L )-... 

Fluorometry is used to determine elements such as boron, silicon, aluminum, beryllium, and zirconium [7], [129], [130], as well as organic compounds [131] (e.g., vitamins [132]). An important application in the field of trace analysis is the determination of aromatic hydrocarbons in wastewater [133], Here, the limits of detection can be improved considerably [64] if fluorescence is excited by a laser (LIF). This is very important for the measurement of polycyclic aromatic hydrocarbons [134],... 

Alumina (Chromarods A) was as good as silica gel (Chromarods S 111) for the separation of neutral lipids, but silica gel produced better separation and higher FID response for polar lipids Chromarods S-III (silica gel-coated quartz rods) were used to quantify neutral and polar lipids in cooked beef Nile red solution used for detection of lipids densitometry by reflectance fluorometry detection limit of assay was 25-100 ng for each lipid... 

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