Native fluorescence detection, applications

Most applications of native fluorescence detection involve the direct detection of a fluorescent analyte of interest. However, native fluorescence can also be used indirectly to detect the presence of nonfluorescent species. For example, the presence of a nonfluorescent enzyme can be detected based on its conversion of substrate into natively fluorescent products. This is often used in conjunction with electrophoretically mediated microanalysis, in which differences in buffer mobilities enable on-column mixing and reaction of enzymes with substrates. One such study exploited the intrinsic fluorescence of nicotinamide adenine dinucleotide (NADH) to investigate differences in the reactivity of individual molecules of lactate dehydrogenase. 

Recently the continuous-addition-of-reagent (CAR) technique [182] was applied for the determination of fluorophores by POCL chemistry [95-99], The applicability of this technique was demonstrated by the determination of natively fluorescent acepromazine in horse plasma [95], the alkaloid harmaline in plasma [96], and other dansylated alkaloids [97], A separation step has also been included and applied to postcolumn detection of PAHs [98] and dansylated P-carboline alkaloids [99],... 

This type of derivatization seems promising for the detection of drugs and metabolites with phenolic groups. A recent application on the determination of chlorophenols in surface water showed that the dansyl derivatives of phenols are readily convertible to the highly fluorescent dansyl-OH and dansyl-OCHs products after postchromatographic irradiation (281). Fluorescence gain factors of up to 8000-fold were obtained for chlorophenol derivatives with a low native fluorescence. 

Fluorescence, however, is often detectable at concentrations up to 10 times lower than those where absorbance is detectable. Native fluorescence is less common than absorption of UVor visible light. In this regard, fluorescence may be thought to bemore selective or less applicable in range than absorption, depending on one s point of view. 

Although not generally used for applications where sensitivity of detection is a limiting factor, UV detection at a wavelength of 280 nm may be utilised. The minimum detection limits for the determination of noradrenaline, DOPA and dopamine have been quoted to be between 5 and 10 ng, i.e. approximately half that achievable using native fluorescence systems (Scratchley et al., 1979). 

Application of fluorescence-detected circular dichroism (FDCD) to the determination of the major pterin (L-monapterin) from Escherichia coli has been published <01H(54)283>. Identification of (6/ )-5,6,7,8-tetrahydro-D-monapterin as the native pleridine in Tetrahymena pyriformis has been reported <01HCA918>. A novel modified pterin has been isolated from... 

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 small inner diameter of the separation capillary used in CE implies a short optical pathway, and the consequent poor concentration sensitivity when conventional UV detectiOTi is used. To overcome this drawback several techniques have been developed some of them consist in application of general approaches that are not specifically addressed to CE analysis of alkaloids. One is the use of LIF detector for analysis of alkaloids with native fluorescence [68, 69] or after their off-line derivatization [64, 88, 112]. Sample pretreatment, a second major approach, is popularly employed in combination with sample extraction and can be conveniently applied in analysis of alkaloids because they can be easily retained in cationic-exchange sorbents in solid-phase extraction (SPE) mode [113, 114]. It may be interesting to focus on more specific aspects to detect very low levels of analytes using limited amoimts of samples to this regard chemiluminescence reactions and the use of online preconcentration methods will be considered. 

LIF has gradually become the most sensitive technique for analyte detection in narrow-bore capillaries e.g. in CE-LIF applications [10]). Also laser fluorometric detection for TLC was reported [122], LIF detection often relies on derivatisation of the target molecules, as most analytes do not show native fluorescence when being excited with commercial lasers. 

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. 

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