Thin-layer measuring fluorescence

An extremely sensitive procedure can detect 0.76 ng of phenoxymethyl penicillin. After hydrolysis, the secondary amine of penicilloic acid is coupled with 9-isothiocyanatoacridine to form a fluorescent compound. Following thin-layer chromatography, fluorescence of the spot is measured, with linearity in the 3 to 30 ng range. 

Densitometry is the instrumental measurement of visible or UV absorbance, fluorescence, or fluorescence quenching directly on the thin layer. Measurements are made either through the plate (transmission), by reflection from the plate, or by reflection and transmission simultaneously, and either single-beam, doublebeam, or single-beam-dual wavelength operation of scanning instruments has been used. 

Finally, and apart from the importance of micelles in the solubilization of chemical species, mention should also be made of their intervention in the displacement of equilibria and in the modification of kinetics of reactions, as well as in the alteration of physicochemical parameters of certain ions and molecules that affect electrochemical measurements, processes of visible-ultraviolet radiation, fluorescence and phosphorescence emission, flame emission, and plasma spectroscopy, or in processes of extraction, thin-layer chromatography, or high-performance liquid chromatography [2-4, 29-33],... 

N-methylcarbamate and N,N -dimethylcarbamates have been determined in soil samples by hydrolyses with sodium bicarbonate and the resulting amines reacted with 4-chloro-7-nitrobenzo-2,l,3-Oxadiazole in isobutyl methyl ketone solution to produce fluorescent derivatives [81]. These derivatives were separated by thin layer chromatography on silica gel G or alumina with tetrahydrofuran-chloroform (1 49) as solvent. The fluorescence is then measured in situ (excitation at 436 nm, emission at 528 and 537nm for the derivatives of methylamine and dimethylamine respectively). The... 

Figure 8.4. Fluorescence excitation spectra of pyrene (sample two of Figure 8.3) measured at A = 392 nm (region of Mi-emission) and J.e = 500 nm (region of A/ -emission) upper panel thick layer (d = 1 cm), lower panel thin layer (single grains).

HPLC-based electrochemical detection (HPLC-ECD) is very sensitive for those compounds that can be oxidized or reduced at low voltage potentials. Spectrophotometric-based HPLC techniques (UV absorption, fluorescence) measure a physical property of the molecule. Electrochemical detection, however, measures a compound by actually changing it chemically. The electrochemical detector (ECD) is becoming increasingly important for the determination of very small amounts of phenolics, for it provides enhanced sensitivity and selectivity. It has been applied in the detection of phenolic compounds in beer (28-30), wine (31), beverages (32), and olive oils (33). This procedure involves the separation of sample constituents by liquid chromatography prior to their oxidation at a glassy carbon electrode in a thin-layer electrochemical cell. 

In the case of enzymes involved in biochemical pathways, the isolation is often based on activity assays. The nature of the activity assay depends on the enzymatic reaction and can involve, for example, the detection of a product on a thin-layer chromatography (TLC) plate (see Chapter 4, Section 1.2.1), the appearance or disappearance of a specific absorbance in a spectrophotometric assay, or a coupled assay involving the oxidation or reduction of a co-factor such as nicotinamide dinucleotide (NAD(H)), which can be measured by changes in fluorescence. 

Fig. 2 Normalized fluorescence lifetime distributions (ELDs) for (a) the cGMP-imprinted and (b) non-imprinted thin-layer polymer film obtained from the fluorescence microscopy measurements (adapted from [47, 66])...

If no fluorophore exists in a given area of the thin-layer plate, then no emission signal can be obtained. The fluorescence of the sample is then an absolute quantity relative to this zero signal and proportional to the number of emitting species present in the sample. However, in practice, the adsorbent does contain trace amounts of fluorescent impurities and thus background noise is observed, but usually at a lower level than experienced in absorption measurements. The low background noise is an important factor in the high sensitivity of fluorescence. 

Semenov et al. [8] determined small amounts of petroleum products in chloroform extracts of non saline water by extracting the sample (200-500mL) followed by thin layer chromatography on alumina. He developed the chromatogram with light petroleum-carbon tetrachloride-acetic acid (35 155 1), and examined the plate in ultraviolet radiation the petroleum products exhibit three zones (pale blue, yellow and brown). Each zone is then extracted with chloroform, the fluorescence of the extracts measured and the results referred to a calibration graph. The sensitivity is O.lmg L. The infrared and fluorescence spectra of the zone obtained with various petroleum products are discussed. 

Figure 12. Relative spectral distribution of a low-pressure mercury vapor lamp with visible light cut-off filter as measured through the emission monochromator of a spectrofluorometer (2 nm slit width). (Typical of lamps used to view fluorescence/ fluorescence quenching on thin-layer chromatographic plates.)...

The literature on fluorescence analysis has also been reviewed periodically since 1949, originally by White C5.) and lately by O Donnell and Solie C ). Only a very brief introduction to the subject as it relates to fluorescence measurements on thin-layer chromatograms will be given in this paper. 

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