Fluorescence quench thin-layer

Color reproduction of the chromatograms can be achieved by color photography — the best, but also the most expensive method of documenting thin-layer chromatograms. It can be used not only to produce true-color reproductions of colored zones but also — with the aid of a Reprostar (Fig. 64) or a UVIS analysis lamp (Fig. 6) — of fluorescent or fluorescence-quenched zones. When photograph-... 

Thin-layer plates were made with silica gel-calcium sulfate and each contained a mixture of zinc silicate and zinc cadmium sulfide as phosphors. Separated components are generally visible under ultraviolet light by fluorescence quenching. This was true, in part, for the pyrethrins, except that some of the separated components possessed a natural fluorescence under the ultraviolet lamps. 

In our previous paper (H), we introduced a novel experimental method to study the mechanistic details of solvent permeation into thin polymer films. This method incorporates a fluorescence quenching technique (19-20) and laser interferometry ( ). The former, in effect, monitors the movement of vanguard solvent molecules the latter monitors the dissolution process. We took the time differences between these two techniques to estimate both the nascent and the steady-state transition layer thicknesses of PMMA film undergoing dissolution in 1 1 MEK-isoproanol solution. The steady-state thickness was in good agreement with the estimate of Krasicky et al. (IS.). ... 

The inclusion of a fluorescent dye into thin-layer plates can be used to detect substances that quench its fluorescence and so result in dark zones when the chromatogram is examined under ultraviolet radiation. Autoradiography can also be used in thin-layer chromatography and electrophoresis when samples are radio-labelled. 

Figure 5. Thin-layer chromatogram of tris (2,3-dibromopropyl)phosphate on fluorescein impregnated silica gel solvent methylene chloride. Scan in fluorescence quench mom. (a) 3.7 /ig TRIS (b) 0.44 ttg TRIS.

Jager, J., Detection and Characterization of Nitro Derivatives of Some Polycyclic Aromatic Hydrocarbons by Fluorescence Quenching after Thin-Layer Chromatography Application to Air Pollution Analysis, J. Chromatogr., 152, 575-578 (1978). 

The Zeiss PMQ 3 chromatogram analyzer is probably the most versatile thin-film scanner available (Fig.3.13). The system can be used for reflectance, transmission, simultaneous reflectance and transmission and fluorescence quenching. It has two direct fluorescence modes, one with filter emission and surface illumination at a direction of 90° to the surface of the plate, and the other with 45° illumination and monochromatic emission. The instrument can be used for scanning thin-layer chromatograms, paper... 

Other techniques that have been used to determine polycyclic aromatic hydrocarbons in soil extracts include ELISA field screening [86], micellar elec-tr okinetic capillary chromatography [ 87], supersonic jet laser-induced fluorescence [88,89], fluorescence quenching [90], thermal desorption gas chromatography-mass spectrometry [81,90,100], microwave-assisted extraction [91], thermal desorption [92], immunochemical methods [93,94], electrophoresis [96], thin layer chromatography [95], and pyrolysis gas chromatography [35]. 

I. Thin-layer chromatography Mangalan et al. [53] used of an HPTLC method for the detection and determination of omeprazole in plasma levels. Plasma was extracted three times with dichloromethane at pH 6.5-7 and the combined extracts were evaporated to dryness at 60 °C. The residue was dissolved in dichloromethane and the solution was analyzed by TLC on aluminium-packed plates precoated with Silica gel 60 F254 with the upper organic layer of butanol-ammonium hydroxide-water (14 1 15) as mobile phase. The spots were observed by fluorescence quenching under UV light illumination at 280 nm the total area of each... 

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.)...

Although TLC-MS (mass spectrometry) has been shown to be technically feasible and applicable to a variety of problems, thin-layer chromatography is generally coupled with spectrophotometric methods for quantitative analysis of enantiomers. Optical quantitation can be achieved by in situ densitometry by measurement of UV-vis absorption, fluorescence or fluorescence quenching, or after exctraction of solutes from the scraped layer. The evaluation of detection limits for separated enantiomers is essential because precise determinations of trace levels of a d- or L-en-antiomer in an excess of the other become more and more important. Detection limits as low as 0.1% of an enantiomer in the other have been obtained. 

An evanescent wave biosensor was devised for determination of analytes capable of intercalation in dsDNA in a FIA system. A polyethylene lensed optical fiber is coated with a thin polymeric layer containing dsDNA which is immobilized there. The fiber is placed in a FLA system immersed in a solution of ethidium bromide (144), which undergoes intercalation in the dsDNA. The fluorescence signal of 144 is thus enhanced about a 1000-fold relative to the evanescent wave fluorescence measurement without the coating and is dependent on the concentration in solution. If an analyte is present in the same solution, it competes with 144 for intercalation in the DNA and causes fluorescence quenching, which can be measured and correlated to the analyte concentration. This method was applied to determination of various analytes, including 4, 6-diamidino-2-phenylindole dihydrochloride (145)247. 

The DNP-amino acids, after separation into individual spots on the chromatographic plate, can be eluted from the scraped off area by adding 4 ml of water to the material in a small tube. The tube is heated at 50° in a water bath for 15 minutes and centrifuged to clear the solution. The color is read against known standards at 360 nm. Direct estimation of DNP-, PTH-, and DANS-amino acids separated on the thin-layer plate can be performed by fluorescence and fluorescence quenching techniques (P8). It is also possible to convert unmodified amino acids, separated on a silica gel G chromatographic plate, into DNP-amino acids by in situ conversion as was described in Section 4.7.18. The DNP-derivatives can then be developed in the second dimension and the spots analyzed quantitatively. 

Frequently, colorless or nonfluorescent spots can be visualized by exposing the developed plate to iodine vapor. The iodine vapor interacts with the sample components, either chemically or by solubility, to produce a color. Thin-layer plates and sheets are commercially available that incorporate a fluorescent dye in the powdered adsorbent. When held under ultraviolet light, dark spots appear where sample spots occur due to quenching of the plate fluorescence. 

Fluorescence can be used for detection in two different ways. Firstly, the separated compounds could be labeled with a fluorescent label (if not fluorescent as such), or UV absorbing compounds could be visualized as dark bands in matrices to which a fluorescent chromophore was added (fluorescence quenching). A variation of the second method was worked out by Eisinger [201] for polyacrylamide gel the gel was placed between a quartz and a fluorescent glass plate and then illuminated with UV light. Ultraviolet absorbing compounds appear as dark bands on the glass plate. When electrophoresis is done on sihca gel thin layer or cellulose, addition of a fluorescent indicator to the sorbent can be made use of in the conventional way. 

The pH-sensitive chemistry consists of a cellulosic material to which hydroxypyrene trisulfonate (HPTS) is covalently bonded. The C02-sensitive material is a fine emulsion of a hydrogen-carbonate buffer (plus HPTS) in a two-component silicone. The oxygen-sensitive chemistry is simply a solution of chemically modified decacyclene (which is strongly quenched by oxygen) in a one-component silicone. To make it insensitive toward halothane (an inhalation narcotic), it is covered with a thin layer of black PTFE, which also serves as an optical isolation. The fluorescence intensities of the three sensing spots can be related to PO2, pH, and PCO2 via modified Stem-Volmer or Henderson-Hasselbalch algorithms. 

Many fluorescence sensors are based, not on direct fluorescence, but on the quenching of fluorescence. Molecular oxygen, for example, is one of the best col-lisional quenchers. Oxygen can quench the fluorescence from polycyclic aromatic hydrocarbons complexes of ruthenium, osmium, iridium, and platinum and a number of surface-adsorbed heterocyclic molecules. An oxygen sensor can be made by immobilizing the fluorophore in a thin layer of silicone on the end of a fiber-optic bundle. Sensors for SO-, haliilcs, H-O-, and several other molecules have been ba.sed on fluorescence quenching. 

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