Detection by fluorescence excitation

M. Orrit and J. Bernard, Single pentacene molecules detected by fluorescence excitation in a p-terphenyl crystal, Phys. Rev. Lett. 65, 2716-2719 (1990). 

Short-wavelength UV radiation (A = 254 nm) is employed for excitation. This allows aromatic organic compounds, in particular, to be detected by fluorescence quenching. Uranylacetate may also be excited at A = 366 nm. 

Free porphyrin acids, including isomers, are separated depending on their polarity on a reverse-phase system [9, 10]. The detection by fluorescence is highly specific because both their excitation and their emission are at relatively long wavelengths. 

Drug residues in foods that strongly fluoresce can be more efficiently detected by fluorescence detectors. Typically, fluorescence sensitivity is 10-1000 times higher than that offered by a UV detector for strong UV-absorbing materials (125). Using a fluorescence detector, it has been possible to detect the presence of even a single analyte molecule in an LC flow cell. This type of detection is very versatile because of its ability to measure the intensity of the fluorescent radiation emitted from analytes excited by UV. 

Beyer and Welge43 have photolyzed NO by use of very far ultraviolet radiation. Photodissociation was detected by fluorescence between 1100 and 1500 A. The primary processes depended on the exciting wavelength and formed electronically excited oxygen and... 

Fig. 8 Serum PC molecular species distribution after 6 weeks of treatment for both the placebo control group (A) and the drug-treated group (B). The molecular species were derivatized with 7-methoxycoumarin-3-carbonyl azide prior to analysis and detected by fluorescence with excitation at 340 nm and emission at 370 nm. (Reprinted from Ref. 91 with the permission of the Journal of Lipid Research.)...

Fig. 9 Reversed-phase chromatogram of frozen pea extract. The C, 8-column was subjected to an isocratic elution by acetone water (70 17 13). The chlorophylls and pheophytins were detected by fluorescence, with the excitation wavelength at 413 nm and the emission wavelength at 669 nm. (From Ref. 107.)...

Fine structure experiments are often carried out with synchrotron sources, since the initial electron state is better defined for photoemission than for electron excitation. When core-hole decay is detected by Auger or secondary electron emission, the technique is surface sensitive. Core-hole decay can also be detected by fluorescence, or by adsorption of the incident photon beam. These methods are not intrinsically surface sensitive, but they are useful when the source atoms are exclusively located at the surface. 

The monobromobimane derivatives of cysteine, y-glutamylcysteine, and glutathione were separated on a Merck Supersphere RP-18 column (4.0 mm X 250 mm). The mobile phase was methanol-water (18 82) containing 0.25% acetic acid adjusted to pH 3.8 with NaOH. The flow rate was 1.0 mL/min. Detection was by fluorescence, excitation and emission wavelengths 370 and 485 nm, respectively. 

The fluorimetric determination of -aminolevulinic acid is based on derivati-zation with acetylacetone and formaldehyde. The fluorescent derivative was separated at ambient temperature from other reaction components on a Unisil NQ C18 column (4.6 mm x 250 mm). The mobile phase was composed of methanol-water-glacial acetic acid (600 400 10, v/v). Detection is by fluorescence (excitation, 370 nm emission, 460 nm). 

In these multichromophoric cyclodextrins the fluorophores are randomly oriented. Excitation of one of the naphthoate fluorophores is followed by efficient dipole-dipole excitation energy transfer between the seven fluorophores, with a Forster radius of 14 A. This process is not detectable by fluorescence intensity measurements, as neither the intensity nor the decay law are affected by energy transfer between identical fluorophores (also called homotransfer. The dynamics of energy hopping are on the other hand reflected in the fluorescence anisotropy. To avoid depolarization by rotational motion of the fluorophores, experiments were conducted in a low temperature and optically clear rigid glass (9 1 ethanol-methanol at 110 K). 

Polymer films applied to wool as shrinkproofing treatments can also be selectively stained with optical brighteners and then detected by fluorescence microscopy. The levelness of their application can also be tested in this way the wool sample is treated with a 0.5% solution of the optical brightening agent Ciba Uvitex BHT 180% for 30 min at 0- 2 °C (cooled with ice) at a liquor ratio of about 1 100, then rinsed three times for half a minute each with cold water and dried. After UV excitation the polymer film can be recognized by its strong blue fluorescence. ... 

Figure 11.12 Comparison, following a chromatographic separation, of UV and fluorescence detection. Aflatoxins, which are carcinogenic contaminants present in certain hatches of grain cereals, are the subject of analysis by HPLC. Detection by fluorescence is much more sensitive to Gj and B2 than with UV detection (reproduced courtesy of SUPELCO). Below left, schematic of the different components of a LC-detector based upon fluorescence. This detector is able to find rapidely, for each compound eluted, the best coupling of excitation/emission without interrupting the chromatography underway (reproduced courtesy of a document from Agilent Technologies).

The H + NO2 OH + NO reaction provides an excellent example of the use of laser fluorescence detection for the elucidation of the dynamics of a chemical reaction. This reaction is a protot5q)e example of a radical-radical reaction in that the reagents and products are all open-shell free radical species. Both the hydroxyl and nitric oxide products can be conveniently detected by electronic excitation in the UV at wavelengths near 226 and 308 nm, respectively. Atlases of rotational line positions for the lowest electronic band systems of these... 

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