Fluorescence acid-stable

Aspartame and its degradation products aspartylphenylalanine, aspartic acid, and phenylalanine can also be separated after reaction with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide (sodium or potassium cyanide) in borate buffer (50 mM, pH 8). This reaction affords highly fluorescent and stable l-cyano-2-substituted-benz[/]isoindole (CBI) derivatives that can be detected at 420 nm excitation and 490 nm emission. The CBI derivatives are separated on a TSK ODS-120T column using a gradient of 30-80% B (acetonitrile water, 9 1 v/v) in A (50 mM acetate buffer, pH 6.0) (75). 

For those without native fluorescence, two common approaches have been employed, namely, derivatization and indirect fluorescence. Derivatizing agents should be pure, low fluorescent, and stable, as well as react quickly and uniquely with the analytes and, thus, formed compounds should be strongly fluorescent and stable. These include dansyl chloride, fluorescamine, 4-clair-7-nitro-benz-2-oxa-l,3-diazole (NBD), o-phthaldialdehyde (OPA), fluorescein isothiocyanate (FITC), and naphthalene-2,3-dicarboxaldehyde (NDA), which have been used for the analyses of amino acids, peptides, proteins, thiols, and sugars with LOD in 1-100 nM range. Compared to LINF, approaches based on derivatization provide the advantages of relatively low cost and versatility in instrumentation, but they may suffer from contamination and loss of temporal information. 

As the stationary phase, modified silica gel RP-18 with acid-stable fluorescence indicator was intended. 

Wedge-shaped crystals, mp 247-248°. Slightly sol in water. alcohol, pyridine. Insoluble in ether and in aq acid soin, but Completely sol in aq alkaline soin. Possesses two acidic groups, one a phenolic and the other a carboxyl having pK values of 9.75 and 5.50. respectively. Characteristic blue fluorescence, max at pH 3 to 4. The fluorescence disappears on reduction with hydrosulfite and is restored to the original intensity with HjO.. Is adsorbed on zeolite from aq solns at pH 4 to 5 and can be eluted with 25% KCI butanol extracts of neutral eluates also show characteristic blue fluorescence which is increased by a trace of acetic acid. Stable to boiling with di) alkali (lJV> but upon being heated with 0.5N add for a few minutes it is converted to the lactone. 

Figure 4.13 pH-Titration of protein Hsp47 monitored by intrinsic tryptophan fluorescence spectroscopy (excitation 295 nm). Data is also indicative of a two stage transition between an Alkali stable state and an Acid stable state via a transient intermediate state (see Fig. 4.8). Fluorescence spectroscopy also reveals that Hsp47 will undergo reversible pFI-driven trans-conformational changes (see Chapter 7) (Reproduced from Momma et al., 2008). /em(iC) is fluorescence emission intensity.

One fluorescent indicator used is an alkali earth wolframate, which is acid stable and fluoresces pale blue or white. It is indicated by the letters and numbers UV254s/ F254s, meaning it is activated by 254 nm UV light and is acid stable. The other widely used fluorescent indicator is manganese-activated zinc silicate, which fluoresces green and is not acid stable. It is indicated by the letters and numbers UV254/... 

There are many published TLC protocols that can be used to separate flavonoids that have been extracted from tissue samples and juice. Methods include the use of silica, reverse phase or polyamide plates in a wide variety of solvent systems. Many of these solvent systems are published in a chapter on flavonoids in Kirchner s book on TLC (1967). The flavonoid spots on the developed plates can be visualized in a variety of maners, the most common method being the direct visualization under UV light using plates that have an acid-stable fluorescent indicatior added for visualization at 254 nm. The visualized spots can then be scraped and quantified by UV absorption. Another standard visualization method utilizes a spray of 2% sodium borohydride freshly prepared in methanol followed by fumigation with hydrogen chloride gas (Horowitz 1957). 

Xanthene Dyes. This class is best represented by Rhodamine B. It has high fluorescent brilliance but poor light and heat stabihty it may be used in phenohcs. Sulfo Rhodamine is stable and is useflil in nylon-6,6. Other xanthenes used in acryhcs, polystyrene, and rigid poly(vinyl chloride) are Solvent Green 4, Acid Red 52, Basic Red 1, and Solvent Orange 63 (see Xanthene dyes). 

Silica gel and aluminium oxide layers are highly active stationary phases with large surface areas which can, for example, — on heating — directly dehydrate, degrade and, in the presence of oxygen, oxidize substances in the layer This effect is brought about by acidic silanol groups [93] or is based on the adsorption forces (proton acceptor or donor effects, dipole interactions etc) The traces of iron in the adsorbent can also catalyze some reactions In the case of testosterone and other d -3-ketosteroids stable and quantifiable fluorescent products are formed on layers of basic aluminium oxide [176,195]... 

Properties of luciferin. The crystals are microscopic needles, which melt with decomposition at 205-210°C (Bitler and McElroy, 1957). It is a quite stable luciferin compared with some other luciferins, such as Cypridina luciferin and the luciferins of krill and dinoflagellates. It is not significantly affected by lOmM H2SO4 and lOmM NaOH at room temperature in air. The absorption spectral data of luciferin are shown in Fig. 1.3 (McElroy and Seliger, 1961). The molar absorption coefficient of the 328 nm peak in acidic solutions and that of the 384 nm peak in basic solutions are both 18,200 (Morton et al., 1969). Luciferin is fluorescent, showing an emission maximum at 537 nm in both acidic and basic conditions, although the intensity of the fluorescence is lower in acidic solution than in basic solution (fluorescence quantum yields 0.62 in basic condition, and 0.25 in acidic condition Morton et al., 1969). The chemical synthesis... 

Perhaps most encouraging in these discoveries was the observation that NDA/CN worked equally well for derivatization of dipeptides and higher homologues of the primary amino acid series. Again, a stable, fluorescent, isolatable derivative was obtained. One of the most important initial findings was the high fluorescence efficiency of the CBI adduct (12). Tables 1 and 2 list the efficiencies for a representative group of mono-, di-, and tripeptides and a limited comparison of the CBI efficiencies with the more traditional OPA (8) and dansyl (9) derivatives, respectively. 

It can be advantageous to heat the chromatogram to 160 °C for 15 min before treating with nitrous fumes and to place it in the reagent chamber while still hot [1]. Heating to 260 °C has even been recommended for the purpose of reducing the fluorescent background [14], whereby the layer is previously immersed in 1 percent Ludox solution (silidc acid sol) to increase its stability [2]. The fluorescence of the substances detected usually remains stable for at least 2 weeks [2]. 

Upon absorption of UV radiation from sunlight the bases can proceed through photochemical reactions that can lead to photodamage in the nucleic acids. Photochemical reactions do occur in the bases, with thymidine dimerization being a primary result, but at low rates. The bases are quite stable to photochemical damage, having efficient ways to dissipate the harmful electronic energy, as indicated by their ultrashort excited state lifetimes. It had been known for years that the excited states were short lived, and that fluorescence quantum yields are very low for all bases [4, 81, 82], Femtosecond laser spectroscopy has, in recent years, enabled a much... 

A competitive fluorescence-polarization immunoassay method was described for the monitoring of 12 drugs including valproic acid [18]. Samples (serum or plasma) were deproteinated. Fluorescence from the fluorescein-labeled analyte used as tracer was excited at 488 nm and polarization of light emitted at 531 nm was measured. The calibration was stable for 4 weeks and the coefficient of variation was below 10%. A single measurement took 8-10 min. 

Rye, H. S, Yue, S, Wemmer, D. E, Quesada, M. A, Haugland, R. P, Mathies, R. A. and Glazer, A. N. (1992). Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes -Properties and applications. Nucleic Acids Res. 20, 2803-2812. 

An alternative approach used to investigate the covalent binding of B[a]P to mouse skin has been to release the hydrocarbon-DNA adducts from the isolated DNA by acid hydrolysis (60.61). Since, in the case of BPDE-DNA adducts, they are acid labile and the tetraols produced are not only more fluorescent than the adducts themselves but also more easily extracted from the large excess of unmodified bases, this provides a convenient approach. The method does, however, require the certainty that hydrolysis of the adducts will occur and that the products will be stable or, if degradation occurs, that they can still be recognized. 

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