Fluorescence impurity

Benzoin (2-hydroxy-2-phenyiacetophenone) [119-53-9] M 212.3, m 137 , Crystd from CCI4, hot EtOH (8mL/g), or 50% acetic acid. Crystd from high purity benzene, then twice from high purity MeOH, to remove fluorescent impurities [Elliott and Radley Anal Chem 33 1623 1961]. Sublimes. 

Diaminoanthraquinone [131-14-6] M 238.3, m 310-320°. Crystd from pyridine. Column-chromatographed on AI2O3 / toluene to remove a fluorescent impurity, then recrystd from EtOH. 

Diaminonaphthalene [771-97-1] M 158.2, m 199°, pK 3.54 (in 50% aq EtOH). Crystd from water, or dissolved in O.IM HCl, heated to 50°. After cooling, the soln was extracted with decalin to remove fluorescent impurities and centrifuged. 

Raman spectra (for both the solid state and aqueous solution) provide better fingerprints for heparins than their i.r. spectra.79 However, the application of Raman spectroscopy to glycosaminoglycans is less routine than with i.r., both for instrumental reasons and because of possible interference from traces of fluorescent impurities.77... 

Poly(styrene) and PMMA were synthesized from their respective monomers using azobisisobutyronitrile-initiated radical polymerization in benzene. Four freeze-pump-thaw cycles were used to degas the monomer solutions and polymerization was carried out for 48 hours at 60°C. The polymers were purified by multiple reprecipitations from dichloromethane into methanol. Films of these polymers were prepared and found to be free of any fluorescent impurity. 

Fluorescence spectroscopy is probably the most sensitive method for determining the purity of an IL. It has been observed that the fluorescence of ILs can be effectively reduced to zero by a systematic purification of precursor salts. For the lowest fluorescence response, particular care should be taken while carrying out s)mthesis. For convenience it is possible to judge the degree of fluorescence impurity in the IL by exposing a sample to the UV light from a hand scanner at 380 nm. If the IL has significant impurities, it will fluoresce visibly [40]. 

By introducing the complexing moiety —CH2N(CH2C02H)2 into fluorescein or umbelliferone metallochromic indicators termed calcein or fluorexone, and calcein blue or umbellicomplexone are formed. These indicators exhibit green (and blue) fluorescence in dilute alkali which disappears at pH 12 but reappears on the addition of calcium, strontium, or barium. Problems arise from the difficulty of freeing the indicators from fluorescent impurities, from self-fluorescence, and from the quenching effect of copper ions. 

Again, transfer 3.0 mL of the pH 7.5, ethidium bromide-Tris buffer solution into the cuvette. Add 15 /xL of the unknown DNA solution. Mix well and place in the spectrofluorimeter. Record the fluorescence intensity, Z7 [. The measured fluorescence is due to both DNA and RNA (if present). The amount of fluorescence due to ethidium-RNA can be eliminated by digesting the RNA with ribonuclease. To the cuvette containing pH 7.5, ethidium bromide-Tris buffer and unknown DNA, add 2 fiL of ribonuclease solution. Mix well and incubate the cuvette at 37°C for 20 minutes in a water bath. After incubation, measure the fluorescence intensity, (Atotal). Compare this with the original measurement taken on the unknown DNA (Atota ). Is RNA present in your DNA sample Calculate the concentration of DNA in your unknown solution as described in the Analysis of Results. The possibility exists that the solution of ribonuclease may contain a fluorescent impurity. What control experiment should be done to correct this situation ... 

One of the main values of fluorescence as a technique for probing protein conformation is that it is highly sensitive and very economical of material. This means, however, that small traces of fluorescent impurities in the solvent or on the cell are readily detected and, if care is not taken, can lead to misinterpretation of the spectra. The essential aim in using this technique, therefore, must be to obtain a fluorescence emission spectrum for a protein that is guaranteed free from all too easily generated artifacts. 

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. 

Method. Hie metal chelates are prepared by extracting the metal ion from aqueous solution with 20-, 20- and 10-ml volumes of chloroform after addition of an appropriate amount of a solution of DDTC [0.22S g of the sodium salt in 75 ml of water and 25 ml of an ammonia-ammonium nitrate (1 1) buffer, 3 M in total ammonia]. The exact volumes which are used depend on whether the metal is uni-, bi- or tri-valent. The combined chloroform extracts are diluted to at least 50 ml for chromatography. The metal chelates are separated on plates of silica gel G or N which have been activated at 110 °C for 1 h. The Rp values of a number of DDTC metal chelates in a variety of solvent systems are listed in Table 4.31. The dried plates are sprayed with a solution consisting of 1 10 4A/ Pd(II), 7.0 10 5Af calcein and 0.02Mphosphate buffer [dihydrogen phosphate-hydrogen phosphate (1 1)]. This solution must be allowed to stand for 12 h in order to ensure that equilibrium is attained. For quantitative work with low concentrations, the solution of DDTC should be washed with chloroform before use. This removes fluorescent impurities which may cause interference in the chromatography. 

Until recently we were unable to determine k for 1(4) and 1(6) via this method, since this not only requires a time resolution better than 10 ps, but especially since the quenching of the donor fluorescence, that accompanies the electron transfer, makes the measurements extremely sensitive to the presence of minor, fluorescent impurities. After careful recrystallization a sample of 1(6) was now obtained for which the level of impurity fluorescence is sufficiently low to detect the very short lived ( 3-4 ps) residual donor fluorescence. A typical fluorescence decay as observed for this sample in ethylacetate via picosecond time correlated single photon counting (Bebelaar, 1986) is shown in Fig. 3. Via a biexponential reconvolution procedure the lifetime of the short component was determined to be 3.5 0.5 ps, while that of the impurity background is comparable to the lifetime of the isolated donor (-4500 ps) and thus probably stems from one or more species lacking the acceptor chromophore. Similar results were obtained in tetrahydrofuran (3 1 ps) and in acetonitrile (4 lps). 

Either the sample or impurities may absorb the laser radiation and reemit it as fluorescence. If this occurs, Raman spectra can be obscured by a broad, strong fluorescence band. The intensity of the latter could be as much as 104 greater than the Raman signal. There are several ways to minimize this problem. If impurities in the sample are causing fluorescence, the sample should be purified or irradiated by high-power laser beams for a prolonged time so that fluorescent impurities are bleached out. 

Fluorescence or traces of fluorescent impurities can prevent acquisition of RR spectra. 

Figure 9.13. FT-Raman spectra of a mildly fluorescent, impure sample of ortho dinitrobenzene before (A) and after (B) correction for instrumental response. Modulation at A is caused by the laser rejection filter. (Adapted from Reference 4, p. 104.)...

Example Investigations into the fluorescence of 8-hydroxyquinoline (1), 4-iodo-8-hydroxyquinoline (2) and 2-methyl-4-iodo-8-hydroxyquinoline (3) are described in this paper. Recrystallization of 1 and 2 afforded analytically pure samples, but vacuum sublimation of the methyl derivative (3) was necessary to remove fluorescent impurities. ... 

Local structure around fluorophores, presence of fluorescent impurities... 

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