Wavelength, exdtation

Multicolor FRET silica nanopartides by single wavelength exdtation. Nano Letters, 6,84-8. 

THF = tetrahydrofuran. ACN = acetonitrile, p.s. = particle size. i.d. = internal diameter, o.d. = outer diameter. MeOH = methanol. MS = mass spectrometry. DAD = diode array detector, n.a. = not available. THC = tetrahydrocurcumin. = exdtation wavelength. X = emission wavelength. 

The substrate and two products are separated on a //.Bondapak TM C]8 column (3.9 mm x 300 mm). The mobile phase consisted of 0.05 M acetate buffer (pH 4.7), 10 mM Na2S205, 1 mM EDTA, and 25% methanol. The column effluent was monitored by fluorimetry with exdtation, and emission wavelengths being 295 and above 340 nm, respectively. 

The spectral characteristics of BODIPY FL lA somewhat mimic the green luminescence of fluorescein, thus the FL designation in its name. The excitation maximum for the probe occurs at 504 nm, and its emission at 510 nm. The extremely small Stokes shift makes it difficult to avoid problems of exdtation-light interference in critical emission measurements unless suboptimal excitation wavelengths below its excitation maximum are used. The molecule has an extinction coefficient in methanol (containing 1% sodium acetate and 1% 2-mercaptoethanol) of about 79,000 M icm at 504 nm. 

Also, one can put into evidence energy transfer mechanism by recording the fluorescence exdtation spectrum of the complex (donor-acceptor) (3an is set at a wavelength where raity the accqitor emits) and comparing it to the absorption spectrum of the donor alone. In presence of energy transfer between the two molecules, a peak characteristic of the abstxption of the donor will be di ktyed in the fluorescence exdmbcHi spectrum. 

Amino acid derivatives are best extracted from silica gel layers with methanol or with a mixture of methanol/ 25% NH3 (95 5) (7,10,17] or chloroform/methanol/ acetic acid (7 2 2) [77]. Peptides are extractable with acetone/water (1 1) [78], and amine derivatives are extractable with less polar solvents, e.g. ethyl acetate, benzene/acetic acid (99 1), benzene/triethylamine (95 5) [10,17], or with dioxane if, as well as fluorescence, radioactivity is to be measured by liquid scintillation counting [80]. Chloroform is suitable for the extraction of Dns-amino adds from polyamide sheets [40]. Exdt-ation and emission wavelengths are, if possible, adjusted to those of the individual Dns-deiivatives however, for almost every compound, exdtation can generally be achieved using the 365 nm mercury line. 

Secrist et al. [512] have studied the fluorescence characteristics of l- N-ethenoadenosines in detail. They have a high fluorescence quantum yield 0.56 in 0.25 M phosphate buffer. Of the three absorption maxima, that at 300 nm is normally used for exdtation. Since it is shifted to lower wavelength with decreasing pH, optimization is necessary according to the pH of the eluent used. Wagner el al. [509] recommend exdtation of fluorescence at 270—275 nm for an eluent of pH 4.3. Fluorescence emission is measured at 410 nm. 

The sample cells used in fluorescence spectroscopy have all four sides dear, since in most instruments the fluorescence photons are detected at an angle of 90° to the propagation direction of the exdtation beam. The 90° geometry is used in order to minimize any interference in the fluorescence hght detection by the exdtation light beam. In most cases the cuvettes are made from fused silica (quartz) but if the exdtation and fluorescence wavelengths are above 300 nm plastic disposable cuvettes may be used. 

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