Intensity of fluorescence

The intensity of fluorescence. If, is proportional to the amount of the radiation from the excitation source that is absorbed and the quantum yield for fluorescence... 

The intensity of fluorescence therefore, increases with an increase in quantum efficiency, incident power of the excitation source, and the molar absorptivity and concentration of the fluorescing species. 

Standardizing the Method Equations 10.32 and 10.33 show that the intensity of fluorescent or phosphorescent emission is proportional to the concentration of the photoluminescent species, provided that the absorbance of radiation from the excitation source (A = ebC) is less than approximately 0.01. Quantitative methods are usually standardized using a set of external standards. Calibration curves are linear over as much as four to six orders of magnitude for fluorescence and two to four orders of magnitude for phosphorescence. Calibration curves become nonlinear for high concentrations of the photoluminescent species at which the intensity of emission is given by equation 10.31. Nonlinearity also may be observed at low concentrations due to the presence of fluorescent or phosphorescent contaminants. As discussed earlier, the quantum efficiency for emission is sensitive to temperature and sample matrix, both of which must be controlled if external standards are to be used. In addition, emission intensity depends on the molar absorptivity of the photoluminescent species, which is sensitive to the sample matrix. 

Detection and result The chromatogram was freed from mobile phase and dipped for 1 s in solution I and after drying for 1 min in a stream of cold air it was dipped in a solution of liquid paraffin — -hexane (1 + 2) in order to stabilize and increase the intensity of fluorescence by a factor of 1.5—2.5. The derivatives which were pale yellow in daylight after drying fluoresce pale blue to turquoise in long-wave... 

Factors such as dissociation, association, or solvation, which result in deviation from the Beer-Lambert law, can be expected to have a similar effect in fluorescence. Any material that causes the intensity of fluorescence to be less than the expected value given by equation (2) is known as a quencher, and the effect is termed quenching it is normally caused by the presence of foreign ions or molecules. Fluorescence is affected by the pH of the solution, by the nature of the solvent, the concentration of the reagent which is added in the determination of inorganic ions, and, in some cases, by temperature. The time taken to reach the maximum intensity of fluorescence varies considerably with the reaction. 

Images were acquired with 5-s exposure time after having fixed the focus. The total intensity of fluorescence was estimated by calculations the integral of the gray-level histogram. 

Type of experiment Integral intensity of fluorescence with respect to control (a.u.) Frequency shift with respect to control [Hz]... 

Figure 1.5. Femtosecond spectroscopy of bimolecular collisions. The cartoon shown in (a illustrates how pump and probe pulses initiate and monitor the progress of H + COj->[HO. .. CO]->OH + CO collisions. The huild-up of OH product is recorded via the intensity of fluorescence excited hy the prohe laser as a function of pump-prohe time delay, as presented in (h). Potential energy curves governing the collision between excited Na atoms and Hj are given in (c) these show how the Na + H collision can proceed along two possible exit channels, leading either to formation of NaH + H or to Na + H by collisional energy exchange.

Fig. 8. Intensity of fluorescence and rate of photoanodic dissolution of colloidal CdS as function of the concentration of added methyl viologen...

The fluorescence intensity of fluorescent proteins is pH dependent and most fluorescent proteins are less fluorescent at lower pH mainly because of a reduction in absorbance. Since the absorbance of the acceptor determines the FRET efficiency, changes in the acceptor absorbance spectrum due to pH variations can be wrongly interpreted as changes in FRET efficiency. Thus, a pKa well below physiological pH is recommended to prevent artifacts due to pH changes inside cells. This is especially challenging if the fluorescent proteins are to be targeted to acid cellular compartments, for example, endosomes, lysosomes, or plant vacuoles. 

In a later work, Stokes established the relationship between the intensity of fluorescence and the concentration, pointing out that the emission intensity depended on the concentration of the sample (analyte), but that attenuation of the signal occurred at higher concentrations as well as in the presence of foreign substances. He actually was the first to propose, in 1864, the application of fluorescence as an analytical tool, based on its sensitivity, on the occasion of a conference given previously in the Chemical Society and the Royal Institution, and entitled On the Application of the Optical Properties to the Detection and Discrimination of Organic Substances [5],... 

Measurement of fluorescence intensity can be used for quantitative analysis of fluorescent compounds where the intensity of fluorescence is proportional to the concentration of the compound. Because of their high sensitivity and selectivity, analytical techniques based on fluorescence detection are commonly used. If a target compound is fluorescent then direct detection of the fluorescence emitted is possible using a fluorimeter (Figure 4.7). 

The optimal enhancement effect is observed when the localized surface plasmon resonance is tuned to the emission wavelength of a locally situated fluorophore [86]. This is consistent with the model suggesting a greatly increased efficiency for energy transfer from fluorophores to surface plasmons [78]. Since resonance energy transfer is involved, the important factors affecting the intensity of fluorescence emission must also be the orientation of the dye dipole moments relative to the... 

In the microscopic technique, photobleaching FRET (5), the intensity of fluorescence excitation is increased to cause photobleaching of the donor fluorochrome, and the decay kinetics are measured in the absence or presence of an acceptor fluorochrome. If the acceptor is in close proximity to the donor, then the availability of excited-state donors for photobleaching is reduced, thus making the photobleaching process slower. 

As a function of the dye content intercalated, the relative intensity of fluorescence, the maximum wavelength in the fluorescence spectrum of lax d-spacing are illustrated in Fig. 2(a) for the pyronine Y-saponite composite (referred to as PY-SA), and Fig. 2(b) for the rhodamine 590-saponite composite (referred to as R590-SA). 

The fluorescence of the composite showed a somewhat different profile from that of the dye solution. The intensity of fluorescence, especially, was considerably reduced, and the value of also shifted towards higher wavelengths by increasing the quantity of intercalated dye. Representative data of fluorescence excited at 387 nm for the PY-SA composite and at 354 nm for the R590-SA composite are shown in the figures. It was noted that the changes of d-spacing, relative intensity and involved almost the same tendencies as a... 

In atomic fluorescence spectroscopy an intense excitation source is focused on to the atom cell. The atoms are excited then re-emit radiation, in all directions, when they return to the ground state. The radiation passes to a detector usually positioned at right-angles to the incident light. At low concentrations, the intensity of fluorescence is governed by the following relationship ... 

The intensity of fluorescence is proportional to the concentration of atoms, and hence the concentration of the element in the sample, so a plot of concentration against fluorescence will yield a straight Hue. There are several different types of atomic fluorescence as follows ... 

A prospective sensor made of a couple 159 consisting of p-cyclodextrin 11 and calix[4]arene 18 bearing a fluorophoric substituent was reported by Bugler and coworkers [68]. The compound forms fibers which change into vesicles upon complexation, forcing the fluorophore out of the cyclodextrin cavity. As a consequence, the intensity of fluorescence is reduced. In another approach to... 

The increased lifetimes and intensity of fluorescence over those of Kropp and Windsor are probably related to the care exercised by Gallagher in compound preparation. It is not clear at this time why there is such marked differences in concentration dependencies however. One possibility though, is that their deuterium-rich solution was substantially less rich than expected. 

The primary process in the vacuum ultraviolet photolysis of methylene iodide has been studied by Style and Ward,12 who observed that irradiation with light of wavelength 1250-2000 A. excites the fluorescence spectrum of iodine. Attempts to observe any appreciable delay between light absorption and fluorescence were unsuccessful, and the intensity of fluorescence was directly proportional to the light intensity and the pressure of methylene iodide. It was concluded that the excited I2 was produced in the primary process,... 

The third rule of practical value concerns the intensity of fluorescence obtained and its variation with frequency of exciting light. The rate of emission of fluorescence is by definition equal to the rate of light absorption, measured in quanta, multiplied by the quantum efficiency of fluorescence, i.e.,... 

Equation (2) also shows how the intensity of fluorescence varies when the frequency of the exciting light varies. For a given solution the fluorescence intensity is proportional to 7oe0 and for many substances in solution the fluorescence efficiency () is approximately independent of the excitation frequency. Thus, if the intensity of exciting light is kept constant as the frequency is varied, the fluorescence intensity will be proportional to e, the molecular extinction coefficient of the solute. Hence the true excitation spectrum frequently corresponds closely to the absorption spectrum of the compound (see Fig. 2). Spectrofluorimetry can thus be used to measure the absorption spectra of fluorescent solutes, but at concentrations far lower than could be measured directly with an absorption spectrophotometer. It has the further advantage that the... 

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