Phosphorescence is light energy produced by a particular type of chemical reactionwhere the excess chemical energy of the reactants is given off as light energy. Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence.
What are the factors affecting fluorescence and phosphorescence?
- Nature of Molecule. Having conjugated double bonds.
- Temperature/Viscosity. The viscosity of the medium varies as the temperature changes, affecting the number of collisions between fluorophore molecules and solvent molecules.
Why phosphorescence takes much longer time than fluorescence?
The reason phosphorescence lasts longer than fluorescence is because the excited electrons jump to a higher energy level than for fluorescence. The electrons have more energy to lose and may spend time at different energy levels between the excited state and the ground state.
Both fluorescence and phosphorescence are based on the ability of a substance to absorb light and emit light of a longer wavelength and therefore lower energy. The main difference is the time in which it takes to do so.
Why does fluorescence occur at longer wavelengths?
Because the energy associated with fluorescence emission transitions (see Figures 1-4) is typically less than that of absorption, the resulting emitted photons have less energy and are shifted to longer wavelengths.
What factors affect fluorescence?
Three important factors influencing the intensity of fluorescence emission were theoretical analyzed, including the absorption ability of excitation photons, fluorescence quantum yield, and fluorescence saturation & fluorescence quenching.
How does temperature affect fluorescence?
The intensity of fluorescence decreases with the increase of temperature. The temperature quenching of the fluorescence is accompanied by an energy transfer from tyrosine to tryptophan. The optimum enzyme activity is observed at about 40 degrees C, at 75 degrees C the activity ceases.
Does pH affect fluorescence?
Increasing the pH caused a corresponding increase in the maximum fluorescence intensity from 77.74 (units) in the acidic sample to 146.13 at neutral pH and 232.69 at alkaline pH.
Why phosphorescence is more intense in solid samples?
In phosphorescence, there is a change in electron spin, which results in a longer lifetime of the excited state (second to minutes).
Does fluorescence have a shorter wavelength than phosphorescence?
Phosphorescence involves the transition from the single ground energy state to excited triplet state and involving a change of spin state. The emitted photon (light) has lower energy than the absorbed photon and emission occurs at a longer wavelength than fluorescence.
Why is phosphorescence generally not observed at room temperature?
Phosphorescence is usually not seen in fluid solutions at room temperature because there are many deactivation processes that have faster rate constants, such as nonradiative decay and quenching processes. These processes effectively compete with photon emission in liquid solutions, thus reducing phosphorescence.
Why is phosphorescence forbidden?
In phosphorescence, a spin change must occur to go from an excited triplet state to the ground singlet state. “Forbidden” in practice means that these transitions are very weak (but often still observable). Does visible light cause fluorescence the same way UV does?
Why does phosphorescence glow?
Phosphorescence is a type of photoluminescence related to fluorescence. When exposed to light (radiation) of a shorter wavelength, a phosphorescent substance will glow, absorbing the light and reemitting it at a longer wavelength.
What is the basic difference between fluorescence and phosphorescence?
The difference is that the glow of fluorescence stops right after the source of excitatory radiation is switched off, whereas for phosphorescence, an afterglow with durations of fractions of a second up to hours can occur [6,7].
What is phosphorescence physics?
phosphorescence, emission of light from a substance exposed to radiation and persisting as an afterglow after the exciting radiation has been removed.
Why is fluorescence so sensitive?
Fluorescence is more sensitive because of the different ways of measuring absorbance and fluorescence. Light absorbance is measured as the differ- ence in intensity between light passing through the refer- ence and the sample.
How does concentration affect fluorescence?
However, too concentrated a solution decreases the fluorescence intensity, as shown in Figure 3.22(a). Further increases in concentration induce change in the shape of the fluorescence spectrum because the fluorescence at shorter wavelengths is absorbed by other molecules of the same species (Figure 3.22(b)).
How does light affect fluorescence?
Fluorescent objects reflect light as well as absorb the energy of the light, turning some of it into heat, and the majority of the light is emitted as the fluorescent colour. The electrons in the fluorescent pigments absorb light energy and are temporarily promoted into higher-energy orbitals.
Does fluorescence intensity depend on concentration?
In addition, the fluorescence intensity depends directly on concentration of fluorophore in the sensing volume, molecular extinction coefficient, and the quantum yield.
Why does fluorescence decrease with concentration?
If the concentrations are quite high, the fluorescence intensity will decrease because of the inner filter effect. This effect is due to the absorbance of the excitation light by the sample itself.
Does viscosity affect fluorescence?
The fluorescence lifetimes increase following an increase in viscosity, consistent with a slower rate of interconversion between the twisted and the planar conformers, arising from a restriction of rotation at higher viscosities.
Does Beer’s law apply to fluorescence?
The Beer-Lambert law applies to absorbance, not fluorescence. However, in the same way that absorbance is directly proportional to the concentration of the chromophore, fluorescence intensity is directly proportional to fluorophore concentration..
Why is fluorescence measured at 90 degrees?
The sample emits a wavelength, which travels to the detector. The detector is usually set at a 90-degree angle to the light source to avoid any interference from the transmitted excitation light.
What is the unit of fluorescence intensity?
The intensity of the fluorescent signal is usually relative to other measurements or to a refence measurement taken by an instrument. Consequently, fluorescence plate readers measure the light signal emitted by a sample in Relative Fluorescent Units (RFU).
How do you calculate fluorescence intensity?
Record the number of Value 0 (red) and Value 1 (green) pixels. The percent area of signal is calculated by dividing the number of red pixels by the total number of red and green pixels, multiplied by 100. Repeat steps for all images being imaged and analyzed. Record all results in excel.