Molecular Fluorescence Spectroscopy
Vibrational relaxation
A nonradiative relaxation in which energy of the excited species is decreased by an amount equal to the quantity of vibrational energy transferred.
Internal conversion
A nonradiative relaxation of a molecule from a low energy vibrational level of an excited electronic state to a high energy vibrational level of a lower electronic state
Phosphorescence
A photoluminescence process in which an excited triplet state is produced via electromagnetic radiation and the transition is from the excited triplet state to the ground singlet state.
Excitation spectrum
A plot of fluorescence or phosphorescence emission intensity as a function of the excitation wavelength.
Inner-filter effect
A result of excessive absorption of the incident beam (primary absorption) or absorption of the emitted beam (secondary absorption).
AR: Explain why adsoption on the solid surface induces a higher quantum yield of fluorescence
Adsoption on the solid surface promotes less collision in which the analyte assumes a rigid structure. Due to this, the rate of nonradiative relaxation decreases which gives time for the fluorescence emission to happen
AR: Explain why molecular fluorescence spectroscopy has an inherent sensitivity compared to absoprtion spectroscopy
Because fluorescence intensity is directly proportional to the intensity of the source (F=kcPo); hence just by using a highly powerful light source (e.g mercury arc lamp), you can increase the fluorescence intensity and thus increase sensitivy to even very low concentration. Unlike with absoprtion spectroscopy which takes the ratio of transmittance, hence the intensity of the source wont matter. .
Analytical operation commonly used in Molecular Fluorescence spectroscopy
Direct method( Analyte + complexing agent--> fluorescent complex; or Indirect method ( Analyte + fluorescing agent ---> less fluorescent intensity e.g. Al-complex of Alizarin garnet +Flouride (quencher) --> decrease in relative intensity); External calibration and standard addition can be done.
AR: Explain the theory behind the Stokes shift phenomenon
Fluorescence almost always occurs from the lowest vibrational level of the excited electronic state to various vibrational levels of the ground electronic state. Such transitions involve less energy than the excitation energy.
Instruments used in Molecular Fluorescence Spectroscopy
Fluorometer (Two filters as wavelenght selectors); Spectrofluorometer ( Two monochromator); Hybrid (excitation filter and emission monochromator); Filter is more preferable since it yields a higher intensity excitation source; Detector- photmultiplier tube, CCD, photodiodes.
Applications of Molecular Fluorescence Spectroscopy
Inorganic( determination of metal ions); Organic and Biochem ( Determination of proteins, amino acids, nucleic acid, and many others); Used in DNA labelling; Study kinetics and chemical equilibria; and many others
AR: Explain chemiluminiscence
It is produced when a chemical rxn yields an electroanalytically excited molecule which emits light aas it returns to ground state. Example: Luminol +Hydprogen Peroxide (in presence of Fe(II))--> photon; and, Ozone + NO -->excited NO2--> light
AR: Explain why phosphorescence emission stays longer than fluorescence emission
Phophorescence transition from the triplet state to the ground state emits a less intense but long lasting light due to lesser rate of relaxation since triple to singlet is a a harder transition the singlet-singlet in fluorescence emission
AR: Explain the effect of solvent visosity to the quantum yield of fluorescence
The more viscous the solvent is the more effective it is for the analyte to fluoresce. High viscosity means less collision and more rigidity and hence less nonradiative relaxation
Intruments used in Chemiluminiscence spectroscopy
Reaction flask and a detector (photomultiplier tube) only
Stokes shift
The difference in wavelength between the radiation used to excite fluorescence and the wavelength of the emitted radiation
AR: Explain the effect of temperature on the quatum yield of fluorescence
The higher the temperature is, the less effective it is to fluoresce. High temperature increases collison between the excited state to the solvent molecule and thus increases the rate of nonradiative relaxation.
AR: Explain the effect of Structure rigidity to the quatum yied of fluorescence
The more rigid the structure is, the more effective it is to fluoresce.Compounds that fluoresce have structures that slow the rate of nonradiative relaxation to the point where there is time for fluorescence to occur
Quatum yield of fluorescence
The ratio of the number of fluorescing molecules to the total number of excited molecules.
AR: Common structural features of fluorescing agent
They have structural rigidity. Organic compounds containing aromatic rings often exhibit fluorescence. Rigid molecules or multiple ring systems tend to have large quantum yields of fluorescence while flexible molecules generally have lower quantum yields.
Nonradiative relaxation
This occurs when excited species collide with molecules, such as the solvent, and in doing so lose energy without emission of electromagnetic radiation.
Defination of fluorescence
a photoluminescence process in which atoms or molecules are excited by absorption of electromagnetic radiation and then relax to the ground state, giving up their excess energy as photons. The transition is from the lowest lying excited singlet state to the ground singlet state.
