Fluorescence spectroscopy
Komal Choudhary
Lecturer
School of Biotechnology
DAVV Indore
Fluorescence spectroscopy
 Luminescence
It is the emission of light from any substance, and
occurs from energetically excited states.
It is divided into two categories
(1) Fluorescence
(2) phosphorescence
 Non radioactive relaxation- excitation energy is
dissipated as heat(vibration) to the solvent.
 It includes internal conversion(IC) intersystem
conversion(ISC).
 Singlet state
 Triplet state
 Jablonski energy diagram tell us about the relaxation
mechanism for exited state molecule.
 Once a molecule has absorbed energy in the form of
electromagnetic radiation there are numbers of routs
by which it can return to ground state.
 If the photon emission occurs between same spin
state(i.e. S1-S0), this is called fluorescence emission.
(release of energy is faster)
 If the spin state of the initial and final energy level are
different(i.e. T1-S0), this is called phosphorescence
emission. (release of energy is slower)

 Fluorescence spectroscopy or spectrofluorometry, is
a type of electromagnetic spectroscopy which analyzes
fluorescence from a sample.
 Fluorescence is a phenomenon where by molecule
after absorbing radiation emit the radiation of longer
wavelength (compound absorb UV radiation, emit
visible light).
 stoke’s shift= λab-λemt
 Best results are obtained from
those compounds which
showing large shifts.
Components of spectroflorimeter
 Light source-xenon or mercury lamps
 Slits
 Two monochromator(prism or differaction
grating)
(1) excitation monochromator
(2) emission monochromator
 Sample holder(quartz cuvette)
 Detector-photocell (red-sensitive PMT)
Factors which affect fluorescence or
quench the emission of fluorescence are
 Solvent polarity
 pH
 Temperature
 Viscosity
 Presence of oxygen
 Concentration of the sample molecule
 Intensity of incident light
Fluorescent probes
 Ethidium bromide
 Flourescein
 Densyl chloride
 1-Anilinonapthalene 8-sulphonate(ANS)
 4-methylumbelliferone
 Acridine
 GFP,RFP
 Extrinsic fluorescence
 Intrinsic fluorescence
Applications
 Used in both qualitative and quantitative(major)
estimation.
 Assays of vitamin B in food stuff ,NADH, hormones,
drugs, pesticides, Carcinogens, chlorophyll,
cholesterol, metal ions etc.
 enzyme assays and kinetic analysis.
 Protein structure analysis.
 Membrane structure analysis.
 Microspectrofluorimetry (used to detect malignant
cell in biopsy tissue)
 FACS.