2. INTRODUCTION
Fluorescence spectroscopy
also known as
spectrofluorometry is a type of
electromagnetic spectroscopy
that analyses fluorescence from
a sample.
It involves using a beam of light
i.e. ultraviolet light, that excites
the electrons in molecules of
certain compounds and causes
them to emit light.
3. Luminescence
Emission of photons from electronically excited
states
Two types of luminescence:
1. Fluorescence :-
Relaxation from singlet excited state
2. Phosphorescence :-
Relaxation from triplet excited state
Types of emission :-
1. Fluorescence - return from excited singlet state
to ground state; does not require change in spin
orientation.
2. Phosphorescence - return from a triplet excited
state to a ground state; electron requires change in
spin orientation
4. PRINCIPLE
The principle states that the
luminescence caused by photons
exciting a molecule, raises it to an
electronic excited state.
It’s brought about by absorption of
photons in the singlet ground state
promoted to a singlet-excited state.
It analyses fluorescence from a molecule
based on its fluorescent properties.
5. 1. Light source: Xenon arc lamp, mercury vapor lamp and tungsten lamp
2. Monochromators: These help to separate light bands.
Excitation and emission monochromators
3 .Sample cells: Cuvettes are small tube-like structures used to hold the sample for analysis. They are
made of color corrected fused glass. All the sides of the cell have polished surfaces. The path length of the
sample holder is 10mm.
4. Detectors: Photomultiplier PMT tubes are employed as detectors. These are efficient and accurate for
radiations with low intensity
WORKING COMPONENTS:
7. APPLICATIONS:
● Fluorescence spectroscopy is suitable for the analysis of solid powders, crystals, films,
liquids, and other samples. Quartz cell (liquid sample) or solid sample holder (powder or
sheet sample) can be selected according to the sample.
● Fluorescence analysis can qualitatively and quantitatively analyze substances based on
the characteristics and intensity of fluorescence produced by the photoluminescence
of the substance.
8. Biological field
● Fluorescence spectroscopy is mainly
used to determine the content of
certain components in biological
samples, analysis of biotechnology
and immunotechnology, such as the
determination of deoxyribose and
deoxyribonucleic acid, DNA,
antibodies, antigens, and other
aspects of research.
● In the field of drug analysis,
fluorescence spectroscopy analysis
can be used to identify the active
components of drugs, study
pharmacokinetics, clinical
pharmacological efficacy analysis, and
so on. it is widely used in the analysis
of antimicrobial drugs, analgesics,
sedatives and haemostatic drugs.
DRUG ANALYSIS
9. APPLICATIONS IN FOOD :
● Fluorescence analysis is mainly used in this field to analyze and detect minerals
and metal elements, amino acids, vitamins, fungal contamination, additives,
preservatives, harmful substances in food packaging, pesticide residues, etc. in
food
● Its combination with HPLC, TLC, FIA, and other technologies can improve the
direction of various substances in food
11. DISADVANTAGES :
● The major disadvantage of fluorescence spectroscopy is that not all molecules are
fluorescent.
● It has limitations related to loss of recognition capability and photostability.
● Susceptible to interference because of the changes in pH and oxygen levels of the
sample.
● It is susceptible to the auto-fluorescence of the sample.
● Issue related to potential toxicity, due to the foreign material in the biological
media.
● The short lifespan of fluorophores is another disadvantage of fluorometry.
