This slide discusses on Fluorescence Spectroscopy, principle, types and applications

1. Fluorescence Spectroscopy
Presented By
NIZAM ASHRAF
1st year M.Sc. Marine Microbiology
OST-2018-22-15

2. • Fluorescence is the emission of light by a substance that has absorbed
light or other electromagnetic radiation.
• Fluorescence spectroscopy (fluorimetry or spectrofluorometry) is a
type of electromagnetic spectroscopy which analyzes fluorescence
from a sample.
• It involves the use of a beam of light, usually ultraviolet light, that
excites the electrons in molecules of certain compounds and causes
them to emit light of a lower energy; typically, visible light.
• A complementary technique is absorption spectroscopy.
• Devices that measure fluorescence are called fluorometers or
fluorimeters.

4. Theory
• Fluorescence spectroscopy is a fast, simple and inexpensive method
to determine the concentration of an analyte in solution based on its
fluorescent properties.
• It can be used for relatively simple analyses, where the type of
compound to be analyzed (‘analyte’) is known, to do a quantitative
analysis to determine the concentration of the analytes.
• Fluorescence is used mainly for measuring compounds in solution.

5. • In fluorescence spectroscopy, a beam with a wavelength varying between
180 and ∼800 nm passes through a solution in a cuvette.
• We then measure from an angle the light emitted by the sample.
• In fluorescence spectrometry both an excitation spectrum (the light that is
absorbed by the sample) and/or an emission spectrum (the light emitted
by the sample) can be measured.
• The concentration of the analyte is directly proportional with the intensity
of the emission.
• There are several parameters influencing the intensity and shape of the
spectra. When recording an emission spectrum the intensity is dependent
on the:
• Excitation wavelength
• Concentration of the analyte solvent
• Path length of the cuvette
• Self-absorption of the sample

7. Instrumentation
Filter Fluorometers Spectro Fluorometers
Use filters to isolate the incident light
and fluorescent light.
Use diffraction grating
monochromators to isolate the incident
light and fluorescent light.

9. • The light from an excitation source passes through a filter or
monochromator (1), and passes through the sample. Here some of it
is absorbed, making some of the molecules in the sample fluoresce.
• A part of the fluorescent light is then focused on a filter or
monochromator (2), which often is placed at a 90° angle to the
excitation light.
• The light is then detected by a detection device.
• Various light sources may be used as excitation sources; (lasers,
photodiodes; xenon arcs and mercury vapour lamps).

10. • A laser only emits light of high irradiance at a very narrow wavelength
interval, typically under 0.01 nm, which makes an excitation
monochromator or filter unnecessary.
• The disadvantage is that wavelength of a laser cannot be changed
much.
• A mercury vapour lamp is a line lamp, meaning it emits light near peak
wavelengths contrary to the Xe arc, which have a continuous emission
spectrum with intensity in the range from 300-800 nm.

11. • Filters and/or monochromators may be used in fluorimeters. A
monochromator transmits light of an adjustable wavelength with an
adjustable tolerance.
• The monochromator can then select which wavelength to transmit.
• For allowing anisotropy measurements, the addition of two
polarization filters are necessary:
• One after the excitation monochromator or filter, and
• One before the emission monochromator or filter.
• No monochromator is perfect and it will transmit some stray light,
ie; light with other wavelengths than the targeted.
• An ideal monochromator would only transmit light in the specified
range and have a high wavelength-independent transmission.

12. • The detector can either be single-channeled or multi-channeled.
• The single channeled detector can only detect the intensity of one
wavelength at a time, while the multi-channeled one detects tin
intensity of all wavelengths simultaneously, making the emission
monochromator or filter unnecessary.
• When measuring fluorescence spectra, the wavelength of the
excitation light is kept constant, preferably at a wavelength of high
absorption, and the emission monochromator scans the spectrum.
• For measuring excitation spectra, the wavelength passing through the
emission filter or monochromator is kept constant and the excitation
monochromator is scanned.

13. • An excitation (also called an absorption spectrum) and an emission
spectrum of a fluorescent compound can be made.
• The absorption spectrum tells us which incoming wavelengths are
absorbed by the solution, with the emission or fluorescence spectrum
we can see which wavelengths are emitted after absorbing the
incoming light.

14. Applications
• It is used in biochemical, medical, and chemical research fields, for
analyzing organic compounds.
• Used in differentiating malign skin tumors from benign.
• In analytical chemistry, fluorescence detectors are used with HPLC.

15. Thank You