Fluorimetry-Principle and Applications
Fluorometry- Fluorometry is a type of spectroscopy and is also called fluorescence
spectroscopy. It is used to identify and determine the analyte concentrations in a sample.
The mechanisms involve the excitation of an ultraviolet light beam in the molecules of a specific
analyte and enable them to emit visible light. At a wavelength, it is the molecular absorption of
light energy and its almost instantaneous re-emission at a longer, different wavelength.
Principle of Fluorimetry:
The phosphorescence and fluorescence are processes of the photon emission, which arise from
electronically excited states throughout molecular relaxation. These photonic mechanisms
cause the polyatomic fluorescent molecules (fluorophores) between vibrational and electronic
states. Fluorophores play an essential role in the fluorescence spectroscopy.
Fluorophores are the components that produce fluorescence in molecules. The samples that
have been excited electronically after absorption of UV (200 nm to 400 nm), visible (400 nm to
800 nm), or NIR (700 nm to 1100 nm) radiation. The excitation method is very rapid from the
ground state to the excited state on the order of 10 to 15 seconds.
The molecule is quickly relaxed after excitation, to the lowest vibrational point of the excited
electronic state. The quick process of vibrational relaxation takes place on the time scale of
femtoseconds to picoseconds. The emission and excitation spectra of fluorescence,
respectively, reflect the vibrational level structures in the ground and the excited electronic
states.
Applications of Fluorimetry:
The application of fluorometry is significant as a potent and valuable tool for studying the
physical and chemical behavior of macro-molecules.
Fluorescence spectroscopy used in environmental analysis.
Fluorescence spectroscopy is used where the sample is scared and complex to process.
Fluorescence spectroscopy used in food analysis.
It is used to determine several types of analytes in serum.
Each form of fluorescence activity is to assist to apply fluorescent probes in polymer
systems.
Fluorescence spectroscopy used in dairy processing.
Disadvantage-
The main disadvantage of fluorescence spectroscopy is that only fluorescent molecules can
be analyzed.
It has limitations associated with loss of photostability and the ability to recognize
Fluorophores have a short lifespan.
It is also sensitive to autofluorescence from the solution.
May be susceptible to interference from changes in sample pH and oxygen levels
Colorimeter- Definition, Principle, Parts, Uses, Examples
,Definition- Colorimeter refers to a device used in colorimetry that aids in the absorption of a
particular wavelength of light by a specific sample solution. It is employed to measure how much
light transmits and absorbs as it passes through a liquid. These are used to identify the color
and establish the concentration of a solution. By comparing a solute’s color intensity in a
solution to that of a reference solution with a known solute concentration, one can estimate the
concentration of colored solute in that solution.
Principle- It is a photometric technique which states that when a beam of incident light of
intensity Io passes through a solution, the following occur:
A part of it is reflected which is denoted as Ir
A part of it is absorbed which is denoted as Ia
Rest of the light is transmitted and is denoted as It
Therefore, Io = Ir + Ia + It
To determine Ia the measurement of Io and It is sufficient therefore, Ir is eliminated. The amount
of light reflected is kept constant to measure Io and It.
The operation of the colorimeter is based on Beer-Lambert’s law which states that the amount
of light absorbed by a color solution is directly proportional to the solution’s concentration and
the length of a light path through it.
A ∝ cl A = ∈cl
Where,
A = Absorbance/ Optical density of the solution ∈ = Coefficient of absorption
c = Concentration of solution l = Length of the path
A light ray of a certain wavelength, which is specific for the assay is in the direction of the
solution. The light passes through a series of different lenses and filters. The coloured light
navigates with the help of lenses, and the filter helps to split a beam of light into different
, wavelengths allowing only the required wavelength to pass through it and reach the cuvette
of the standard test solution.
When the beam of light reaches’ cuvette, it is transmitted, reflected, and absorbed by the
solution. The transmitted ray falls on the photodetector system where it measures the
intensity of transmitted light. It converts it into the electrical signals and sends it to the
galvanometer.
The electrical signals measured by the galvanometer are displayed in the digital form.
Parts-
Light Source: The source of light should produce energy with enough intensity to cover the entire
visible spectrum (380-780 nm). Commonly, Tungsten lamps are used as a light source for
measurement in the visible spectrum and near-infrared ranges. Halogen deuterium is suitable for
measurement in the UV range (200-900 nm).
Slit: It reduces unwanted or stray light by allowing a light beam to pass through.
Condensing lens: Parallel beam of light emerges from condensing lens after the light passes
through slit incidents on it.
Monochromatic: It filters the monochromatic light from polychromatic light, which absorbs
unwanted light wavelengths and permits only monochromatic light. These are of three types:
prism, grating, and glass
Prism: It facilitates the refraction of light when it passes from one medium to another.
Glass: It selectively transmits light in certain ranges of wavelengths
Gratings: These are made of graphite, which separates light in different wavelengths.
Fluorometry- Fluorometry is a type of spectroscopy and is also called fluorescence
spectroscopy. It is used to identify and determine the analyte concentrations in a sample.
The mechanisms involve the excitation of an ultraviolet light beam in the molecules of a specific
analyte and enable them to emit visible light. At a wavelength, it is the molecular absorption of
light energy and its almost instantaneous re-emission at a longer, different wavelength.
Principle of Fluorimetry:
The phosphorescence and fluorescence are processes of the photon emission, which arise from
electronically excited states throughout molecular relaxation. These photonic mechanisms
cause the polyatomic fluorescent molecules (fluorophores) between vibrational and electronic
states. Fluorophores play an essential role in the fluorescence spectroscopy.
Fluorophores are the components that produce fluorescence in molecules. The samples that
have been excited electronically after absorption of UV (200 nm to 400 nm), visible (400 nm to
800 nm), or NIR (700 nm to 1100 nm) radiation. The excitation method is very rapid from the
ground state to the excited state on the order of 10 to 15 seconds.
The molecule is quickly relaxed after excitation, to the lowest vibrational point of the excited
electronic state. The quick process of vibrational relaxation takes place on the time scale of
femtoseconds to picoseconds. The emission and excitation spectra of fluorescence,
respectively, reflect the vibrational level structures in the ground and the excited electronic
states.
Applications of Fluorimetry:
The application of fluorometry is significant as a potent and valuable tool for studying the
physical and chemical behavior of macro-molecules.
Fluorescence spectroscopy used in environmental analysis.
Fluorescence spectroscopy is used where the sample is scared and complex to process.
Fluorescence spectroscopy used in food analysis.
It is used to determine several types of analytes in serum.
Each form of fluorescence activity is to assist to apply fluorescent probes in polymer
systems.
Fluorescence spectroscopy used in dairy processing.
Disadvantage-
The main disadvantage of fluorescence spectroscopy is that only fluorescent molecules can
be analyzed.
It has limitations associated with loss of photostability and the ability to recognize
Fluorophores have a short lifespan.
It is also sensitive to autofluorescence from the solution.
May be susceptible to interference from changes in sample pH and oxygen levels
Colorimeter- Definition, Principle, Parts, Uses, Examples
,Definition- Colorimeter refers to a device used in colorimetry that aids in the absorption of a
particular wavelength of light by a specific sample solution. It is employed to measure how much
light transmits and absorbs as it passes through a liquid. These are used to identify the color
and establish the concentration of a solution. By comparing a solute’s color intensity in a
solution to that of a reference solution with a known solute concentration, one can estimate the
concentration of colored solute in that solution.
Principle- It is a photometric technique which states that when a beam of incident light of
intensity Io passes through a solution, the following occur:
A part of it is reflected which is denoted as Ir
A part of it is absorbed which is denoted as Ia
Rest of the light is transmitted and is denoted as It
Therefore, Io = Ir + Ia + It
To determine Ia the measurement of Io and It is sufficient therefore, Ir is eliminated. The amount
of light reflected is kept constant to measure Io and It.
The operation of the colorimeter is based on Beer-Lambert’s law which states that the amount
of light absorbed by a color solution is directly proportional to the solution’s concentration and
the length of a light path through it.
A ∝ cl A = ∈cl
Where,
A = Absorbance/ Optical density of the solution ∈ = Coefficient of absorption
c = Concentration of solution l = Length of the path
A light ray of a certain wavelength, which is specific for the assay is in the direction of the
solution. The light passes through a series of different lenses and filters. The coloured light
navigates with the help of lenses, and the filter helps to split a beam of light into different
, wavelengths allowing only the required wavelength to pass through it and reach the cuvette
of the standard test solution.
When the beam of light reaches’ cuvette, it is transmitted, reflected, and absorbed by the
solution. The transmitted ray falls on the photodetector system where it measures the
intensity of transmitted light. It converts it into the electrical signals and sends it to the
galvanometer.
The electrical signals measured by the galvanometer are displayed in the digital form.
Parts-
Light Source: The source of light should produce energy with enough intensity to cover the entire
visible spectrum (380-780 nm). Commonly, Tungsten lamps are used as a light source for
measurement in the visible spectrum and near-infrared ranges. Halogen deuterium is suitable for
measurement in the UV range (200-900 nm).
Slit: It reduces unwanted or stray light by allowing a light beam to pass through.
Condensing lens: Parallel beam of light emerges from condensing lens after the light passes
through slit incidents on it.
Monochromatic: It filters the monochromatic light from polychromatic light, which absorbs
unwanted light wavelengths and permits only monochromatic light. These are of three types:
prism, grating, and glass
Prism: It facilitates the refraction of light when it passes from one medium to another.
Glass: It selectively transmits light in certain ranges of wavelengths
Gratings: These are made of graphite, which separates light in different wavelengths.
