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SPECTROSCOPY
BIOMEDICAL OPTICS
BY: SUCCESS KAMUHANDA
Spectroscopy Definition

● The study of the emission and absorption of light and other radiations by
matter is known as spectroscopy.
● They mainly process the wavelength of the radiation.
● Spectroscopy also deals with the study of the interactions between particles
like protons, electrons, and ions.

● Spectroscopy can also be used to study the interaction with other particles
related to the function of their collision energy. Spectroscopic analysis plays
an important role in quantum mechanics, theory of relativity and quantum
electrodynamics.
Principle of Spectroscopy

● Spectroscopy is mainly used to identify and elucidate the elements and

compounds of atoms and molecules.
● They are measured by examining the absorbed or emitted radiant energy
by the sample or object.

● Here, the beam of electromagnetic radiation like infrared rays, UV rays..etc

are passed on the sample and the response of the sample is measured
through the wavelength of the electromagnetic spectrum applied from
the external energy source.
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What is a Spectrometer
● A scientific instrument that is
mainly used to analyse the
wavelength of
electromagnetic radiations by
measuring and separating the
spectral components based
on their physical
phenomenon is known as a
spectrometer.

● The spectrometer is
commonly used for molecular
spectroscopy. The
spectrometer mainly consists
of radiation source and
detection and analysis
equipment.
● The emission spectrometers are used to excite molecules of a sample to
higher energy states and analyze the radiation emitted while molecules return
to their original energy state.
● Usually, the absorption spectrometers will pass the radiations of known
wavelengths. So, it is possible to determine the changes in the radiation
wavelength.

● The process of Fourier-transform spectrometers are similar to the

absorption spectrometers, but they are using broadband radiations and the
computer will analyze the output. Mass spectrometers are mainly used for
analysing the atomic or molecular components in a sample based on their
masses.
Electromagnetic Radiations (EMR)
● It is a form of energy and it consists of both magnetic and electric
field.
● It is composed of an electric and a perpendicular magnetic vector,
each one oscillating in plane at right angles to the direction of
propagation.
● Electromagnetic spectrum is mainly range from shorter wavelengths
to longer wavelengths such as gamma rays.
● Wavelengths of the visible region of the spectrum is ranges between
400-700 nm.
● Blue end of the spectrum has shorter wavelengths, while red end has
longer wavelengths.
● In spectrum, energy associated with the given segment is much
related to the frequency and wavelength.
● Frequency (v) of the electromagnetic radiation is the number of
oscillations made by wave in 1 second.
● Frequency is measured in hertz (Hz), Where, 1Hz = 1 cycle per sec.
● Wavelength (λ): It is the length of one complete wave cycle.
● Wavelength is generally measured in centimeters (cm).
● Wavelength is inversely proportional to the frequency.
 Relationship between wavelength and
frequency
V= c/ λ, where, c= speed of light.
E= hv = hc/ λ where, Planck’s constant (h) = 6.6 X 10-34 joules per second.
● Wavenumber: It defines the number of completed wave cycles per
distance. It is measured in 1 cm-1 wavenumber is reciprocal to the
wavelength.
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What is a spectroscope
● An instrument that is used for
measuring the properties of light
in a certain portion of the
electromagnetic spectrum is
known as a spectroscope.
● Spectroscope is also known as
optical spectroscope,
spectrophotometer or
spectrograph.

● Usually, the spectrometer

present in the spectroscope will
produce spectral lines and help
them to measure wavelengths
and intensities of light
Types of Spectroscopy
● Spectroscopy is the collective terms for diverse group of techniques
which involves the interactions of electromagnetic radiation with a
matter of interest are studied.
● Radiation is either get scattered, emitted or absorbed, when the
radiations meets with the matter.
● Based on this there is three principles branches of spectroscopy:
➔ Absorption spectroscopy
➔ Scattering spectroscopy
➔ Emission spectroscopy
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Absorption spectroscopy uses the range in which a material absorbs the
electromagnetic spectrum. It involves atomic absorption spectroscopy and
different molecular techniques that area, such as infrared spectroscopy and radio
region nuclear magnetic resonance (NMR) spectroscopy.
Emission spectroscopy uses the electromagnetic spectrum range in which a
material radiates (emits). The material must consume energy first. This energy may
come from several sources, such as luminescence, which defines the subsequent
emission. Spectrofluorimetry includes molecular luminescence techniques.
Scattering spectroscopy tests the amount of light at specific wavelengths,
incident angles, and polarization angles that a material scatter. The method of
scattering is much quicker than the process of absorption/emission. Raman
spectroscopy is one of the most beneficial applications of light scattering
spectroscopy.
Principle of Absorption Spectroscopy
● Absorption spectroscopy is based on law known as Beer-Lambert law.
● When electromagnetic radiations pass or fall onto homogeneous medium,
some amount of light is reflected, some amount is absorbed and remained
is transmitted.
● Absorption spectroscopy is governed by two laws i.e., Lambert’s law and
Beer’s law, in the combined form it is known as Beer-Lambert law.
● Lambert’s law: It states that when monochromatic light passes through a
transparent medium, the intensity of transmitted light decreases
exponentially as the thickness of absorbing material increases.
● Beer’s law: It states that the intensity of transmitted monochromatic light
decreases exponentially as the concentration of the absorbing
substances increases.
 A= log Io/ I e.c.l
A = absorbance
Io = intensity of incident light
I = intensity of light through the sample.
e = extinction coefficient or absorption coefficient.
c = concentration
l = path length (cm)
Types of Absorption Spectroscopy
Infrared Spectroscopy
● Infrared spectroscopy will mainly deal with the electromagnetic spectrum
lying in the infrared region.
● They mainly work on absorption spectroscopy. IR spectroscopy is mainly
used for identifying the chemical composition of the material.
● Fourier transform infrared (FTIR) spectrometers mainly use IR
spectroscopy techniques.

● The electromagnetic spectrum of infrared is mainly classified into three

types namely, near-infrared, far-infrared and mid-infrared.
● The near-infrared ranges between 14000-4000 cm-1, which will help to
study overtone or harmonic vibrations.

● The mid-infrared ranges from 4000-400 cm-1, which will help to study
the fundamental vibrations and associated rotational-vibrational
structure.

● The mid-infrared ranges from 400-100 cm-1, which will help to study
microwave regions that have low energy and may be used for rotational
spectroscopy.
UV Spectroscopy

● Ultraviolet spectroscopy is also known as absorption

spectroscopy or reflectance spectroscopy.
● The electromagnetic spectrum of the ultraviolet region lies
adjacent to the infrared region.

● UV spectroscopy is mainly used for bacteria culture, drug

identification and to check nucleic acid purity.
Mass Spectroscopy

● Mass spectroscopy is mainly useful for studying the protein-protein

interaction.
● So, Mass spectroscopy can be used for identifying biomolecules or
proteins present in biological samples.

● The detector of these mass spectroscopies will analyse the substance

based on mass and charge ratio. Here, ion deflection is mainly based on
mass, velocity and charge.
Raman Spectroscopy
● Usually, Raman spectroscopy works based on the absorption of photons.
The Raman spectroscopy will analyze the material based on the scattering
of photons at a higher or lower frequency.
● With photons incident on the molecules or atoms, they may either gain
energy or lose energy based on the vibration or rotation of the molecules.
● If most of the incident photons get scattered by the sample without the
changes in frequency, then the scattering process is known as Rayleigh
scattering.

● Usually, the Raman spectra will be the monochromatic visible laser. The
scanning optical monochromator with a phototube is used as a detector
for analysing the radiation
Fluorescence Spectroscopy
● Fluorescence Spectroscopy is one of the important types of
electromagnetic spectroscopy. They are mainly used for the fluorescence
of a sample.
● Usually, UV lights are used in fluorescence spectroscopy.
● Fluorescence spectroscopy is mainly used for analysing organic
components in biochemical, medical, and chemical research fields.
● By using microfluorimetry, fluorescence spectroscopy can be adopted for
the microscopic level.

● By using the Atomic Fluorescence Spectroscopy (AFS) techniques, we

can find the compound present in air or water, or other media.
FTIR Spectroscopy

● FTIR Spectroscopy is also known as Fourier-transform infrared

spectroscopy.
● This technique is obtained by an infrared spectrum of absorption or
emission of a solid, liquid or gas.

● FTIR spectroscopy is widely used for analysing nano and biological

materials, water content determination in plastics and compositions,
detectors in chromatography...etc.
Applications of Spectroscopy
1. Spectroscopy is mainly used for studying the structure of molecules
and atoms. Spectroscopy will use a large wavelength to investigate
the structure and electron configurations of atoms and molecules.
2. Spectroscopy can also be used for finding the unknown chemical
composition of materials. Spectroscopy emission spectrum will help
to concentrate on a few parts per million of a trace element in a
material.
3. The study of the spectral emission lines will help astronomers to study
distant galaxies. This will help to analyse the universe in all directions.
Astronomers will also use the doppler shift of spectral lines for observations.
Usually, a doppler shift will occur when the source of radiation like stars,
nebula moves relative to an observer.
Examples of Spectroscopy Applications
● Monitoring diffused oxygen content in freshwater and aquatic
ecosystems.
● Determining the atomic structure of a sample.
● Determining the metabolic structure of a muscle.
● Studying spectral emission lines of distant galaxies.
● Altering the structure of drugs to improve the effectiveness.
● Space exploration
● Characterization of proteins
● Respiratory gas analysis in hospitals
ANY QUESTIONS?

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