Infrared spectroscopy, also termed vibrational spectroscopy, is a

technique that utilizes the interaction between infrared and the

sample.
Principle of IR spectroscopy/ Vibrational
spectroscopy
 The wavelength utilized for the analysis of organic
compounds ranges from 2,500 to 16,000 nm, with a
corresponding frequency range from 1.9×1013 to
1.2×1014 Hz.
 These rays don’t have enough energy to excite the
electrons, but they do, however, cause the vibrational
excitation of covalently bonded atoms or groups.
 The vibration observed in the atoms is characteristic of
these atoms and thus helps in the detection of the
molecules.
 The infrared spectrum is the fundamental measurement
obtained in infrared spectroscopy.
 The spectrum is a plot of measured infrared intensity versus
wavelength (or frequency) of light.
 IR Spectroscopy measures the vibrations of atoms, and
based on this; it is possible to determine the functional
groups.
Steps of IR spectroscopy/ Vibrational
spectroscopy
 The IR spectrometer is turned on and allowed to warm up for
30 minutes.
 The unknown sample is taken, and its appearance is
recorded.
 The background spectrum is collected to remove the
spectrum obtained from natural reasons.
 A small amount of sample is placed under the probe by
using a metal spatula.
 The probe is set in place by twisting it.
 The IR spectrum of the unknown sample is obtained. The
process is repeated, if necessary, to get a good quality
spectrum.
 The absorption frequencies that indicate the functional
groups present are recorded.
 The obtained spectrum is analyzed to determine the
probable identification of the unknown sample.
Uses of IR spectroscopy/ Vibrational
spectroscopy
Infrared spectroscopy has been widely used for the
characterization of proteins and the analysis of various solid,
liquid, and gaseous samples. 4. Mass spectroscopy
Mass spectroscopy is a type of spectroscopic technique that helps
to identify the amount and type of chemicals present in the
sample by analyzing the mass to charge ratio of the ions.
Principle of Mass spectroscopy
 Mass spectroscopy is based on the principle that when a
sample is bombarded with electrons, the molecules in the
compounds are ionized into ions.
 The separation of ions is dependent on their mass to charge
ratio. For most ions, the charge is one which means that
the ratio is simply the molecular mass of the ion.
 The ions are then subjected to electric and magnetic fields
which causes deflection of the ions. Ions with a similar
charge to mass ratio show similar deflection.
 The relative abundance of each of such ions is then detected
with the help of the detectors.
 The mass spectrum is formed by plotting the relative
abundance of the ions against the ratio of mass to charge.
 The spectrum can then be used for the determination of the
elemental configuration of the sample, the masses of the
particle or molecules, and the chemical structure of the
sample.
Steps of Mass spectroscopy
 200 µl of the sample is mixed with 1.8 ml of 65% nitric acid.
 The mixture is then added to the water bath at 50°C
overnight.
 The tubes are then cooled down to room temperature, and
the sample is diluted by adding 8 ml distilled water to
obtain nitric acid concentration below 20%.
 The sample is then added to the spectroscope and run.
 The results are obtained through the software on the
computer in the form of the mass spectrum.
Uses of Mass spectroscopy
 Mass spectroscopy is a valuable tool to quantify known
materials.
 It also allows the identification of unknown compounds and
determination of the structure and chemical composition of
various substances.
15. Molecular spectroscopy
Molecular spectroscopy is a type of spectroscopy that utilizes the
interaction between molecules and electromagnetic radiation to
determine the structural composition of samples.
Principle of Molecular spectroscopy
 Molecular spectroscopy involves the interaction of materials
with electromagnetic radiation in order to produce an
absorption pattern (i.e. a spectrum) from which structural
or compositional information can be deduced.
 The molecular spectrum is formed when the molecules move
from one energy state to another as a result of interaction
with different electromagnetic radiation.
 The mechanisms involved are similar to atoms, but the
process is more complicated.
 The interaction between different nuclei and electrons takes
place in molecules that are not observed in atoms.
 These interactions can be electronic, rotational, or
vibrational. Based on the type of interactions, different
spectrometric techniques can be applied for the analysis of
the compounds.
 Similarly, either the absorption or emission spectrum can be
utilized based on the type of interactions being exploited.
Steps of Molecular spectroscopy
 Different types of spectrometer systems like emission
spectrometer and Fourier-transform spectrometer can be
used for molecular spectroscopy.
 All these systems include a source of radiation, a sample,
and a system for detecting and analyzing the sample.
 The sample is added to the spectrometer where the
radiation source focuses on it.
 Two samples of known and unknown concentrations are
taken in a transport vessel, also termed as a cuvette.
 The vessels are then placed, one after the other, in the
spectrophotometer that is provided with light source and
detectors.
 The spectrophotometer is operated that passes light of a
particular wavelength through the sample.
 The photosensitive detectors present in the
spectrophotometer detect the light passing through the
sample, which is then converted into digital values.
 A graph of the absorbance measured against the
concentration of the sample is plotted, which can then be
used for the determination of the unknown concentration
of the sample.
Uses of Molecular spectroscopy
 Molecular spectroscopy allows the analysis of the atomic and
molecular structures of various compounds.
 It also helps in the determination of gas composition as well
as the composition of other compounds.
UV spectroscopy is a type of absorption spectroscopy where UV
lights are absorbed by the electrons that causes them to excite to
a high energy state.
Principle of UV spectroscopy
 In UV spectroscopy, the UV rays passed to the sample are
absorbed by the electrons, which increases the energy of
the system.
 This causes the excitation of an electron from a lower
energy state to a higher energy state.
 This excitation forms an absorption spectrum that can be
detected by the detectors in the spectrometer.
 The amount of photon (radiation) absorbed results in an
absorption spectrum which can then be measured in terms
of absorbance.
 The absorbance of a sample is dependent on the number of
excited electrons which in turn is dependent on the
concentration of molecules in the sample.
Steps of UV spectroscopy
 Two samples of known and unknown concentrations are
taken in a transport vessel, also termed as a cuvette.
 The vessels are then placed, one after the other, in the
spectrophotometer that is provided with light source and
detectors.
 The spectrophotometer is operated that passes light of a
particular wavelength through the sample.
 The photosensitive detectors present in the
spectrophotometer detect the light passing through the
sample, which is then converted into digital values.
 A graph of the absorbance measured against the
concentration of the sample is plotted, which can then be
used for the determination of the unknown concentration
of the sample.
Uses of UV spectroscopy
 UV spectroscopy is a technique used for the detection of
impurities in organic substances.
 This can also be used for the quantitative determination of
compounds that can absorb UV radiation.
  It can also be used for the study of the kinetics of a reaction
where the UV rays are passed through the reaction cell,
and the changes in absorbance are studied.
21. Ultraviolet and visible (UV/Vis)
spectroscopy
Ultraviolet and visible spectroscopy is an absorption spectroscopy
technique which uses the radiation in the UV range and the
adjacent visible range of the electromagnetic radiation.
Principle of UV/Vis spectroscopy
 UV/Vis spectroscopy is based on the principle that materials
produce an absorption spectrum which is a range of
absorbance resulting from the radiation absorbed by the
material at different frequencies.
 The absorption spectrum of materials depends on the atomic
and molecular composition of that material.
 The frequency of light radiation absorbed by a material is
dependent on the energy difference between the two
energy states of the molecules.
 The absorption results in the formation of the absorption
line, which, together with other lines, form an absorption
spectrum.
 The incident light in this spectrometer is in the range of UV
and visible spectrum of the electromagnetic spectrum.
 Thus, when a photon with sufficient energy reaches an
object, the energy is absorbed by the electrons causing
them to bump into a higher energy state.
 The amount of photon (radiation) absorbed results in an
absorption spectrum which can then be measured in terms
of absorbance.
 The absorbance of a sample is dependent on the number of
excited electrons which in turn is dependent on the
concentration of molecules in the sample.
Steps of UV/Vis spectroscopy
 Solvent liquid and the sample solution are taken in two
transport vessels, also termed as cuvettes.
 The vessel with solvent liquid is then placed in the
spectrometer to determine the light loss due to scattering
and absorbance by the solvent. Any absorbance observed
in this process is to be subtracted from the absorbance of
the sample.
 The cuvette with the sample solution is then placed in the
spectrometer.
  The absorbance of the sample is noted in different
frequencies which usually ranges from 200-800 nm.
 A similar spectrum is formed from the different
concentrations of the samples.
 A graph of the absorbance measured against the
concentration of the sample is plotted, which can then be
used for the determination of the unknown concentration
of the sample.
Uses of UV/Vis spectroscopy
 Qualitative analysis may be performed in the UV/Vis regions
to identify certain classes (proteins and nucleic acids) of
compounds both in the pure state and in biological
mixtures.
 This type of spectroscopy is used for the quantification of
biological samples either directly or via colorimetric assays.

16. Mossbauer spectroscopy

Mossbauer spectroscopy is a technique based on the Mossbauer
effect discovered by Rudolf Mössbauer which utilizes the
spectrum formed by the absorption or emission of nuclear gamma
rays in solid particles.

IR spectroscopy can be used for the detection of functional
groups which helps in the identification of molecules and
their composition.
Applications of IR spectroscopic techniques allow identifying
molecular changes due to bodily changes, understanding of the
molecular mechanism of various diseases, and identifying specific
3.
spectral biomarkers that can be used in diagnosis.
Atomic absorption spectroscopy
Atomic absorption is an analytical technique utilizing the principle
of spectroscopy for the quantitative determination of chemical
elements.
Principle of Atomic absorption spectroscopy
 Atomic absorption spectroscopy utilizes the principle that
free electrons generated in an atomizer absorb radiation of
different wavelengths.
 The free electrons absorb UV or visible light, causing the
electrons to transfer to higher energy orbits.
 During this process, the absorption spectrum is released,
which is detected by the photodetectors.
  The absorption spectrum formed allows the quantification of
free electrons in the gaseous state of the matter.
 The amount of photon (radiation) absorbed results in an
absorption spectrum which can then be measured in terms
of absorbance.
 The absorbance of a sample is dependent on the
concentration of molecules in the sample.
Steps of Atomic absorption spectroscopy
 The liquid sample is mixed with a particular volume of spirit
which is added to a flask which is then vaporized into a gas
by a fuel-rich acetylene-nitrous oxide flame.
 A lamp is set with the necessary wavelength as a light
source.
 The gas formed from the liquid sample is then passed
through a detector that detects the absorbance of the
atoms in the gas.
 A similar process is performed for the detection of
absorbance of solvent bank and standard solution.
 A graph is plotted for the absorbance against the
concentration of the molecules in the sample.
Uses of Atomic absorption spectroscopy
 Atomic absorption spectroscopy can be used for the
quantitative and qualitative determination of metallic
elements in biological systems.
 This also helps in the detection of metals as an impurity in
alloys and other mixtures.
 Atomic absorption spectroscopy has been utilized for the
purification of environmental samples like water and soil.
 Detection of metals in pharmaceutical products and oil
products can also be done by this method.