JIMMA UNIVERSTY

COLLAGE OF NATURAL SCIENCE

DEPPARTMENT OF CHEMISTRY

Instrumental Techniques in Environmental

Analysis (EnvChem 553)

BY: TAGAY ALEMU

INSTRUCTOR : SHIMELIS ADDISU (PhD)

APRIL, 2024
JIMMA , ETHIOPIA

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 Basic Principles of Spectroscopy
 Spectroscopy deals with the production, measurement, and
interpretation of spectra arising from the interaction of
electromagnetic radiation with matter
 Interaction with Matter: When electromagnetic radiation
interacts with matter, it can be absorbed, transmitted or scattered
 The nature of this interaction depends on the energy levels of the
atoms or molecules in the material.

 LIGHT
Properties Light assumed as particles of energy that move
through space with wavelike properties.

Light has a dual nature: particulate and wave like.

 Phenomena associated with light propagation, such as
interference, diffraction, and refraction, are easily explained using
the wave theory of electromagnetic radiation.
 The interaction of light with matter, which is the basis of
absorption and emission spectroscopy, best understood in terms
of the particulate nature of light.

 Electromagnetic Radiation: Spectroscopy involves the use of

electromagnetic radiation, which includes a wide range of
wavelengths, from radio waves to gamma rays.
 Different regions of the electromagnetic spectrum are used for
different types of spectroscopy.
 The wave properties of electromagnetic radiation are described
in terms of the wave’s frequency, wavelength, and amplitude.
Amplitude
Wavelength C = λν
 Shorter wavelength means greater frequency
 Frequency
 Frequency is defined as the number of cycles per second, and
is expressed as sec-1 or Hertz (Hz).
express as: E = hν
 E is energy
 h is Planck's constant, (h = 6.62607 x 10-34 J s)
 ν is frequency

Velocity
 The velocity of wave in general is expressed as: Velocity = λν
For Electromagnetic wave, the velocity in vacuum is 2.99 × 108m/s.
 ENERGY STATES OF MATTER
Quantum Nature of Matter

 The energy content of matter is quantized.

 The potential or internal energy content of an atom or molecule does
not vary in a continuous manner
 Atoms and molecules, under normal conditions, exist
predominantly in the ground state, which is the state of lowest
energy.
 Ground state atoms and molecules can gain energy, their higher
energy states, referred to as excited states.
 Ground State
Lowest Energy Configuration

In the ground state, atoms and molecules exist in their most

stable and lowest energy arrangement.

Equilibrium State

The ground state represents the equilibrium condition where

the system has minimum potential energy.

Fundamental Properties

Characteristics like bond lengths, angles, and electron

distributions are defined in the ground state.
 Excited States

 Higher Energy Configurations

 When atoms or molecules absorb energy, they can be promoted
to higher electronic energy levels, known as excited states.

 Instability

 Excited states are inherently unstable, as the system seeks to

return to the more stable ground state configuration.

 Potential Energy Surfaces

 The transitions between electronic energy levels can be visualized

using potential energy surfaces, which map the energy landscape.
Electronic Energy Levels:
In atoms, electrons occupy specific energy levels or orbitals
around the nucleus.
These energy levels are quantized, meaning that only certain
discrete energy values are allowed.
Vibrational Energy Levels:
 These vibrational motions have quantized energy levels.
 When a molecule absorbs energy, infrared radiation,& its
vibrational energy levels can be excited, changes in the
molecular structure and properties.

Rotational Energy Levels:

 Molecules can also rotate around their center of mass. The rotation
of a molecule is quantized, meaning it can only occur at certain
energy levels.
Electronic Transitions:
• When atoms or molecules transition between different
electronic energy levels, they can emit or absorb
electromagnetic radiation, in characteristic spectra.
• The energy of the absorbed or emitted photons corresponds to
the energy difference between the initial and final energy
levels.
 Atomic absorption spectroscopy

 Atomic absorption spectroscopy (AAS) is an analytical technique

used to determine the concentration of specific elements in a
sample by measuring the absorption of light by atoms in the
ground state.
 It is widely employed in various fields, including environmental
analysis, pharmaceuticals, and metallurgy.
 Here are the details regarding the instrumentation, principles, and
applications of atomic absorption spectroscopy:
 Instrumentation:
Light Source:
 A hollow cathode lamp (HCL) is the most common light source
used in AAS.
 The HCL contains the element of interest in its cathode, and
when a high voltage is applied, it emits characteristic
wavelengths of light corresponding to that element.

Monochromator:
 A monochromator is used to isolate the specific wavelength of
light emitted by the HCL.
 It ensures that only the absorption line of interest reaches the
sample, minimizing interference from other elements present in
the sample.
Sample Introduction System:
 The sample introduction system delivers the sample into the
atomizer, where it is converted into gaseous atoms.
 Common methods of sample introduction include flame
atomization, (graphite furnace), and hydride generation.
Atomizer:
 The atomizer converts the sample into atomic form by
vaporizing, and the analyte.
 In flame atomization, the sample is nebulized and introduced into
a flame,
 Electro thermal atomization involves the heating of a solid sample
placed on a graphite furnace, leading to volatilization and
atomization.
 Detector
 The detector measures the amount of light absorbed by the
sample.
 The most common detector used in AAS is a photomultiplier
tube (PMT), which converts the light signal into an electrical
signal that can be quantified.
 Principles
Absorption of Light:
 When a beam of light passes through a sample the atoms can absorb
specific wavelengths of light corresponding to their electronic
transitions from the ground state to higher energy levels.

Beer-Lambert Law:

 which states that the absorbance of a sample is directly proportional

to the concentration of the absorbing species and the path length of
the light through the sample.
Calibration Curve:

 To quantify the concentration of the element in the sample, a

calibration curve is constructed using standard solutions with
known concentrations.
 Applications
 Environmental Analysis:
 AAS is used to measure trace elements in environmental
samples, such as water, soil, and air.
 Pharmaceutical Analysis:
 AAS is employed in pharmaceutical industries to determine the
elemental composition of drugs, detect trace impurities, and
ensure the quality and safety of pharmaceutical products
 Metallurgical Analysis:
 AAS is utilized in metallurgical analysis to determine the
concentration of metals in ores, alloys, and industrial materials.
 Clinical Analysis:
 AAS is applied in clinical laboratories to measure essential
elements, such as calcium, iron, and zinc, in biological
samples.
 Food and Beverage Analysis:
 AAS is used to analyze the elemental composition of food and
beverages, including the determination of heavy metals,
nutritional elements, and contaminants.
 UV-Vis spectroscopy
Is a widely used analytical technique that measures the
absorption of ultraviolet and visible light by molecules
It provides information about the electronic structure,
concentration, and chemical properties of a sample
The transition is from the highest occupied molecular orbital
(HOMO) to the lowest unoccupied molecular orbital
(LUMO).
 Types of Electronic Transition
 σ → σ* transition:
This transition takes place when σ electrons are
excited to the corresponding antibonding σ* orbital.
eg.methane (which has only C-H bonds, and can only
undergo σ → σ* transitions).
 n → σ* transition
 In this type of transition, an electron of unshared electron pair on
hydrogen atom get excited to π* antibonding orbital
 π → π* transition
 promotion of π electrons to π* antibonding orbital. It requires
smaller energy and occurs at longer wavelengths.
 n → π* transition
 Non bonding electron gets promoted to an antibonding sigma
orbital. Need less energy than σ →. σ* transitions
 Instrumentation

 Light Source:
 UV-Vis spectrophotometers use a deuterium lamp for the
ultraviolet region (190-400 nm) and a tungsten-halogen lamp for
the visible region (400-800 nm).
 These lamps provide a broad spectrum of light for the analysis.

Monochromator:
 A monochromator is used to isolate specific wavelengths of light
from the light source. It selects a narrow range of wavelengths
and directs them towards the sample.
 Sample Compartment:
 The sample compartment holds the sample for analysis.
 The sample should be optically transparent to the wavelength of
light being used.
Detector:
 A photomultiplier tube (PMT) is used as the detector in UV-Vis
spectroscopy.
 These detectors convert the light intensity into an electrical signal
that can be measured and analyzed.
Data Acquisition and Analysis:
 The detector output is processed by a computer or a data
acquisition system, which records the absorbance or transmittance
values.
 Principles
 Absorption of Light: When a molecule absorbs light in the UV-Vis
range, it undergoes electronic transitions.
 These transitions involve the promotion of electrons from lower
energy orbitals (ground state) to higher energy orbitals (excited
state).

 Beer-Lambert Law:
 The absorption of light by a sample is governed by the Beer-
Lambert Law, which states that the absorbance of a sample is
directly proportional to the concentration of the absorbing species,

 Calibration Curve:
 To quantify the concentration of a compound in a sample, a
calibration curve is constructed using standard solutions with
known concentrations.
 Applications:
 Quantitative Analysis: UV-Vis spectroscopy is commonly used
for quantitative analysis of compounds in areas such as
pharmaceuticals, environmental analysis, and chemical research.

Qualitative Analysis: UV-Vis spectroscopy is employed for

qualitative analysis to identify and characterize compounds based
on their absorption spectra.

 Environmental Analysis: UV-Vis spectroscopy helps in the analysis

of environmental samples, such as water and air, for the presence of
pollutants, organic compounds, and heavy metals.
Biochemical Analysis:
 UV-Vis spectroscopy is widely employed in biochemistry to study
biomolecules like proteins, nucleic acids, and enzymes.
 It aids in determining their concentration, structure, and
interactions.

Pharmaceutical Analysis:
 UV-Vis spectroscopy is utilized in pharmaceutical industries to
assess the purity and concentration of active pharmaceutical
ingredients, monitor drug stability, and perform dissolution
testing.
 FT-IR. SPECTROSCOPY
FT-IR. is a powerful analytical technique
used to identify and characterize chemical
compounds based on their infrared absorption
spectra
 It provides valuable information about
molecular structure, functional groups,
and chemical bonding.
Instrumentation:
Infrared Source: A broadband infrared source emits a range of infrared

radiation, typically spanning the mid-infrared region of the

electromagnetic spectrum about 2.5-25 μm).
 A silicon carbide rod, and Nernst glower are common sources
used in FT-IR spectrometers.
 Interferometer: The interferometer is a key component of an FT-IR
spectrometer.
It consists of a beam splitter, a moving mirror, and a fixed mirror.
Modulates the intensity of infrared radiation, creating an interferogram.

Sample The sample compartment holds the sample for analysis.

 The sample can be in forms of , liquids, solids, gases, or thin films.
 Different types of sample holders are available to accommodate
different sample types.

Detector: A detector, such as a liquid nitrogen-cooled mercury cadmium

telluride detector or a deuterated triglycine sulfate detector, is used to
measure the intensity of the infrared radiation after it passes through the
sample.
Data(Acquisition and Analysis) The electrical signal from the
detector is processed by a computer or data acquisition system.

Principles of FT-IR
 Infrared Absorption: When infrared radiation passes through a
sample, certain wavelengths of the radiation are selectively
absorbed by the sample's molecules.

Interferometry: In an FT-IR spectrometer, the interferometer

creates an interferogram by splitting the incoming infrared radiation
into two beams, one passing through the sample and the other
through a reference mirror.

Fourier Transform: The interferogram obtained from the

interferometer is Fourier transformed to generate a spectrum.
 Mathematically converts the time-domain interferogram into a
frequency-domain spectrum,
 which represents the absorption intensity as a function of
wavenumber or wavelength.
 Applications of FT-IR :
Chemical Analysis:
o FT-IR spectroscopy is extensively used for the identification and
characterization of organic and inorganic compounds.

Pharmaceutical Analysis:
o FT-IR spectroscopy plays a crucial role in pharmaceutical
analysis.
o It is used for the analysis of drug formulations, identification of
drug substances, detection of impurities, and monitoring drug
stability.
Polymer Analysis:
 FT-IR spectroscopy is employed in the analysis of polymers to
study their structure, composition, and chemical properties

Environmental Analysis:
 FT-IR spectroscopy is utilized in environmental analysis to
identify pollutants, assess air quality, analyze water samples,
and study soil composition.
Biomedical Research:
 FT-IR spectroscopy is applied in biomedical research to study
biomolecules, such as proteins, nucleic acids, lipids, and
carbohydrates.
 Gas chromatography (GC)
 Gas chromatography is a powerful analytical technique used to
separate, identify, and quantify a wide range of volatile chemical
compounds.
 This versatile method is essential for research, quality control,
and environmental monitoring across various industries.
 Fundamentals of Gas Chromatography
Sample Separation
The core principle of gas chromatography is to separate the
components of a complex sample mixture for individual analysis.

Temperature Control
The sample is vaporized and flows through a heated column where
the components separate based on their boiling points and
interactions with the stationary phase.

Detection
As the separated components exit the column, they are detected and
measured, providing quantitative and qualitative data about the
sample.
 Principles of Gas Chromatography
1 Volatility
The sample must be in a gaseous state to be separated effectively
This is achieved by heating the sample to ensure its volatility.

2 Partitioning
The sample vapors are partitioned between the mobile gas
phase and the stationary phase based on their affinity and
solubility.
3
Retention Time
Each compound has a unique retention time, allowing for
identification based on the time it takes to elute from the column.
Stationary Phase
 The stationary phase can be either a solid adsorbent or a liquid coating.
 In packed columns, the stationary phase is typically a solid material,
such as silica or alumina, coated onto an inert support material.
 In capillary columns, the stationary phase is a thin liquid film
immobilized on the inner walls of a fused silica capillary.

 Mobile Phase (Carrier Gas)

 The carrier gas is typically an inert gas, such as helium, nitrogen, or

hydrogen.
 It carries the sample through the column and does not interact with
the analytes.
 The choice of carrier gas depends on factors such as column
efficiency, detector compatibility, and safety considerations.
 Sample Injection:
 The sample is introduced into the gas chromatograph by injection.
 There are several injection techniques available, such as split, split
less, and on-column injections.
 Sample Separation:
 As the sample components interact with the stationary phase, they
experience different degrees of interaction and partitioning.
 Components with stronger affinity for the stationary phase will
spend more time interacting with it and will exhibit slower
migration through the column, resulting in longer retention times.
 Weaker interacting components will elute from the column faster.
 Detection
 The separated components exit the column and enter a detector,
which detects and quantifies the analytes.
 Several types of detectors are commonly used in GC, such as
flame ionization detector (FID), thermal conductivity detector
(TCD), electron capture detector (ECD), and mass spectrometer
(MS).
 Detectors provide signals that are proportional to the
concentration of the analytes, allowing for their identification and
quantification.
 Benefits of Gas Chromatography

1.High separation efficiency –

 GC can separate complex mixtures into individual components
with high resolution.

2.Sensitivity and selectivity –

 GC can detect and quantify trace-level analytes with excellent
sensitivity and selectivity.
3.Wide applicability –
 GC is used for analysis of a diverse range of volatile and semi-
volatile organic compounds in various fields like chemistry,
environmental science, and forensics.
 Factors Affecting Separation in GC
1 Column Temperature
2 Carrier Gas Flow Rate
 Increasing the column temperature
 Adjusting the carrier gas flow
improves separation by increasing
rate impacts the rate of analyte
analyte diffusion and volatility, but
transport through the column,
can also lead to loss of resolution
affecting retention time and
for some compounds.
resolution.

3 Stationary Phase Polarity

4 Sample Composition
 Choosing a stationary phase with the
 The complexity and
appropriate polarity is crucial for compatibility of the sample
effective separation, as it determines the matrix can influence the
interaction between the analytes and the separation, as some components
column. may interfere with the analytes
of interest.
High performance liquids chromatography

It separates the analytes based on their differential interactions

with a stationary phase and a mobile phase.
The stationary phase is typically a packed column containing
small particles with a high surface area, while the mobile phase
is a liquid solvent or a mixture of solvents
 Stationary Phase:
 The stationary phase is a solid or liquid material that is packed
into a column.
 It can be polar or nonpolar, depending on the sample and the
separation requirements.
 The stationary phase provides a surface for the analytes to interact
with and separates them based on their affinity for the stationary
phase.
 Mobile Phase:
The mobile phase is a liquid solvent or a mixture of solvents
that flows through the column.
It carries the sample components through the stationary phase.
The composition of the mobile phase can be adjusted to
optimize the separation based on the analytes characteristics

Sample Injection:
 The sample, which contains the analytes of interest, is
introduced into the HPLC system through an injection port.
 The sample is typically dissolved in a compatible solvent to
ensure proper interaction with the stationary phase.
 Separation Mechanisms:
 HPLC can utilize various separation mechanisms, including
adsorption, partition, ion exchange, size exclusion, and
affinity.

Adsorption Chromatography:
 In this mechanism, the analytes interact with the stationary phase
through adsorption.
 Components with stronger interactions spend more time interacting
with the stationary phase and are retained longer, resulting in
slower elution.
 Partition Chromatography:
 Partition chromatography is based on differential partitioning of
the analytes between the stationary phase and the mobile phase.
 The analytes distribute themselves between the two phases based
on their solubility and affinity, leading to separation

Ion Exchange Chromatography:

 Ion exchange chromatography separates analytes based on their
charge interactions with the stationary phase.
 The stationary phase consists of charged sites that attract or repel
analytes with opposite or similar charges, respectively.
THANK YOU !!

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