Module III

Instrumental Methods:
• UV-visible, Atomic Absorption Spectroscopy,
• Flame Photometry with Reference to Principle,
• Instrumentation,
• Calibration, Working
• Applications in Environmental Analysis,
• Chromatography and its Types,
• Mass Spectroscopy and Gas Chromatography with Reference to Principle,
• Instrumentation,
• Calibration, Working
• Applications in Environmental Analysis
Analytical Chemistry:
Definition:
• “The science and art of determining composition of material in terms
of element or compound content”

•– Concerned with chemical characterization of matter

•– Involves Qualitative and Quantitative determination

Qualitative analysis
• It may be define as “Which analyte present in the given sample”.
• This has the goal to identify the various component present in the
sample on the basis of physical and chemical properties such as
functional group, element composition and melting point etc.

Quantitative analysis
• It may be define as “How much amount of analyte present in the
sample”.
• This has the goal to determine the quantity of each component
present in the sample.
 Classification of Analytical Methods

Analytical
Methods

Chemical Methods Physical Methods

(Classical Methods) (Instrumental
Methods)
 Classical Methods

• Gravimetric method

• Volumetric method
– Acid-base titrations
– Oxidation-reduction titrations
– Precipitation titrations
– Complexometric titrations
• Gravimetric methods in analytical chemistry determine an
analyte's quantity by measuring the mass of a solid substance
derived from it, often through precipitation gravimetry (forming a
precipitate like silver chloride from chloride ions) or volatilization
gravimetry (collecting a gaseous product like carbon dioxide from
a solid sample).
• After precipitation or volatilization, the solid is filtered, washed,
dried, and accurately weighed to calculate the analyte's
concentration using stoichiometry.
Instrumental Methods:
• Now a days instrumental method of analysis is widely used.
• These method are extremely sensitive, providing precise and detailed
information from small of material.
a. Electro analytical method: it is the modification of methods in which the
reaction process and endpoint are determine by an electric measurement.
b. Spectroanalytical methods: It is widely used for quantitative as well as
qualitative analysis. They give result in the form of an interpredectable
spectrum.
c. Chromatography methods: The method is used for the determination of
complex mixture or mixture of compound by separation of individual. For ex.
Gas, HPLC/TLC
 Instrumental
Analysis

Spectral methods Electro analytical methods Separative Miscellaneous

(Optical methods) methods
methods
 Spectral Methods

• Visible spectroscopy
• Colorimetry, Nesslerimetry
• UV-visible spectrometry
• I R spectroscopy

• X-ray diffraction

• Atomic absorption spectroscopy

• Atomic emission spectroscopy
• Flame photometry
• Polarimetry
• Refractometry
• Nephlometry/ Turbidimetry
 Electro Analytical Methods

• Conductometry

• Potentiometry, pH metry

• Amperometry
 Separative Methods

• Chromatographic methods

– Column chromatography

– Thin-layer, paper chromatography

– Gas chromatography

– High performance liquid chromatography

 Miscellaneous Methods

Thermal methods:

• Thermogravimetric analysis

• Differential thermal analysis

• Differential scanning calorimetry

The following steps are involved in the analysis of sample:
1. Obtained the sample in pure form
2. Preparation of sample
3. Identification of suitable analytical procedure
 Chemical or physical method
 Condition determined either by analytical problem by the
substance being investigated.
 Requirement, either fast, accurate & economical adaptable to
automation
 Evolution of satisfactory result.
 Module: III
Instrumental Methods
1. Spectroscopy: a). UV-Visible Spectroscopy: Measures the
absorption of ultraviolet and visible light by molecules to identify
and quantify substances.
b). Infrared (IR) Spectroscopy: Analyzes the absorption of
infrared radiation to identify functional groups in organic and
inorganic compounds.
c). Nuclear Magnetic Resonance (NMR) Spectroscopy:
Utilizes the magnetic properties of atomic nuclei to determine the
molecular structure of compounds.
 Instrumental Methods
2. Mass Spectrometry (MS): Determines the mass-to-charge ratio
of ions to identify and quantify molecules in a sample, providing
information about their structure and composition.
3. Chromatography: a) Gas Chromatography (GC): Separates
and quantifies volatile compounds in a gas phase.
b). Liquid Chromatography (LC): Separates and quantifies
compounds dissolved in a liquid phase.
c). High-Performance Liquid Chromatography (HPLC): A
variant of liquid chromatography used for higher resolution and
sensitivity.
 Instrumental Methods
4. Electrochemical Methods: a). Potentiometry: Measures electrical
potential (voltage) to determine ion concentrations or pH.
b). Voltammetry: Measures current as a function of applied voltage
to analyze electroactive species.
5. Atomic Spectroscopy: a). Atomic Absorption Spectroscopy
(AAS): Determines the concentration of specific elements in a sample
by measuring the absorption of light at characteristic wavelengths.
b). Inductively Coupled Plasma Mass Spectrometry (ICP-MS):
Combines ionization and mass spectrometry to detect and quantify
trace elements at extremely low concentrations.
 Instrumental Methods
6. Thermal Analysis: a). Differential Scanning Calorimetry
(DSC): Measures heat flow in a sample to analyze phase transitions,
melting points, and reaction kinetics. b). Thermogravimetric
Analysis (TGA): Measures changes in mass as a function of
temperature to study decomposition and degradation processes.
7. X-ray Techniques: a). X-ray Diffraction (XRD): Determines the
crystal structure of materials by analyzing the diffraction pattern of
b). X-rays.X-ray Fluorescence (XRF): Identifies and quantifies
elements in a sample by measuring the emission of characteristic X-
rays.
 Instrumental Methods

8. Microscopy:
a). Scanning Electron Microscopy (SEM): Provides high-
resolution images and information about the surface morphology of
materials.
b.) Transmission Electron Microscopy (TEM): Offers detailed
images and structural information at the nanoscale.
c). Atomic Force Microscopy (AFM): It is a powerful imaging and
measurement technique used in nanotechnology and materials
science.
1. Spectroscopy Definition:
The study of how matter interacts with electromagnetic radiation (light).
Focus: The theoretical and analytical principle behind measuring light
absorption, emission, or scattering by substances.
Purpose: To understand molecular structure, energy levels, and chemical
composition.
Examples:
• UV–Visible Spectroscopy
• Infrared (IR) Spectroscopy
• Atomic Absorption Spectroscopy (AAS)
• NMR Spectroscopy
Think: “Spectroscopy = the science or theory behind how light interacts with
matter.”
2. Spectrometry Definition: The measurement or quantification
of the light–matter interaction identified by spectroscopy.
Focus: The instrumental process of detecting and measuring
radiation intensity.
Instrument: A spectrometer.
Examples:
• Mass Spectrometry (MS)UV–Vis Spectrometry
• Atomic Emission Spectrometry.
Think: “Spectrometry = the actual measurement process based
on spectroscopy principles.”
3. Photometry Definition: The quantitative
measurement of light intensity, usually based on how
much light is absorbed or transmitted by a sample.
Focus: Measures concentration of substances by light
absorption (Beer–Lambert Law).
Instrument: A photometer.
Example: Colorimetry (a type of photometry used for
colored solutions).
Think: “Photometry = measuring how much light
passes through or is absorbed by a sample.”
4. Chromatography Definition: A separation technique used to
separate components of a mixture based on differences in how
they move through a stationary phase and a mobile phase.
Focus: Physical separation of mixture components before
further analysis.
Types:
• Paper Chromatography
• Thin Layer Chromatography (TLC)
• Gas Chromatography (GC)
• High-Performance Liquid Chromatography (HPLC)
Think: “Chromatography = separating substances before
measuring or analyzing them.”
Technique Main Function What It Measures Instrument
Study of light– Qualitative &
Spectroscopy matter interaction theoretical info – (principle)
Measurement of Intensity or
Spectrometry spectra wavelength Spectrometer
Measurement of
Photometry light intensity Concentration Photometer
Separation of
Chromatography mixtures Composition Chromatograph
UV-Visible Spectroscopy:

Single beam
spectrophotometer

Double beam
spectrophotometer
UV- visible Ultraviolet Visible
Spectroscopy
 UV- visible Ultraviolet Visible Spectroscopy

Principle:
UV-Visible spectroscopy is a technique used to measure the
absorption of ultraviolet and visible light by molecules. The
principle behind it is that molecules absorb light in the UV-Visible
region when the energy of the incident photons matches the energy
difference between electronic energy levels in the molecules.
 UV- visible Ultraviolet Visible Spectroscopy

Instrumentation: a). Light Source: UV-Visible spectrometers typically use a

deuterium lamp for the UV range and a tungsten filament lamp for the visible
range.
b). Monochromator: This device selects a specific wavelength of light to pass
through the sample.
c). Sample Holder: The sample is placed in a cuvette, often made of quartz to
allow UV measurements.
d). Detector: Photodiodes or photomultiplier tubes detect the intensity of
transmitted light.
e). Data Display and Analysis: Modern instruments are computer-controlled and
provide software for data collection, analysis, and presentation.
 UV- visible Ultraviolet Visible Spectroscopy

Calibration: Calibration is performed using known standards of

the substance of interest at various concentrations. A calibration
curve is generated by plotting the absorbance (A) against the
concentration (C) of the standard solutions. This curve is then
used to determine the concentration of an unknown sample based
on its absorbance.
 UV- visible Ultraviolet Visible Spectroscopy

Working: The light source emits a broad spectrum of light, which

passes through a monochromator to select a specific wavelength of
light. The selected wavelength of light is directed through the
sample solution in a cuvette.
The detector measures the intensity of light that passes through the
sample (I) and compares it to the intensity of the incident light (I₀).
Absorbance (A) is calculated as A = -log(I/I₀). A higher
absorbance indicates a greater absorption of light by the sample.
 UV- visible Ultraviolet Visible Spectroscopy

1. Applications in Environmental Analysis: UV-Visible

spectroscopy is widely used in environmental analysis for various
purposes, including:
2. Water Quality Monitoring: Determining concentrations of
pollutants like heavy metals, organic compounds, and nutrients in
water samples.
3. Analysis of Air Pollutants: Measuring the concentration of gases
like ozone and nitrogen dioxide in the atmosphere.
4. Monitoring Soil Contamination: Detecting the presence and
concentration of contaminants in soil samples.
 UV- visible Ultraviolet Visible Spectroscopy

5. Characterization of Natural Organic Matter: Studying the

composition of dissolved organic matter in natural waters.
6. Analysis of Pesticides: Identifying and quantifying pesticides
in environmental samples.
7. Monitoring of Algal Blooms: Detecting and quantifying
pigments produced by algae in water bodies.
Classification of Chromatography:

• Chromatography is an important biophysical technique that is used to separate,

identify, and analyse the components of a mixture.
• The classification of chromatography methods can be done based on various
factors, like the stationary phase, the separation mechanism, and the techniques
employed.
Phases for separation techniques
Sample Types and Sensitivity Ranges
Thin-Layer Chromatography
Preparative Thin-Layer Chromatography
Preparative Thin-Layer Chromatography
 O O
H3C H O
H3C
O O
O O
Gel Chromatography
Column Chromatography
Column Chromatography
Ion Exchange Chromatography
Advantages of Gas Liquid Chromatography (GLC)

 Detects trace amounts of analytes with high sensitivity.

 Excellent resolving power for complex mixtures.
Accurate and precise quantitative analysis.
Wide range of analytes can be analyzed.

Disadvantages of Gas Liquid Chromatography (GLC)

 Limited to Volatile Compounds
 Sample Preparation
 Limited Detection for Non-Volatile Compounds
 Inert Gas Costs
 Column Maintenance
 Limited Information on Chemical Structure
Applications:
Applications in different environmental matrices

• Air quality: GC is used to monitor air for pollutants like VOCs, which can
indicate smog formation or industrial contamination. It helps determine if air is
safe to breathe and can track pollution sources.
• Water quality: The technique is vital for analyzing drinking water, surface water,
and wastewater for contaminants such as pesticides, industrial chemicals, and
pharmaceuticals. It helps ensure the safety of water sources and monitor for
emerging contaminants.
• Soil and sediment: GC is used to investigate organic pollutants in soil and
sediment, such as polychlorinated biphenyls (PCBs) and PAHs, which can result
from industrial activities or agricultural practices.
Types of pollutants identified
•Volatile Organic Compounds (VOCs): These are a major class of pollutants
found in air and water, and GC is used to identify them.
•Pesticides and herbicides: GC can detect and quantify pesticide residues in
soil, water, and food, helping to evaluate agricultural practices and ensure food
safety.
•Persistent Organic Pollutants (POPs): This includes compounds like PCBs,
which are stable and can persist in the environment for a long time. GC is a
primary tool for monitoring their presence.
•Polycyclic Aromatic Hydrocarbons (PAHs): Found in the environment from
sources like incomplete combustion, PAHs are analyzed by GC in air, water, and
soil.
•Emerging contaminants: GC, especially in combination with mass
spectrometry (GC-MS), is used to detect newer pollutants like pharmaceuticals
and endocrine-disrupting compounds in environmental samples
Mass spectroscopy Instrumentation
Mass spectroscopy principle: Mass spectrometry (MS) is an
analytical technique used to determine the mass-to-charge ratio
(m/z) of ions in a sample. It is based on the principles of ionization,
mass analysis, and detection. Here's an overview of the principle of
mass spectrometry
Mass spectroscopy Working
Quick Summary:
•Spectroscopy → Science/theory (interaction with light).
•Spectrometry → Measurement of that interaction.
•Photometry → Measures light intensity to find concentration.
•Chromatography → Separates mixture components before further
analysis.
Point of Comparison Spectroscopy Spectrometry Photometry Chromatography
Study of interaction between Measurement and analysis of Measurement of light intensity
Separation technique used to
1. Definition matter and electromagnetic spectra obtained from absorbed or transmitted by a
divide components of a mixture.
radiation. spectroscopy. solution.

To understand the structure, To quantify and identify elements To separate, identify, and
To determine concentration of
2. Objective bonding, and composition of or compounds based on their sometimes quantify mixture
analytes using light absorption.
substances. spectra. components.

Based on detection and Based on partitioning of solutes

Based on absorption, emission, or Based on Beer–Lambert’s law
3. Basic Principle quantification of energy or mass between stationary and mobile
scattering of light. (absorbance ∝ concentration).
signals. phases.

Quantitative (measures Both qualitative and quantitative

Qualitative (explains how matter Quantitative (measures the extent
4. Nature of Technique concentration from light (separates and measures
interacts with light). of that interaction).
intensity). components).

Electromagnetic radiation (UV, IR, Electromagnetic or particle-based No radiation — relies on solvent

5. Type of Radiation Used Usually visible or UV light.
visible, X-ray, etc.). (e.g., ions in mass spectrometry). flow or carrier gas.

Spectroscope or Spectrometer (e.g., Mass, UV–Vis, Chromatograph (e.g., HPLC, GC,

6. Instrument Used Photometer or colorimeter.
spectrophotometer. Atomic Emission). TLC apparatus).

Usually gaseous, liquid, or Same as spectroscopy; may Clear liquid samples allowing light Any mixture in liquid or gas form
7. Sample Requirement
dissolved solid samples. require ionized or vaporized form. passage. depending on technique.
Spectrum showing relation
Numerical spectral data — peaks, Absorbance or transmittance Chromatogram showing
8. Data Obtained between wavelength and
mass-to-charge ratio, etc. values used for concentration. separated component peaks.
intensity.
Elemental analysis, isotope Concentration measurement in Separation of mixtures in
Structural and functional group
9. Common Applications identification, molecular mass solutions (e.g., metals, pharmaceuticals, food, water, and
analysis in chemistry.
determination. biomolecules). forensic analysis.

UV–Vis, IR, Raman, NMR, Atomic Mass Spectrometry, UV–Vis Colorimetry, Flame Photometry, Paper, Thin Layer (TLC), Gas (GC),
10. Example Techniques
Absorption Spectroscopy. Spectrometry, AES, ICP–MS. UV–Visible Photometry. Liquid (HPLC) Chromatography.