Analysis of perilous elements in

wheat flour and pulse using

Gas chromatography-mass spectroscopy

Thesis submitted to the University of Lucknow

For the award of the degree of
Master of Science
In
Forensic Science

By
Gigyasha Singh

Under the supervision of

Dr. Keya Pandey

Department of Anthropology
University of Lucknow
Lucknow-226007
October 2023

[1]
CERTIFICATE

[2]
 ACKNOWLEDGMENT
Acknowledgment for a few might be just a trifle thing written on a piece of paper. Here I get a
great chance to express my token of thanks to people who in a way helped and supported me
to complete this record. I am grateful to The Director, Joint Director, staff, and officials of State
Forensic Science Laboratory, Lucknow. I would like to thank my supervising guide faculty Dr.
Keya Pandey ma’am (Head of Department) for her guidance all along with a deep sense of
gratitude, I wish to thank every one of my family members and friends for continuous support
and indulgence throughout.

Gigyasha Singh
(Roll No: 2110014205060)
Master of Science - Forensic Science

[3]
 SELF DECLARATION
I, Gigyasha Singh hereby declare that all the provided data of this experiment is done by me.
Strict norms, rules and protocols have been followed. Experiments are conducted under the
supervision of guides and staff members.

Gigyasha Singh
(Roll No: 2110014205060)
Master of Science - Forensic Science

[4]
 TABLE OF CONTENT
SR. List of Content PAGE
NO. NO.
1 Title of dissertation 1
2 Certificate 2
3 Acknowledgment 3
4 Self-Declaration 4
5 Table of content 5
6 Abstract 6
7 Chapter 1: Introduction 7-11
8 Chapter 2: Literature review 12-13
9 Chapter 3: Materials and Methodology 14-21
10 Chapter 4: Result and Discussion 21-24
11 Conclusion 25
12 References 26-27

Table of Figures

1 Electronic Weighing balance 15

2 pH meter 16
3 Centrifuge 17
4 Homogeniser 17
5 Vortex mixer 18
6 Gas chromatography 19
7 mass spectrometer 21

Table of Graphs

1 Graph of wheat flour sample 24

2 Graph of pulse flour sample 25

[5]
 Analysis of perilous elements in wheat flour and pulse using
Gas chromatography-mass spectroscopy

Abstract
With the advent of modern civilization, a large number of chemicals are introduced into
the primary components of our environment such as air water, and soil. Long-term
accumulation of such chemicals ultimately dissipates to groundwater and even contaminates
the crop and vegetables. Such contamination may yield some health problems by entering our
food chain. Among the different hazardous chemicals pesticides in general play some crucial
role as they have now become an integral part of the agricultural sector in the context of control
of pests and maintaining the food security of large human populations. Pesticides, in contrast
to other contaminants, are added consciously and directly to agricultural fields, crops, and
vegetables. Depending on the nature of the pesticides applied together with their load and
residence time the risk of hazard or the damage manifested. The unregulated use, the toxicity
of parent and degraded products, long-term residence to the environment, damage to non-target
species, and spread over long distances affecting species or people even not exposed directly
are some conventional problems associated with pesticides. It is a great challenge, therefore,
to monitor the pesticide load both at the point of application and afterward not only in the
agricultural field or products but also in water (surface and ground) and soil, which eventually
ends up in the food chain affecting humans. Further, the determination of pesticide load
becomes crucial due to the lack of systematic protocol, variability in load, and variability of
the matrix. Therefore, it requires precise analytical detection using advanced techniques. This
work analyses the presence of various such pesticides in different food materials using
advanced detection techniques of gas chromatography-mass spectroscopy.
Keywords:
Carcinogens, Gas chromatography-mass Spectroscopy, Perilous substances, Pesticides,
Potentially hazardous substances

[6]
 Chapter 1:

Introduc on
Forensic science is a multidisciplinary field that involves the application of scientific principles
and techniques to investigate and solve crimes. Forensic science refers to the use of scientific
methods and knowledge in the examination of physical evidence related to criminal
investigations. It plays a critical role in the criminal justice system[1][14]
Forensic science, the application of the methods of the natural and physical sciences to
matters of criminal and civil law. Forensic science can be involved not only in
investigation and prosecution of crimes such as rape, murder, and drug trafficking but
also in matters in which a crime has not been committed but in which someone is
charged with a civil wrong (see tort), such as wilful pollution of air or water or causing
industrial injuries.[2]

Almost any science can be a forensic science because almost any science can
contribute to solving a crime or evaluating a civil harm. In fact, with few exceptions,
forensic sciences are no different in what they study than traditional sciences. The only
difference is that forensic scientists apply the methods and techniques of established
sciences to legal matters.Forensic science encompasses various specialized disciplines,
including forensic biology, forensic chemistry, forensic toxicology, forensic anthropology,
forensic odontology (dentistry), forensic pathology, digital forensics, and more. Each discipline
focuses on specific types of evidence. Evidence Analysis: Forensic scientists analyse a wide
range of evidence, including DNA, fingerprints, bloodstains, firearms, drugs, trace evidence,
and more. They use scientific techniques to examine and interpret this evidence, providing
objective and reliable information for criminal investigations. Forensic experts are often
involved in crime scene investigations. They collect, document, and preserve evidence from
crime scenes, ensuring that it is handled properly to maintain its integrity for analysis. Forensic
scientists often testify as expert witnesses in court, presenting their findings and interpretations
to help judges and juries understand the evidence and its significance. The field of forensic
science adheres to strict legal and ethical standards to ensure the validity and reliability of
findings. This is essential for maintaining the integrity of the criminal justice system. Forensic
science continues to evolve with advancements in technology and research. Techniques such
as DNA analysis have revolutionized the field and contributed to solving cold cases and
exonerating wrongfully convicted individuals. Forensic science plays a crucial role in solving
crimes, identifying suspects, and ensuring justice is served. It combines scientific expertise
with legal principles to provide objective and reliable evidence in the criminal justice system.[2]

With the fastest growing economy, the world is facing a range of pollutants, beyond expulsion
of perilous substances that have significant health and environmental impacts. Some of the
perilous substances include Particulate matter, Carbon Monoxide, Ozone, Nitrogen Dioxide,
Sulfur Dioxide, and Persistent Organic Pollutant. Particulate matter refers to tiny particles or
droplets in the air, which can include dust, pollen, soot, and liquid droplets. These particles can
be inhaled into the lungs and have adverse health effects, especially fine particulate matter
(PM2.5) and coarse particulate matter (PM10). Carbon monoxide is a colourless, odourless gas
that can be produced by incomplete combustion of carbon-containing fuels. Exposure to high
levels of CO can be life-threatening as it interferes with the body's ability to transport. Ground-
level ozone is a major component of smog and can lead to respiratory problems and other health
issues. It forms as a result of chemical reactions between nitrogen oxides (NOx) and volatile
[7]
organic compounds (VOCs) in the presence of sunlight. Nitrogen dioxide is a reddish-brown
gas produced by combustion processes. It can irritate the lungs and lower resistance to
respiratory infections. Sulfur dioxide is a colorless gas with a pungent odour, primarily released
from the burning of fossil fuels containing sulfur. It can cause respiratory problems and
contribute to the formation of acid rain. Persistent organic pollutants are toxic chemicals that
adversely affect human health and the environment. They are a global issue due to their long-
lasting presence and potential for bioaccumulation in ecosystems. These pollutants pose
significant health and environmental risks and are the focus of various regulatory efforts to
mitigate their impacts. Governments and organizations need to continue monitoring and
addressing these perilous substances to protect public health and the environment.[3][4]

1.1. Perilous Substances[3][4][5]

Perilous Substances in the context of forensic chemistry refer to chemicals that pose a high risk
to employees working in laboratories and forensic settings. These substances can include
chemicals known to cause cancer or known carcinogens. Exposure to carcinogens can have
long-term health effects, making them particularly perilous in forensic laboratories where
professionals handle potentially harmful substances. Some of them are known to have
reproductive toxins, and handling of such chemicals should be done with care. These
substances can also include substances with a high degree of acute toxicity. These chemicals
can be immediately harmful upon exposure, requiring strict safety protocols when working
with them in forensic investigations. The selection of perilous substances is crucial in forensic
chemistry to ensure the safety of laboratory personnel and to prevent long-term health risks
associated with exposure to perilous chemicals. These substances are handled with specialized
safety measures and protective equipment to minimize risks in forensic laboratories.
Perilous substances are potentially affecting India in various ways, including health risks,
environmental degradation, and occupational hazards. The exposure to perilous chemicals in
India has led to a significant increase in health-related issues. This includes various diseases,
respiratory problems, and long-term health effects among workers and communities living near
industrial areas. These substances contribute to environmental pollution and contamination.
Improper disposal and management of these chemicals can lead to soil and water pollution,
affecting ecosystems and wildlife. India has been grappling with the environmental
consequences of toxic waste and perilous material. Workers in various industries, such as
manufacturing and chemical processing, are at high risk due to occupational exposure to
perilous substances. Occupational safety measures and regulations are crucial to protect the
workforce. Exposure to these substances has contributed to public health concerns, including
a rise in deaths due to perilous chemical exposure. India has witnessed an increase in the
number of fatalities associated with these chemicals, emphasizing the need for stricter
regulations and safety measures. India's import of perilous waste material can have severe
environmental and health impacts if not managed properly. This issue highlights the importance
of regulating and monitoring the import of perilous materials into the country.
In fact, perilous chemicals are present everywhere – in the air, consumer products, water, soil
and at the workplace. They can cause several diseases, including mental, behavioural and
neurological disorders, cataracts and asthma. Labors across India are facing a health crisis due
to occupational exposure to toxic chemicals such as lead, mercury, manganese, chromium,
cadmium, benzene, pesticides and asbestos. Large numbers of Indian workers lose their lives
following exposure to these perilous substances, succumbing to poisoning, cancer, disorders of

[8]
the lung, kidney and liver or, suffering fatal injuries due to fires and explosions involving these
agents. The lives of workers and their families are also severely affected when workers develop
debilitating and long-term diseases and injuries due to exposure to and incidents involving
chemicals and other perilous substances.India’s chemical manufacturing and processing
industry, which manufactures both basic chemicals and chemical-based substances – like
petrochemicals, fertilizers, paints, pesticides, bulk drugs and pharmaceuticals – is one of the
country’s most diverse industrial sectors, responsible for the creation of more than 70,000
commercial products containing or made from chemicals.

1.2. Legal Framework

Although India has over 15 Acts and 19 rules governing different aspects of the chemical
industry, none of the laws have been designed exclusively for the chemical sector. Environment
(Protection) Act, 1986, is currently the umbrella act for the chemical industry, according to the
National Chemical Management Profile for India report, launched in 2005-06 by the Ministry
of Environment, Forest and Climate Change (MoEFCC) and the Central Pollution Control
Board (CPCB). In the three years up to 2020, there were on average over four major chemical
accidents every month at Indian chemical manufacturing plants. “The 152 recorded accidents
[during the three years] are underestimated as most cases go unregistered,” said Gopal Krishna
of Delhi-based non-profit organization ToxicsWatch, which researches and reports on the
health risks from perilous substances in India. .[16]
Legal framework is needed and a national chemical policy for India has been pending since
2012, and scientists say the country now needs a comprehensive legal framework to effectively
regulate the production, use and safety of chemicals. Krishna says India does not have adequate
and appropriate knowledge about the chemicals that are being used in the country. “So, several
chemicals that are banned internationally continue to be used here,” he warns. According to
2021 estimates by the World Health Organization (WHO), deaths due to exposure to perilous
chemicals worldwide rose 29 per cent in 2019 compared to 2016.Two million people lost their
lives due to exposure to perilous chemicals in 2019, compared to 1.56 million in 2016,
according to the Geneva-based global health body. The alarming estimates were released by
Tedros Adhanom Ghebreyesus, WHO director-general, during the Ministerial Dialogue held in
July 2021 at the Berlin Forum on Chemicals and Sustainability: Ambition and Action towards
2030.
A report from the UN Special Rapporteur on Human Rights and Perilous Substances and
Wastes, presented at the 39th session of the Human Rights Council in Geneva in 2018, found
that around the world, workers are dying as a result of toxic exposure in the workplace at a rate
of one every 30 seconds. With 2.78 million deaths per year attributable to unhealthy or unsafe
workplaces, the report warned that women, children, migrant workers, and the poor are
especially vulnerable to work-related fatalities, injuries, and ill health and that the solutions to
solve this global crisis of public occupational health require the participation of business,
governments, and international organisations. Most deaths due to exposure to perilous
chemicals are preventable, according to the WHO. They can be prevented by reducing or
removing chemical exposure.

1.3. Pes cides

One of the major concerns is the use of pesticides in agriculture leading to an unescapable risk
of pesticide poisoning. Scientists estimated that about 385 million people, particularly farmers

[9]
and agriculture workers, are poisoned by pesticides every year around the world. These
incidents result in around 11,000 deaths per year, nearly 60 % of which occur in India,
according to the research. Two of the pesticides blamed for these deaths – monocrotophos [13]
and oxydemetonmethyl [13] – Class I pesticides by the WHO, and exposure to even small
amounts of these pesticides can be lethal. Data from India’s Directorate of Plant Protection,
Quarantine and Storage shows that Class 1 insecticides account for 30% of the insecticides
used in India. Many foreign countries have banned these deadly pesticides. Phosphamidon [13]
is banned in 49 countries, Phorate [13] in 37, Triazophos [13] in 40, and Monocrotophos is
banned in 60 countries.
The Indian government and authorities now seem to have concluded that it is time for India to
follow the lead of other countries and introduce a ban on these pesticides. As a result, the
government is considering banning as many as 27 pesticides and has appointed an expert
committee to look into this issue, Narendra Singh Tomar, Union Minister for Agriculture and
Farmers’ Welfare, told the Rajya Sabha in July 2021.Meanwhile, Ladakh soon intends to ban
the use of chemicals, fertilisers and pesticides in the region, according to a recent
announcement by the UT Secretary Ravinder Kumar. In summary, particularly perilous
substances have the potential to impact India by posing health risks to the population,
contributing to environmental problems, creating occupational hazards, and raising public
health concerns. Effective regulations, waste management, and safety measures are essential to
mitigate these effects. [10][6]

1.4. Forensic science and perilous substances[7][8][9]

Forensic science has extensively researched perilous substances, especially in the context of
toxicology and chemical analysis. A study on "Reference Materials in Clinical and Forensic
Toxicological Analysis" by F. Pragst discusses the use of reference materials in urine for
calibration and control of immunoassays in the detection of illicit and therapeutic drug abuse.
Another study from the University of Central Florida Libraries - "Chemical Safety - CHS 4591
- Forensic Science" provides information on databases and resources related to toxicology and
perilous chemicals, which are crucial in forensic research. John Jay College of Criminal Justice
Library - "Forensic Science: Drugs & Other Chemicals": This guide offers reference books and
resources for toxicology and other forensic topics, which are valuable for research in this field.
John Jay College of Criminal Justice Library - "Forensic Science: Reference Books": This
resource includes reference books for toxicology and special forensic topics, which can be used
as a starting point for research in the area of perilous substances in forensics. "Biological,
Chemical, Forensic, and Analytical Sciences: Drug ...": This source mentions the Perilous
Substances Databank, which provides comprehensive, peer-reviewed toxicology data for
thousands of chemicals. This data is valuable for forensic analysis. "Environmental Forensics
using Stable and Radioactive Isotopes" by N. Kim: This study presents an improved forensic
method that includes stable and radioactive isotope analysis, microbial community analysis,
and other techniques relevant to environmental forensics and perilous substances research.
In particularity to this thesis experimental analytical work has been conducted with the
parameter of gas chromatography -mass spectrometry in wheat flour and pulses from the local
area. The thesis topic bends to forensic analysis with chemistry and toxicological analysis.

[10]
 Chapter 2:
Literature Review

Pesticides are chemical substances or mixtures used to control, repel, or eliminate pests that
can harm crops, humans, livestock, or the environment. Pesticides come in various forms,
including insecticides (for insects) herbicides (for weeds), fungicides (for fungi), and
rodenticides (for rodents). They are widely used in agriculture to protect crops from pests, in
public health to combat disease-carrying vectors, and in households to control pests like insects
and rodents. Pesticides can have a negative impact on the environment. Runoff can contaminate
water bodies, affecting aquatic life. Some pesticides may also harm non-target species.
Pesticides can pose risks to human health, especially for those who handle them without proper
protective measures. Short-term exposure can lead to symptoms like nausea and skin irritation,
while chronic exposure can have more severe health effects.
Pesticides are subject to strict regulation in most countries to ensure their safe use. This
includes setting limits on pesticide residues in food. Integrated Pest Management (IPM)
promotes the use of alternative methods like biological control and crop rotation to reduce
reliance on pesticides. Proper handling, storage, and disposal of pesticides are essential to
minimize risks. This includes using protective gear, following label instructions, and avoiding
overuse.

Ongoing research explores the development of safer and more environmentally friendly
pesticides to reduce their negative impacts.
It's important to balance the benefits of pesticide use in agriculture and public health with their
potential environmental and health risks. Safe and responsible use of pesticides is crucial to
mitigate these risks.
Particularly Perilous Substances refer to chemicals or materials that pose significant risks to
health and safety due to their inherent properties. Carcinogens are substances capable of
causing cancer. Exposure to these materials can lead to the development of malignant tumour
and other cancer-related conditions. Reproductive Toxins: Reproductive toxins are chemicals
that can harm reproductive health, including causing birth defects or negatively impacting
fertility. They are of concern, especially for individuals of reproductive age. Substances with
High Acute Toxicity: These are materials with a high degree of acute toxicity, which means
they can cause severe harm, disability, or even death after a single exposure or a short-term
exposure event. .[10][11]
Particularly Perilous Substances are subject to stringent safety protocols and regulations to
minimize exposure and protect the health and safety of individuals working with or around
these substances. They do not form on their own. Instead, is a classification or designation
given to certain chemicals or materials based on their inherent properties and potential risks to

[11]
 human health and safety. is a regulatory classification rather than a chemical or material
category in itself. The formation of perilous substances involves identifying and categorizing
substances that fall into specific criteria set by regulatory authorities. These criteria typically
include substances that are:
 Select Carcinogens: Chemicals known to be capable of causing cancer.
 Reproductive Toxins: Chemicals that can harm reproductive health, including causing
birth defects or impacting fertility.\
 Substances with High Acute Toxicity: Materials that can cause severe harm, disability,
or even death after a single exposure or a short-term exposure event.
These criteria are defined and regulated by organizations like the Occupational Safety and
Health Administration (OSHA) [12] in the United States. substances are subject to stringent
safety protocols, handling procedures, and regulations to minimize exposure and protect the
health and safety of individuals working with or around them.

In summary, perilous substances is a classification based on the inherent properties and hazards
of certain substances, and they are identified and regulated as such by authorities to ensure safe
handling and use and can have significant adverse effects on both humans and animals. The
specific effects can vary depending on the type of and the level of exposure, but they generally
include the following:
 Human Health Effects: Carcinogenicity: perilous substances that are classified as
carcinogens can increase the risk of cancer development in humans.
 Reproductive Toxicity: Reproductive toxins can harm the reproductive system, leading
to birth defects, infertility, or other reproductive health issues.
 Acute Toxicity: Substances with high acute toxicity can cause severe health effects even
after a short-term exposure, such as organ damage, respiratory distress, or death.
 Irritation: Some perilous substances may cause skin or eye irritation, leading to
discomfort, rashes, or eye problems.
 Systemic Effects: Prolonged exposure to certain perilous substances can result in
systemic health issues, including respiratory problems, headaches, nausea, and other
symptoms.
 Environmental Impact:
 Ecological Hazards: perilous substances can also have detrimental effects on wildlife
and ecosystems. This can include contamination of water bodies, soil, and air, which
can harm aquatic and terrestrial life.
 Bioaccumulation: Some perilous substances can accumulate in the food chain, leading
to higher concentrations in organisms at the top of the food web, including humans who
consume contaminated wildlife or fish.
It's important to note that the severity of these effects depends on factors such as:
 the type of perilous substances,
 the level of exposure
 individual susceptibility
 Proper handling

[12]
 Storage and disposal of perilous substances are essential to minimize risks to both
human health and the environment.

[13]
 Chapter 3
Material and Methodology
SAMPLE COLLECTION:
Samples are collected in nearby grain stores, flour mills and households.
SAMPLE PREPARATION:
Pesticidal analysis follows certain methods and protocols. Here Quencher’s method [15] is
followed for sample preparation. Analysing pesticides in laboratories precise methods to
ensure accuracy and safety.
The QuEChERS method [4], which stands for "Quick, Easy, Cheap, Effective, Rugged, and
Safe," is a widely used sample preparation approach in analytical chemistry, particularly for
the determination of pesticide residues in various matrices, including food and
environmental samples.
Here's an overview:
Quick: QuEChERS is known for its speed. It streamlines the sample preparation process,
allowing for efficient and rapid analysis.
Easy: The method is designed to be user-friendly. It involves straightforward steps that make
it accessible to analysts with varying levels of experience.
Cheap: QuEChERS is cost-effective. It minimizes the need for expensive equipment and
reagents, which is advantageous for labs with budget constraints.
Effective: QuEChERS has proven to be effective in extracting a wide range of pesticides from
complex sample matrices, including fruits, vegetables, and soil.
Rugged: This aspect refers to the method's robustness and ability to handle a variety of sample
types. It can adapt to different sample characteristics and still produce reliable results.
Safe: QuEChERS is designed with safety in mind. It minimizes the use of hazardous chemicals
and reduces the risk to analysts. The technique involves a series of steps, including
extraction, cleanup, and concentration, to prepare samples for analysis using techniques like
gas chromatography (GC) and liquid chromatography (LC).QuEChERS is particularly
effective for samples with high water content, such as fruits and vegetables. It has become
a standard method in many laboratories for pesticide residue analysis due to its efficiency
and practicality.

Highlighted Elemental Compounds [13]

 ACEPHATE
acephate is an organophosphate foliar and soil insecticide of moderate persistence
 CHLORONEB
a particular fungicide

[14]
  DICHLORVOS
a highly volatile organophosphate, 2,2-dichlorovinyl dimethyl phosphate (ddvp),
widely used as a fumigant to control household pests and to protect stored product
from insects.

 DIPHENYLAMINE
(chemistry) an aromatic amine used in the manufacture
of plastics, dyes, explosives, pesticides, fungicides and pharmaceuticals

 ETRIDIAZOLE
a particular fungicide
 FENOBUCARB
(chemistry) a carbamate insecticide used agriculturally on rice and cotton.

 METHAMEDAPHOS
 MOLINATE
 OMETHOATE
 PROPOXUR
 TECNAZENE

SAMPLE ANALYSIS INSTRUMENTATION:

Methods for pesticide analysis include:
 Chromatographic Methods: Gas chromatography (GC) and liquid chromatography (LC) are
widely used. They separate and quantify pesticides based on their chemical properties. GC
is suitable for volatile compounds, while LC is versatile for a broader range.
 Mass Spectrometry (MS): Coupled with chromatography, MS is used for identification and
quantification. It provides highly specific results.
 Spectroscopy: UV-Visible and Infrared spectroscopy can be employed to detect pesticides
based on their absorption of specific wavelengths. Nuclear Magnetic
 Resonance (NMR): NMR spectroscopy helps identify chemical structures, including
pesticide residues.
 Enzyme-Linked Immunosorbent Assay (ELISA): ELISA is an immunological method that
can detect pesticides with high sensitivity and specificity.
 Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS): This is a sample preparation
technique widely used for pesticide residue analysis. It involves extraction and purification
of samples before analysis.
 Accelerated Solvent Extraction (ASE): ASE is another sample preparation method that uses
high-pressure and high-temperature conditions for efficient extraction.
 Atomic Absorption Spectroscopy (AAS): AAS can be used to measure metal-based
pesticides by detecting the absorption of specific wavelengths of light.
 High-Performance Liquid Chromatography (HPLC): HPLC is used for separating and
quantifying pesticides in liquid samples.

[15]
 Tandem Mass Spectrometry (MS/MS): MS/MS offers enhanced selectivity and sensitivity
by using multiple stages of mass spectrometry.
These methods are chosen based on the type of pesticide, sample matrix, required sensitivity,
and regulatory requirements. Laboratories must adhere to rigorous quality control procedures
to ensure the accuracy and reliability of their pesticide analysis results.[17]
In the thesis the instrumentation under went is Gas Chromatography-Mass Spectroscopy along
with certain instruments as vortex, centrifuge, homogenizer etc mentioned below.

Weighing Balance:( Fig. Electronic Weighing Balance)

• A Beam balance (or Beam scale) is a device to measure

weight pr mass. These are also known as mass scales,
weight scales, mass balances, weight balances, or simply
scales, balances or balance.
• The traditional scale consists of two plates or bowls
suspended at equal distances from a fulcrum. One plate
holds an object of unknown mass (or weight), while
known masses are added to the other plates until static
equilibrium is achieved and the plates level off, which
happens when the masses on the two plates are equal. The
perfect scale rest at neutral.
• A spring scale will make use of a spring of known stiffness to determine mass (or weight).
Suspending a certain mass will extend the spring by a certain amount depending on the spring’s
stiffness (or spring constant). The heavier the object, the more the spring stretches, as described
in Hooke’s law.
• Other types of scales making use of different physical principles also exist. Some scales can
be calibrated to read in units of forces (weight) such as newtons instead of units of mass such
as kilograms. Scales and balances are widely used in commerce, as many products are sold and
packaged by mass.
An analytical balance is a class of balance designed to measure small mass in the sub-
milligrams range. The measuring pan of an analytical balance (0.1mg or better) is inside a
transparent enclosure with doors so that dust does not collect and so any air currents in the
room do not affect the balance’s operation. This enclosure is often called a draft shield. The use
of a mechanically vented balance safely enclosure, which has uniquely designed Acryalic air
foils, allows a smooth turbulence- free airflow that prevents balance fluctuation and the
measure of mass down to 1µg without fluctuations or loss of product. Also, the sample must
be at room temperature to prevent natural convection from forming air current inside the
enclosure from forming air currents inside the enclosure from causing an error in reading.
Single pan mechanical substitution balance maintains consistent response throughout the useful

[16]
capacity is achieved by maintaining a constant load on the balance beam, thus the fulcrum, by
subtracting mass on the same side of the beam to which the sample is added. Electronic
analytical scales measure the force needed to counter the mass being measured rather than
using actual masses. As such they must have calibration adjustments made to compensate for
gravitational differences. They use an electromagnet to generate a force to counter the sample
being measured and out puts the result by measuring the force needed to achieve balance. Such
measurement device is called electromagnetic force restoration sensor. [16][17]

pH metre (potential of hydrogen) (figure of pH metre)

A pH meter is a scientific instrument that measure the hydrogen
ion activity in water-based solution indicating its acidity or
basicity expressed as ph. The pH meter measures the difference
electrical potential between a pH electrode and a reference
electrode, and so the pH meter is sometimes referred to as a
“potentiometric pH meter”. The difference in electrical potential
relates to the acidity or Ph of the solution. The Ph meter is used
in many applications ranging from laboratory experimentation to
quality control. [16][17]

CENTRIFUGE
Centrifugation is a term used to describe a method of separating mixtures using spinning and
centrifugal force. Several characteristics can separate particles during centrifugation, including
size, shape, density, and viscosity. A centrifuge is a lab instrument for the density-based
separation of fluids, gas, or liquid. (figure of centrifuge)

PRINCIPLE OF CENTRIFUGE
The centrifuge utilizes the sedimentation principle due to gravitational force. The
centrifugation technique uses a centrifugal field to separate particles suspended in a liquid
medium. These are put in the centrifuge’s rotor either in bottles or tubes. Sedimentation is a
process whereby gravity causes suspended particles to separate from fluids. The suspended
substance may consist of powder or clay-like [16][17]

[17]
HOMOGENIZER
A sample is divided into identical pieces using homogenization, which preserves the molecular
composition of the other portions of the sample even when one part of it is removed. It is also
frequently used to mix naturally immiscible materials fully. The purpose of homogenization is
to reduce particle size, breach the cell wall and/or cell membrane, destruction of pathogens,
and facilitate stable emulsions and dispersions. (figure of homogeniser)
PRINCIPLE: Shearing, cavitation, and turbulence- three fundamental physical principles—

work together to produce homogenizing action.

 Shearing: The primary cause of shearing in fluids is friction between fluid molecules
brought on by viscosity. Large particles and droplets are reduced in size by the shear
forces. A big particle or droplet experiences shearing when it becomes trapped between
fluid layers moving at various speeds. Cavitation:
 Cavitation happens when a fluid has a considerable pressure reduction. A pump that
introduces the fluid under greater pressure typically has a homogenizer valve upstream.
This makes it possible for cavities to form from tiny vapor pockets briefly. Shockwaves
are generated when these cavities collapse or implode, breaking the particles and
droplets in the mixture.
 Turbulence: When the fluid reaches a high velocity, it becomes turbulent. The fluid
moves erratically as a result of the high velocity. These unsteady movements are a type
of energy dissipation in which the fluid’s kinetic energy is transformed into internal
energy in the form of eddy currents and a small quantity of heat. The particles are
reduced in size by the eddies created. [16][17]

[18]
VORTEX
A vortex mixer is a simple laboratory device or equipment used for blending laboratory samples
in test tubes, well plates, or flasks. This apparatus is compact, quiet, and equipped with several
inserts that enable the agitation and mixing of samples of various sizes. Rapidly oscillating
motorized drive shafts under the sample platform transmit orbital motion to sample containers
positioned inside the mixer. This results in the turbulent flow- also referred to as a vortex of
the sample fluids. This is the kinetic force that is comparable to stirring but does not involve a
component that protrudes into the sample container. Microplates may also be affixed. (Figure
of vortex mixture) [16][17]

GAS CHROMATOGRAPHY- MASS SPECTROMETER

Gas chromatography differs from other forms of chromatography in that the mobile phase is a
gas and the components are separated as vapours It is thus used to separate and detect small
molecular weight compounds in the gas phase. The sample is either a gas or a liquid that is
vaporized in the injection port. The mobile phase for gas chromatography is a carrier gas,
typically helium because of its low molecular weight and being chemically inert. The pressure
is applied and the mobile phase moves the analyte through the column. The separation is
accomplished using a column coated with a stationary phase.
PRINCIPLE
The equilibrium for gas
chromatography is partitioning,
and the components of the
sample will partition (distribute)
between the two phases: the
stationary phase and the mobile
phase. Compounds that have a
greater affinity for the stationary
phase spend more time in the
column and thus elute later and
have a longer retention time (Rt) than samples that have a higher affinity for the mobile phase.
Affinity for the stationary phase is driven mainly by intermolecular interactions and the polarity
of the stationary phase can be chosen to maximize interactions and thus the separation.

[19]
 Ideal peaks are Gaussian distributions and symmetrical, because of the random nature of the
analyte interactions with the column.
 The separation is hence accomplished by partitioning the sample between the gas and a thin
layer of a non-volatile liquid held on a solid support.
 A sample containing the solutes is injected into a heated block where it is immediately
vaporized and swept as a plug of vapor by the carrier gas stream into the column inlet.
 The solutes are adsorbed by the stationary phase and then desorbed by a fresh carrier gas.
 The process is repeated in each plate as the sample is moved toward the outlet.
 Each solute will travel at its own rate through the column.
 Their bands will separate into distinct zones depending on the partition coefficients, and band
spreading.
 The solutes are eluted one after another in the increasing order of their kid, and enter into a
detector attached to the exit end of the column.
• Here they register a series of signals resulting from concentration changes and rates of elution
on the recorder as a plot of time versus the composition of carrier gas stream.
• The appearance time, height, width, and area of these peaks can be measured to yield
quantitative data.
Gas chromatography is mainly composed of the following parts:
1. Carrier gas in a high-pressure cylinder with attendant pressure regulators and flow
meters. Helium, N2, H, Argon is used as carrier gases.
2. Helium is preferred for thermal conductivity detectors because of its high thermal
conductivity relative to that of most organic vapours. N2 is preferable when a large
consumption of carrier gas is employed. Carrier gas from the tank passes through a
toggle valve, a flow meter, (1-1000 ml/min), capillary restrictors, and a pressure gauge
(1-4 ATM).
3. Flow rate is adjusted by means of a needle valve mounted on the base of the flow meter
and controlled by capillary restrictors.
4. The operating efficiency of the gas chromatograph is directly dependant on the
maintenance of constant gas flow.
5. Sample injection system. Liquid samples are injected by a micro syringe with a needle
inserted through a self-scaling, silicon-rubber septum into a heated metal block by a
resistance heater. Gaseous samples are injected by a gas-tight syringe or through a by-
pass loop and valves. Typical sample volumes range from 0.1 to 0.2 ml.
6. The separation column. The heart of the gas chromatography is the column which is
made of metals bent in U shape or coiled into an open spiral or a flat pancake shape.
Copper is useful up to 250 Celsius.
7. Liquid phases have an infinite variety of liquid phases are available limited only by
their volatility, thermal stability and ability to wet the support. No single phase will
serve for all separation problems at all temperatures.
8. Non-Polar – Paraffin, silicone greases, silicone gum rubber. These materials separate
the components in order of their boiling points.

[20]
9. Intermediate Polarity – These materials contain a polar or polarizable group on a long
non-polar skeleton which can dissolve both polar and non-polar solutes. For example.
diethyl hexyl phthalate is used for the separation of high boiling alcohols.
10. Polar – Carbowaxes – Liquid phases with a large proportion of polar groups.
Separation of polar and non-polar substances.
11. Hydrogen bonding – Polar liquid phases with high hydrogen bonding ex. Glycol.
12. Specific purpose phases – Relying on a chemical reaction with solute to achieve
separations. Ex. AgNO3 in glycol separates unsaturated hydrocarbons.
13. The structure and surface characteristics of the support materials are important
parameters, which determine the efficiency of the support and the degree of separation
respectively. The support should be inert but capable of immobilizing a large volume
of liquid phase as a thin film over its surface.
14. Detectors sense the arrival of the separated components and provide a signal. These are
either concentration-dependent or mass dependant. • The detector should be close to the
column exit and the correct temperature to prevent decomposition.
15. The recorder should be generally 10 mv (full scale) fitted with a fast response pen (1
sec or less). The recorder should be connected with a series of good quality resistances
connected across the input to attenuate the large signals. An integrator may be a good
addition.

APPLICATION
GC analysis is used to calculate the content of a chemical product, for example in assuring
the quality of products in the chemical industry; or measuring toxic substances in soil, air
or water. • Gas chromatography is used in the analysis of: (a) air-borne pollutants (b)
performance-enhancing drugs in athlete’s urine samples (c) oil spills (d) essential oils in
perfume preparation. [16][17]

MASS SPECTROMETER
(Figure of mass spectrometer)

Gas chromatography-mass
spectrometry provides
universal and specific
information on individual
components in the debris
sample, although many GC
peaks remains as
unresolved mixture of two,
and often more, very
similar hydrocarbon components in nearly all petroleumbased accelerants. Today mass
spectrometry is perhaps the foremost tool in the identification of unknown compounds
(James and Martin 1952). It provides information on the identity of every component in the
sample by taking advantage of the common fragmentation pathway for the individual
substance classes, which are truly unique of a particular chemical substance, much like

[21]
fingerprint. In GC/MS, the mass spectrometer scans the masses continuously throughout
the separation.
HOW A MASS SPECTROMETER WORKS?
Step 1: The sample is vaporized and then ionized by a beam bombarded by a high beam of
high energy electrons (usually at 70 eV). The electron knocks out an electron from the
molecule of the injected sample, creating a molecular ion (which is also a radical cation
because it has an unpaired electron and a positive charge). Losing an electron weakens the
bond, while the collision gives it extra kinetic energy. These factors make it more likely for
the molecular ion to break into fragments as it travels through the MS.
Step 2: There is a pair of oppositely charged plates in the ionization chamber. The positively
charged one causes the positively charged radical cation to accelerate into an analyser tube.
Step 3: The analyser tube is surrounded by a curved magnetic field, which causes the path
of the radical cation to be deflected in proportion to its mass-to-charge ratio (m/z). The
flight path of the ion depends on its molecular mass, its charge, and the strength of the
magnetic field. Thus, at a given magnetic field strength, ions of only one specific mass
collide with the detector and are recorded.
Step 4: The strength of the magnetic field is varied in increments to produce a mass
spectrum, which is a plot of m/z (on the x axis) against relative abundance (on the y axis).
If we assume that all ions have a charge of +1, then the peaks give the mass ratios and their
heights give the proportions of ions of different masses.
TANDEM MASS SPECTROMETRY
Tandem mass spectrometry, also known as MS/MS, involves multiple steps of mass
spectrometry selection, with some form of fragmentation occurring in between the stages.
PRECURSOR ION SCAN
The product ion is selected in the second mass analyser, and the precursor masses are
scanned in the first mass analyser. A precursor ion scan cannot be done with time-based
MS instruments. Note that precursor ion is synonymous with parent ion and product ion
with daughter ion, however the use of these anthropomorphic terms is discouraged.

PRODUCTION SCAN
A precursor ion is selected in the first stage, allowed to fragment and then all resultant
masses are scanned in the second mass analyser and detected in the detector that is
positioned after the second mass analyser. The experiment is commonly performed to
identify transitions used for quantification by tandem MS.
NEUTRAL LOSS SCAN
The first mass analyser scans all the masses. The second mass analyser also scans, but at a
set offset from the first mass analyser. This offset corresponds to a neutral loss that is
commonly observed for the class of compounds. Neutral loss scans cannot be done with
time-based MS instruments. In a constant-neutral-loss scan, all precursors that undergo the

[22]
loss of a specified common neutral are monitored. To obtain this information, both mass
analysers are scanned simultaneously, but with a mass offset that correlates with the mass
of the specified neutral. Similar to the precursor-ion- scan, this technique is also useful in
the selective identification of closely related class of compounds in a mixture.
SELECTED REACTION MONITORING
Both mass analysers are set to a selected mass. This mode is analogous to selected ion
monitoring for MS experiments. A very selective analysis mode, which can increase
sensitivity.
FRAGMENTATION TANDEM MASS SPECTROMETRY
Fragmentation of gas-phase ions is essential to tandem mass spectrometry and occurs
between different stages of mass analysis. There are many methods used to fragment the
ions and these can result in different types of fragmentation and thus different information
about the structure and composition of the molecule.
ION SOURCE FRAGMENTATION
If the product ions persist in their non-equilibrium state for a moderate amount of time
before auto-dissociation this process is called metastable fragmentation. Although in-
source fragmentation allows for fragmentation analysis, it is not technically tandem mass
spectrometry unless metastable ions are analysed or selected before auto-dissociation and
a second stage of analysis is performed on the resulting fragments. In-source fragmentation
is often used in addition to tandem mass spectrometry (with post-source fragmentation) to
allow for two steps of fragmentation in a pseudo-MS/MS/MS type of experiment.
POST SOURCE FRAGMENTATION
Most often what is being used in a tandem mass spectrometry experiment. Energy can also
be added to the ions, which are usually already vibrationally excited, through post-source
collisions with neutral atoms or molecules, the absorption of radiation, or the transfer or
capture of an electron by a multiply charged ion. Collision-induced dissociation (CID) also
called collision ally activated dissociation (CAD), involves the collision of an ion with a
neutral atom or molecule in the gas phase and subsequent dissociation of the ion.
For example,
AB+ + M―› A + B+ + M
Where the ion AB+ collides with the neutral species M and subsequently breaks apart.
The details of this process are described by collision theory. [16][17]

[23]
 Chapter 4
RESULT AND DISCUSSION

DATA OBTAINED IN GC-MS FOR WHEAT FLOUR

PEST NAME RT STD NS A1 A1-NS Conc. A2

ACEPHATE 9.13 131625 63431 1192467 1129036 17.15534283 584206
CHLORONEB 10.44 26757127 116525251 188197548 71672297 5.35724908 192991285
DICHLORVOS 6.73 19850181 80408 3054382 2973974 0.299642003 3939362
DIPHENYLAMINE 12.83 74561022 105228 7400627 7295399 0.195689351 4317085
ETRIDIAZOLE 9.41 7368360 46816 13250803 13203987 3.583969024 2074429
FENOBUCARB 12.35 86056171 153852 36828540 36674688 0.852343012 26072435
ISOPROCARB 10.93 7722496 7028 3492906 3485878 0.902785317 2587845
METHACRIFOS 10.22 16556834 32370 6572877 6540507 0.790067352 7273949
METHAMEDAPHOS 6.8 1175375 34577 2082176 2047599 3.484162927 1360628
MOLINATE 11.1 68535799 404452 21885869 21481417 0.626867048 21639673
OMETHOATE 11.98 33951 3907 1568960 1565053 92.19481017 620160
PROPOXUR 12.4 28267097 57728 16769143 16711415 1.182393438 7717312
TECNAZENE 12.06 10371817 19313 3315352 3296039 0.635576004 2975221

WHEAT FLOUR SAMPLE

250000000

200000000
PEAK AREA

150000000

100000000

50000000

PESTICIDAL ELEMENTS
RT: Retention time
STD: Standard (as per government body norms) NS: non-spike
A1:sample 1 of wheat flour A2:sample 2 of wheat flour

[24]
 DATA OBTAINED IN GC-MS FOR PULSE FLOUR
PEST NAME RT STD NS B1 B1-NS Conc. B2
ACEPHATE 9.13 131625 63431 375087 311656 1.6617798 214536
CHLORONEB 10.44 26757127 116525251 120882121 4356870 0.07208461 22214916
DICHLORVOS 6.73 19850181 80408 1487395 1406987 1.89188077 567938
DIPHENYLAMINE 12.83 74561022 105228 3993483 3888255 1.94730014 6051958
ETRIDIAZOLE 9.41 7368360 46816 767487 720671 1.87800184 191400
FENOBUCARB 12.35 86056171 153852 21311311 21157459 1.98556147 17878151
ISOPROCARB 10.93 7722496 7028 2062679 2055651 1.99318556 1675813
METHACRIFOS 10.22 16556834 32370 3675816 3643446 1.98238758 2570677
METHAMEDAPHOS 6.8 1175375 34577 818210 783633 1.91548136 447433
MOLINATE 11.1 68535799 404452 13777963 13373511 1.94129002 11908558
OMETHOATE 11.98 33951 3907 436034 432127 1.98207938 183498
PROPOXUR 12.4 28267097 57728 6846239 6788511 1.98313585 5362125
TECNAZENE 12.06 10371817 19313 2002934 1983621 1.98071529 1900545

PULSE FLOUR SAMPLE

140000000

120000000

100000000
PEAK AREA

80000000

60000000

40000000

20000000

PESTICIDAL ELEMENTS

RT: Retention time

STD: Standard (as per government body norms)
NS: non-spike
B1:sample 1 of PULSE flour B2:sample 2 of PULSE flour
**Peak Area Is Area of Given Elements Highest Peak also determining its quantitative trace.

[25]
 Challenges in the field include backlogs of evidence processing, quality control, and
the need for ongoing research to improve techniques and ensure accuracy.
 Compound to be analysed should be stable under GC operation conditions.
 They should have a vapor pressure significantly greater than zero.
 Typically, the compounds analysed are less than 1,000 Da, because it is difficult to
vaporize larger compounds.
 The samples are also required to be salt-free; they should not contain ions.
 Very minute amounts of a substance can be measured, but it is often required that the
sample must be measured in comparison to a sample containing the pure, suspected
substance known as a reference standard.
 The entire experimental analysis enhances over presence of perilous substances in form
of pesticidal elements in food items.
 A proper sample should have the criterion that the analyte must be converted to suitable
form for analysis and the technology must provide precise analyte signal that will not
be influenced by sample matrix. Extraction of analyte / solute from sample matrix may
fulfil such criteria for solute determination that enables preconcentration and removal
via recovery of solute from even complex sample matrices.

[26]
 CONCLUSION
Perilous substances are not typically associated with food in the same way they are in a
laboratory or industrial setting. The presence of perilous substances in food is not a common
concern, as food safety regulations are in place to ensure that food products are safe for
consumption. The focus of food safety is on preventing contaminants, pathogens, and other
hazards rather than perilous substances. In the context of food safety, substances that are of
concern include biological pathogens (bacteria, viruses, etc.), chemical contaminants
(pesticides, heavy metals, food additives, etc.), and physical contaminants (such as foreign
objects). The regulations and monitoring systems for food safety are in place to prevent and
control these types of hazards.
So, while perilous substances are a critical consideration in certain workplace settings, they are
not a common concern in the food industry due to strict regulations and quality control
measures designed to ensure the safety of food products [14]. This thesis studies the average
quantity of perilous substances in food items like flour and pulse when concerns Particularly
Perilous Substances.
The future aspect can be wide as the samples are in mass. Here is wheat flour of confined are
but samples can be of packed materials or of different regional and geographical locations,
which would defiantly prove a wider scope of research to this field.

[27]
 REFERENCES

1. https://www.unep.org/
2. Siegel, Jay A. "Forensic science". Encyclopedia
Britannica, 3 Oct. 2023,
https://www.britannica.com/science/forensic-science.
Accessed 26 October 2023.
3. https://www.who.int/news-room/photo-story/photo-story-
detail/10-chemicals-of-public-health-concern
4. https://www.who.int/teams/environment-climate-change-
and-health/air-quality-and-health/health-impacts/types-of-
pollutants
5. https://doi.org/10.1016/j.trac.2015.04.012 Evolu on and
applica on of QuEChERS METHOD
6. https://www.corpseed.com/knowledge-centre/import-of-
hazardous-waste-material-to-india
7. https://link.springer.com/chapter/10.1007/978-3-642-
56986-9_6
8. https://guides.lib.jjay.cuny.edu/forensicscience
9. https://hud.libguides.com/c.php?g=89520&p=577471
10. https://www.wisconsin.edu/ehs/hazmat/phs/
11. https://www.vumc.org/safety/chem/managing-particularly-
hazardous-substances
12. https://www.osha.gov/
13. http://npic.orst.edu/
14. https://nij.ojp.gov/
15. https://www.ohchr.org/en/hr-bodies/hrc/regular-
sessions/session39/regular-session
16. https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Sup
plemental_Modules_(Analytical_Chemistry)/Instrumentation_and_Analy
sis/Chromatography/Gas_Chromatography
17. https://microbenotes.com/

[28]
[29]