Assignment_Chromatography-Techniques.pdf

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Assignment
Chromatography Techniques
Course Outline: CHE 208 (Chemistry for Biologists – II)
SUMMER 2025
Section-03
Group-01
SUBMITTED TO
Tanbir Ahammed (TAN)
Lecturer
Department of Genetic Engineering & Biotechnology
East West University (EWU)
SUBMITTED BY
SL. No. Name of Students ID No Topic
1 Tiaba Akhter Fardin 2023-2-77-071 Introduction
2 Md Safiul Kamal 2024-1-77-009 Type of Chromatography &
Classification Based on the
Physical State of the Mobile
Phase
3 Md Al Imran Ratul 2023-2-77-067 Stationary Phase & Separation
method of Chromatography
4 Meherin Jabin Soha 2024-1-77-009 Applications of
Chromatography in Real Life
5 Bitta Sarkar 2024-1-77-050 The Essential Guide to
Chromatography: it’s
advantages and disadvantages
& Conclusion

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Contents
SL. No. Name of Contents Page No.
01. Introduction to Chromatography 03
02. Basic Principles of Chromatography 04
03. Types of Chromatography 05-16
04. Applications of Chromatography in Real Life 16-23
05. The Essential Guide to Chromatography: it’s advantages and
disadvantages 24-27
06. Conclusion 27
07. References 28

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Introduction to Chromatography
Historical Background and Discovery In 1903, a Russian scientist named Tsvet explored the
study of pigments from the plants and how they interacted with a new compound -- a glass
column filled with calcium carbonate and then analyzed the results from the plant pigments to
construct a new, pioneering method. His method showcased the pigments beautifully separated
and exhibited in stunning, distinct bands, so to him, the results were nothing short of
magnificent. His key discoveries catalogued him as the pioneering scientist of Tsvet
Chromatography a decade later. Since, he has elaborately detailed the simplistic model method.
As time passed, the former model evolved and developed striking new theories including but not
limited to gas and liquid chromatography and thin layer chromatograph.
Definition and Principle of Chromatography
In the most basic terms, chromatography is a technique of breaking down and analyzing the
individual constituents of a compound. The technique operates on the following simple concept:
certain components in the mixture will adhere more strongly to one component of the system
(the stationary phase) while being more readily carried along by a liquid or vapor (the mobile
phase). As a consequence of these interactions, in the course of motion, each component of the
mixture moves along with the system at different velocities, which leads to the separation of the
components from one another during the course of the journey through the system.
Importance in Chemical and Biological Sciences
For a wide range of scientific disciplines, chromatography is a crucial component of the
discipline. It is used in the chemical discipline to analyze the purity of compounds and to isolate
chemical products from a chemical reaction. In the life sciences, it is indispensable in the study
of complicated biological molecules, including proteins, amino acids, and DNA. In the medicine
sector, chromatography is used extensively to determine the purity of pharmaceutical products
and to identify any unwanted foreign contaminants. This technique is also used in the
environmental sector to study water and air pollutants and in the food industry to analyze the
presence of flavors, dyes, and preservatives.

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Basic Principles of Chromatography
Stationary Phase and Mobile Phase
The method is carried out in these two fundamental components. The stationary phase is the
phase that does not move. This can be a solid, such as a sheet of paper, a layer of a silica gel, or
liquid held on a solid support within a column. The mobile phase is a liquid or gas that ascends
through the system, along with the sample. The complete separation process occurs due to the
differing degrees of interaction of the sample's components within the two phases.
Adsorption vs. Partition Chromatography
The manner in which separation occurs can be outlined in a few key types. In adsorption
chromatography, the components of the mixture separate when they adsorb to the surface of a
solid, such as silica. The more strongly a compound adsorbs, the slower it moves. In contrast,
partition chromatography works differently. In this case, separation is enhanced by the differing
degrees of solubility of a compound with two liquid phases. A compound that is more soluble in
the mobile phase travels with greater speed, thus, separating from the other compounds that tend
to reside within the stationary phase.
The Concept of Retention Factor (Rf Value)
In methods such as paper chromatography or thin layer chromatography (TLC), the distance a
compound travels with respect to the mobile phase is measured. This distance is termed as the
retention factor or the Rf value.
The formula from which it has been derived is-
Rf=distance travelled by the solvent front/distance travelled by the compound.
While calculating Rf value the denominator is set to a constant value of ‘1’. Hence the Rf value
tends to lie between 0 and 1 .If the compound has a higher Rf value, it was carried farther by the
mobile (moving) phase, while, if the compound has a lower Rf value, then it was more strongly
held by the stationary (non-moving) phase.

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Types of Chromatography
Chromatography is a method for separating and analyzing that serves to isolate single
components from a complex mixture.
The core tenet is the differential distribution of substances across two phases:
Stationary phase: The fixed phase, which can either be a solid or a liquid that is supported on a
solid.
Mobile phase: The phase in motion that transports the mixture through or across the stationary
phase; it can consist of a gas or a liquid.
As the mixture is introduced, each component interacts with the stationary phase in varying ways
(via adsorption, partition, ion exchange, or size exclusion), causing it to travel at a different rate.
This results in distinct zones or peaks, enabling both qualitative identification and quantitative
measurement.
Key Advantages of Chromatography
• High sensitivity and resolution
• Applicable to small or large molecules
• Works with volatile or non-volatile substances
• Suitable for preparative (purification) and analytical purposes
Common Applications
• Pharmaceutical quality control
• Clinical diagnostics (e.g., drug testing, metabolic profiling)
• Environmental analysis (pollutants, pesticides)
• Food and beverage industry (flavors, preservatives)
• Biochemical research (proteins, nucleic acids)

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Classification Based on the Physical State of the Mobile Phase
The most fundamental classification of chromatography is according to the physical state of the
mobile phase:
A. Gas Chromatography (GC)
• Mobile phase: A non-reactive carrier gas like helium, nitrogen, or hydrogen.
• Stationary phase:
• Gas–liquid chromatography (GLC): The stationary phase consists of a high-boiling
liquid that is applied to a solid support.
• Gas–solid chromatography (GSC): This method employs a solid adsorbent, like
activated charcoal or silica.
• Principle: The main criterion for separating components is their boiling point and how
they interact with the stationary phase.
• Operation Highlights:
• The sample must possess volatility and thermal stability.
• The inert gas vaporizes the mixture and transports it through a heated column.
• Detectors (such as flame ionization and thermal conductivity) measure the elution time
and quantity.
• Applications:
▪ Analysis of essential oils, perfumes, and flavors
▪ Environmental testing of air pollutants
▪ Quality control of petroleum products
▪ Detection of drugs and alcohol in forensic science

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B. Liquid Chromatography (LC)
• Mobile phase: Liquid solvent or a mixture of solvents (water, methanol, acetonitrile,
etc.).
• Stationary phase: Usually a solid adsorbent (silica, alumina) or a liquid immobilized on
a solid.
• Principle: Separation depends on polarity, adsorption, partitioning, ion exchange, or
molecular size.
• Major Techniques:
1. Column Chromatography: A gravity- or pressure-driven column packed with stationary
phase.
2. High-Performance Liquid Chromatography (HPLC): Uses high pressure to force
solvents through tightly packed columns for rapid, high-resolution separations.
3. Paper Chromatography: Stationary phase is water trapped in cellulose fibers of filter
paper; mobile phase moves by capillary action.
4. Thin-Layer Chromatography (TLC): Stationary phase is a thin layer of silica or
alumina coated on glass or plastic plates.
5. Ion-Exchange and Size-Exclusion Chromatography: Specialized methods for proteins,
nucleic acids, or charged molecules.
• Applications:
• Purification of natural products, drugs, or plant extracts
• Separation of amino acids, peptides, and proteins
• Analysis of dyes, pigments, and vitamins
• Quality control in pharmaceuticals and food industry.
Feature Gas Chromatography (GC) Liquid Chromatography (LC)
Mobile Phase Inert gas (He, N₂, H₂) Liquid solvent or solvent mixture
Stationary Phase
Liquid film on solid (GLC) or solid
adsorbent (GSC)
Solid adsorbent or liquid on solid
support
Sample
Requirement
Must be volatile and heat stable
Can handle non-volatile, thermally
labile compounds
Typical Operating
Temp
High (oven-heated column) Ambient or slightly elevated

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Stationary Phase & Separation method of Chromatography
The “stationary phase” is one of the essential phases in chromatography. It is a solid substance or
a liquid coated onto a solid support that remains fixed in place within the chromatographic system.
The “separation method” is the underlying principle by which different components of a mixture
are separated in chromatography.
Feature Gas Chromatography (GC) Liquid Chromatography (LC)
Common
Applications
Petrochemicals, fragrances, forensic
testing
Pharmaceuticals, biochemistry, food
analysis
Separation
Method
Adsorption
Chromatography
Partition
Chromatography
Size-Exclusion
Chromatography
Ion Exchange
Chromatography
Affinity
Chromatography
Stationary Phase
Solid
Chromatography
Liquid
Chromatography
Gas
Chromatography

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1. Solid Chromatography:
In gas-solid chromatography (GSC), the stationary phase consists of a column packed with finely
divided solid materials such as silica, alumina, or activated carbon. Separation occurs through
adsorption, where analytes adhere to the solid surface; stronger adsorption leads to longer retention
times. Solid stationary phases are commonly used to separate gases like oxygen, nitrogen, and
carbon dioxide, as well as small volatile hydrocarbons. However, strong interactions between
analytes and the solid surface can sometimes cause peak tailing.
2. Liquid Chromatography:
In gas-liquid chromatography (GLC), the stationary phase consists of a thin liquid film coated onto
an inert solid support or the inner surface of capillary columns. Separation occurs through a
partitioning mechanism, where analytes dissolve into the liquid phase based on their solubility;
compounds with higher solubility exhibit longer retention times. Liquid stationary phases can be
either polar or non-polar, depending on the properties of the liquid used. GLC is commonly
employed to separate organic compounds such as alcohols, amines, acids, and hydrocarbons. [1]
3. Gas Chromatography:
Gas chromatography (GC) relies on stationary phases to separate and analyze components in a
sample. There are two main types of stationary phases: liquid and solid. Liquid stationary phases
are non-volatile, thermally stable, and chemically inert liquids coated onto the column. Solid
stationary phases are particles of adsorbent material, such as silica gel or molecular sieves. For an
analyte to remain on the column for a sufficient amount of time, it must exhibit some level
of solubility with the stationary phase. Capillary columns, or open tubular columns, are commonly
used for their efficiency in gas chromatography. These columns come in three types.
I. Wall-coated open tubular (WCOT) - columns have a liquid stationary phase coated on
the inner wall of the column. These columns have a high sample capacity and provide high
resolution, making them ideal for separating complex mixtures.
II. Support-coated open tubular (SCOT) - columns have a layer of solid support coated
with the liquid stationary phase, which is then bonded to the inner wall of the column. This
design provides increased stability and efficiency compared to WCOT columns.

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III. Porous-layer open tubular (PLOT) - columns are used in gas-solid chromatography and
have solid stationary phase particles coated onto the inner wall of the column. These
columns have high surface area and retain analytes via adsorption, resulting in large
distribution coefficients and efficiencies. [2]
Figure : Different types of capillary columns (https://www.researchgate.net/figure/Different-
types-of-capillary-columns-Modified-from-De-Lloyd-51_fig5_346926603)
Adsorption Chromatography
Adsorption Chromatography involves the analytical separation of a chemical mixture based on the
interaction of the adsorbate with the adsorbent. The mixture of gas or liquid gets separated when
it passes over the adsorbent bed that adsorbs different compounds at different rates. Adsorbent –
A substance which is generally porous in nature with a high surface area to adsorb substances on
its surface by intermolecular forces is called adsorbent. Some commonly used adsorbents are

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Silica gel H, silica gel G, silica gel N, silica gel S, hydrated gel silica, cellulose microcrystalline,
alumina, modified silica gel, etc.
Figure: Adsorption Chromatography (https://byjus.com/chemistry/adsorption-chromatography/)
Types of Adsorption Chromatography:
1) Thin Layer Chromatography: It is a chromatography technique where the mobile phase
moves over an adsorbent. The adsorbent is a thin layer which is applied to a solid support
for the separation of components. The separation takes place through differential migration
which occurs when the solvent moves along the powder spread on the glass plates.
2) Paper chromatography: It is a technique that uses paper sheets or strips as the adsorbent
being the stationary phase through which a solution is made to pass is called paper
chromatography. The solid surface of the paper is the stationary phase and the liquid phase
is the mobile phase.
3) Column chromatography: the technique in which the solutes of a solution are entitled to
travel down a column where the individual components are adsorbed by the stationary
phase. Based on the affinity towards adsorbent the components take positions on the
column. The most strongly adsorbed component is seen at the top of the column.
4) Gas-Solid chromatography: The principle of separation in GSC is adsorption. It is used
for solutes which have less solubility in the stationary phase. This type of chromatography
technique has a very limited number of stationary phases available and therefore GSC is
not used widely. [3]
Partition Chromatography
Partition chromatography definition states that it is a technique mainly used for the separation of
the components present in the mixture into two liquid phases that are the original solvent and the
solvent coating utilized in the column. The stationary phase immobilizes the liquid surface which
ultimately changes into a stationary phase. The components are separated just after the mobile

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phase shifts from the stationary phase. The separation is because of the differences in partition
coefficients.
Figure : Partition Chromatography (https://byjus.com/chemistry/differential-extraction-
chromatography/)
Types of Partition Chromatography:
A. Liquid-liquid Chromatography: In this partition chromatography type, instead of an
adsorption column, a sheet of adsorbent paper is utilized. Based on their differential
migratory velocities, the components are divided. To make the chromatograms visible, they
are stained after separation.
B. Gas-liquid Chromatography: This is also called Gas-liquid partition chromatography
(GLPC) or vapor-phase chromatography. In this gas-liquid partition chromatography, the
separation of the sample mixture is carried by an inert gas with a tube. The stuffing of the
tube is done with finely divided inert solids that are coated with non-volatile oil. According
to the rate determined by both its solubility in oil and its vapor pressure, the migration of
every component takes place. [4]
Size-Exclusion Chromatography
Size-exclusion chromatography, also known as molecular sieve chromatography, is
a chromatographic method in which molecules in solution are separated by their shape, and in
some cases size. It is usually applied to large molecules or macromolecular complexes such

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as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the
sample through the column, the technique is known as gel filtration chromatography. [5]
Figure : Size-Exclusion Chromatography (https://www.priyamstudycentre.com/2022/02/size-
exclusion-chromatography.html)
Types of Size-Exclusion Chromatography :
a) Gel Filtration Chromatography (GFC): Gel Filtration Chromatography is a size-
exclusion chromatography technique performed in aqueous solutions, where molecules are
separated based on their size as they pass through a column packed with porous gel beads
(e.g., Sephadex, Sepharose).
b) Gel Permeation Chromatography (GPC): Gel Permeation Chromatography is a size-
exclusion chromatography technique performed in organic (non-aqueous) solvents, where
polymers and other molecules are separated according to their molecular size as they pass
through porous polymer beads (e.g., polystyrene or silica gels).
Ion Exchange Chromatography
Ion exchange chromatography includes a suite of related methods that purify proteins based on
their charge. The two main types of ion exchange resins are anion and cation, which bind to
negatively and positively charged molecules, respectively. There are several functional groups for
anion and cation exchange chromatography, as well as different resolutions.
Types of Ion-exchange Chromatography:

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i. Cation exchange chromatography: A cation-exchange resin is actually negatively
charged and binds positively-charged proteins.
ii. Anion exchange chromatography: An anion-exchange resins are positively charged
and bind negatively-charged proteins. [6]
i. Figure : Ion Exchange Chromatography (https://goldbio.com/articles/article/2-
common-types-of-ion-exchange-chromatography-anion-vs-
cation#:~:text=Ion%20exchange%20chromatography%20includes%20a,of%20interest
%20and%20purification%20purpose.)
Affinity Chromatography
Affinity chromatography is a type of liquid chromatography, which utilizes the reversible
biological interaction (affinity) between components in the solid stationary phase for separation.
Figure : Affinity Chromatography (https://microbenotes.com/affinity-chromatography/)
Types of Affinity Chromatography:
a. Boronate and Phenyl Borate Affinity Chromatography: Boronate is used as an affinity
ligand in the analysis of hemoglobin A1c (HbA1c) which is the component of glycated

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hemoglobin found in human blood. Cellufine phenyl borate is an affinity ligand used to
purify glycoproteins, glycated protein, and diol compounds.
b. Lectin Affinity Chromatography: Lectin is used as a stationary phase. Lectins are non-
immune proteins that recognize and bind certain types of carbohydrate residues. Used to
separate polysaccharides, glycopeptides, oligosaccharides, and cells that contain particular
carbohydrate structures.
c. Dye-ligand Affinity chromatography: Used to purify blood proteins, protein
pharmaceutical agents, enzymes, and albumin.
d. Immunoaffinity chromatography: Utilizes antibodies to purify peptides, viruses,
hormones, and enzymes.
e. Immobilized Metal Ion Affinity Chromatography: Interaction between immobilized
metal ions and target amino acids, proteins, peptides, and nucleic acids. Metal ions are
immobilized using chelating agents such as iminodiacetic acid, nitrilotriacetic acid, L-
glutamic acid, etc. A powerful tool for analyzing membrane proteins, histidine-tagged
proteins, and phosphorylated proteins.
f. Analytical Affinity Chromatography: It is also referred to as quantitative affinity
chromatography. Utilized for isolating and measuring specific targets. [7]
High Performance Liquid Chromatography
High Pressure or High Performance; Frequently this technique (HPLC), which utilizes high
pressure for pumping liquids and uses more efficient columns, is simply called LC. At times, this
technique is called high-performance liquid chromatography because it does provide high
performance as compared with classical liquid chromatography.
Stationary phase effects: The stationary phase effects of adsorption/normal phase or the
reversed-phase methods are discussed in the next two sections.
A. Adsorption/Normal Phase:
In a liquid–liquid chromatographic system, the interface between the phases can be involved in the
distribution process. So, it is necessary to consider the distribution at
▪ the interface between solid support and the stationary liquid phase.

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▪ the stationary liquid phase.
It is important to recognize that the following three distribution equilibria may have effects on
retention :
• partition between the bulk phases.
• adsorption on the solid surface.
• adsorption on the liquid–liquid interface.
B. Reversed Phase:
Reversed-phase (RP) silicas are remarkably useful in answering many separation problems in
modern liquid chromatography. [8]
Applications of Chromatography in Real Life
Pharmaceuticals:
Chromatography plays an important role in the safety of pharmaceuticals. Pharmaceutical
companies use chromatography to quantify and analyze compounds for contaminants. For
example, chiral compounds have two different forms due to their atoms differing slightly in
space. One form of chiral compounds is known to be toxic. Chromatography can ensure that the
safe form is separate from the dangerous form of the chiral compound.

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Vaccination creation is also an application of chromatography. Chromatography can be used to
determine which antibodies are the best for fighting and neutralizing certain diseases.
For example: In the fight against the Ebola virus outbreak, which tragically claimed over 11,000
lives, so scientist utilized chromatography to develop immunization Zmapp which was
experimental. This process involved to identify the most effective antibodies, which is capable of
neutralizing the deadly virus by isolating and analyzing these antibodies, researchers were able to
make significant result of these outbreak.
Applications of Chromatography in Pharmaceuticals:
GAS CHROMATOGRAPHY
Any time a lab technician must separate volatile materials or identify raw materials in a mixture,
many pharmaceutical professionals reach for gas chromatography (GC). Since GC machines are
capable of analyzing extremely small and light compounds, GC is used in post-production.
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
Efficient and accurate results are what make HPLC the primary chromatography method in the
pharmaceutical industry. HPLC uses a liquid as the mobile phase to ensure the fastest process.
Furthermore, HPLC machines have a column as the stationary phase, which
separates all the compounds accordingly.

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DISCOVERING NEW MEDICINE
These two chromatography methods have allowed professionals to identify a molecule with
remedial properties After finding a particular molecule-or molecules— lab technicians can begin
research by developing new formulas. From there, there will be further research and analysis to
"fine-tune" the medicine before launching it to the market.
THE DEVELOPMENT OF PURE MATERIALS
Similar to the previous point, as lab technicians test different formulas, they likely find a new
medicine that could have significant benefits. That said, they must identify which material is
beneficial by separating the formulas to their purest state. By doing so, technicians can identify
and research why that material is beneficial.
ANALYSIS AND IMPROVING AN EXISTING MEDICINE
Pharmaceutical lab technicians are always analyzing existing medicines. By doing so, they can
safely alter the formulations to improve results and potentially reduce or eliminate side effects.
Food and Beverage
Quality control within the food and beverage industry can be enacted through chromatography.
In the food industry, chromatography is used to separate and analyze additives, vitamins,
proteins, amino acids, and other nutritional compounds in food items. Chromatography can also
be used to determine expiration dates by distinguishing the number of organic acids present as
well as to detect any harmful toxins that may have been added to the food item.

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Chromatography is a technique it is used to separate and identify individual components within a
food sample. There are several different types of chromatography techniques, such as Gas
Chromatography (GC) which is used for volatile substances and Liquid Chromatography (LC)
for non-volatile substances.
RPC is a form of liquid chromatography that is use to analysis of non-polar or weakly polar
compounds in food and drink. In food science, it helps to identify and quantify food, and detect
contaminants and residues such as pesticides, mycotoxins, and heavy metals.
RPC is an important tool in chemistry. It helps detect the safety, quality, and authenticity of food.
It can easily spot harmful chemical substances like pesticides, herbicides, and synthetic dyes in
food products. This is crucial because it protects consumers from unsafe ingredients.
Food fraud is a significant issue. RPC is used to identify flavors in items like wine, coffee, and
dairy to ensure that the taste remains consistent. It can also distinguish between natural and
synthetic additives. Additionally, it can authenticate certain foods by assessing their chemical
structures.
Applications of Chromatography in Food and Beverage
Ensuring Food Safety
• Identifies contaminants such as pesticides, toxins, and heavy metals.
• Detects banned food additives.
Nutritional & Vitamin Analysis
• Identifies and measures vitamins.
• Quantifies essential amino acids and fatty acids Quality Control of Flavor & Aroma
• Helps maintain consistent taste in beverages, dairy, and processed foods.
• Identifies artificial and natural flavors.

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Authenticating Natural Extracts & Functional Foods
• Analyzes bioactive compounds in tea, coffee, and herbal extracts.
• Ensures standardization of health supplements.
Forensics
Chromatography techniques vary based on the type of separation and detection methods, each
offering unique advantages suitable for different purposes. Separation is achieved based on the
size or the interaction of analytes with the mobile and solid phases.
Applications of Chromatography in Forensics
Chromatography in Forensics
Widely used in drug analysis, toxicology, and trace evidence examination due to advancements
in compound identification.
Planar Chromatography (TLC)
Thin-layer chromatography (TLC) is a fast and cost-effective method.
Useful for ink, dye, and material identification in document analysis and forgery detection.
High-Performance Thin-Layer Chromatography-Mass Spectrometry (HPTLC-MS)
Used for rapid detection of drugs in biological samples.
Successfully identified citalopram, midazolam, chlordiazepoxide in forensic toxicology cases.
Gas Chromatography (GC & GC-MS)

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Ideal for analyzing volatile compounds like drugs, poisons, flammable liquids (e.g., in arson
cases).
GC-MS enhances sensitivity and accuracy.
Volatile Organic Compounds (VOCs) in Death Investigations
VOCs produced during decomposition are useful in medicolegal autopsies.
Two-dimensional GC (GC×GC) helps identify VOCs from bacterial activity.
High-Performance Liquid Chromatography (HPLC) & UHPLC-MS/MS
Used for non-volatile and thermolabile substances.
UHPLC-MS/MS provides faster, more sensitive, and high-resolution results.
Applied in drug detection (both illicit and prescription drugs).
Forensic Ballistics
Chromatography helps analyze organic gunshot residue (OGSR) components like explosives and
stabilizers.
UHPLC-MS/MS identifies trace compounds (e.g., ethyl centralite, diphenylamine,
nitroglycerine), aiding in firearm identification and shooting distance estimation.
Environmental Monitoring
Land pollution
Land Pollution is the third biggest type of environmental pollution today.

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Caused by:
• Industrialization
• Urbanization
• Deforestation
• Overfilled landfills
Role of Agriculture
Agriculture is a major contributor to land pollution. Fertilizers, pesticides, and insecticides are
used to grow more crops. These chemicals harm the soil and can affect human health.
Role of Chromatography
Both gas chromatography (GC) and liquid chromatography (LC/HPLC) are used to:
• Detect harmful chemicals in soil.
• Analyze presence of pesticides and fertilizers.
Chromatography also helps in:
• Restoring polluted soil by identifying and removing harmful substances
Role of Agriculture
• Farms release large amounts of organic waste into water bodies.
• Toxins like nitrogen can
• Make water uninhabitable for aquatic life.
• Cause health issues in infants if consumed.
Harmful Contaminants
Includes dangerous chemicals like PFAs (Per- and polyfluoroalkyl substances).
Chromatography in Water Pollution Detection

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▪ Preferred method:
Solid-phase extraction + LC-MS/MS
(Liquid Chromatography–Tandem Mass Spectrometry)
▪ Used to detect and measure pollutants in water accurately.
Air Pollution
▪ 1.Human-made compounds found in:
• Paint
• Gasoline
• Dry-cleaning agents
• Printers
• Aerosol cans
• Released into the air as gas.
▪ 2. Health & Environmental Impact
VOC exposure can cause:
• Liver damage
• Kidney damage
• Heart and lung problems
In the environment:
• VOCs react with nitrogen oxides in sunlight → form ozone.
• Ozone + fine particles + VOCs = Smog
Smog causes:
• Health issues (example: asthma, heart/lung disease)
• Damages to crops, forests, and plant growth.
3. Detection of VOCs

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• Gas Chromatography (GC) is widely used for VOC analysis.
A more advanced method:
• Thermal Desorption GC-MS (Gas Chromatography–Mass Spectrometry)
• Detects, identifies, and quantifies VOCs in the air
The Essential Guide to Chromatography: it’s advantages and
disadvantages
In science, few things are as versatile as chromatography. It plays an integral role in dissecting complex
substances in medicine, chemistry, and even environmental science. The technique’s working principle is
simple: The sample is partitioned into two phases. One phase is fixed, while the other, the mobile phase,
carries the sample. The mobile phase is arguably the most important phase as it dictates the type of
analysis that will be performed and the molecules that will be separated
The Mobile Phase: Liquids and Gases and Chromatography
The mobile phase is the most important phase as it acts as the ‘driving force’ of the entire system. It is the
phase that most determines the effectiveness of the separation
Gas Chromatography (GC)
Gas chromatography use inert gases such as helium or nitrogen. Because the sample must be vaporized,
this technique focuses on compounds that can exist in the gas phase.
Advantages:
The Produces are very sharp and clean peaks with excellent resolution.Fast and highly sensitive,
especially when paired with detectors like mass spectrometry.and extremely reliable for quantitative
measurements, making it a standard in fuel analysis, fragrance testing, and environmental labs.
Disadvantages:
Limited to volatile molecules larger or unstable compounds cannot be analyzed. Because it Sometimes
requires extra preparation (derivatization) to make samples suitable to adding steps to the process.

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Liquid Chromatography (LC)
Liquid Chromatography utilizes solvents such as water, acetonitrile, and methanol. These solvents allow
the LC method to be applicable to a wider range of compounds in different physical states
Advantages:
Works with non-volatile, polar, and heat-sensitive molecules. Gradient methods (changing solvent
composition during a run) allow separation of very different compounds in one analysis. And Operates
under mild conditions, protecting fragile samples like proteins or vitamins.
Disadvantages:
Separation is slower because diffusion in liquids is less efficient, although HPLC minimizes this problem.
Requires large amounts of high-purity solvents, which is costly and generates waste.Instruments are
sophisticated and need skilled operation.
Chromatography Advantages & Disadvantages:
Technique Advantages Disadvantages
Thin-Layer & Paper
Chromatography (TLC/PC)
Inexpensive and quick, easy to perform often
used for teaching and checking reactions
Limited resolution and not
suitable for quantitative
analysis
Column Chromatography
Useful for isolating compounds, simple and
relatively low cost.
Very slow process which can
take hours or days, resolution
is lower compared to modern
method.
High-Performance Liquid
Chromatography (HPLC)
Provides high resolution and accurate results
and widely used in pharmaceuticals, food, and
biochemistry which reliable for quantitative
studies.
Equipment and maintenance
are costly, consumes large
amounts of solvent which also
raises environmental concerns.
Ion Exchange
Chromatography (IEC)
Essential for separating proteins, amino acids,
and nucleotides, also applied in water
treatment.
Require strict control of pH
and salt conditions, resins may
degrade under harsh
environments.

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The Future of Chromatography in Research and industry -
Chromatography moving very quickly to become faster, smarter, and more efficient. Scientists are
pushing the boundaries of what this technique can do and several big trends those are shaping its future:
Hyphenated Techniques: Modern research rarely relies on chromatography alone. Instead, it’s
increasingly combined with advanced detectors like mass spectrometry. Systems such as GC-MS or LC-
MS don’t just separate complex mixtures they also identify the exact compound that present in a single
automated run. This saves time and provide much more reliable results which is why these systems have
become standard in pharmaceutical labs, forensic science, and environmental monitoring.
Miniaturization: There is a strong push to make chromatography more portable and eco-friendlier. New
[ lab-on-a-chip] devices and microfluidic systems can perform separations using just a few drops of
solvent and very small samples. These portable tools are opening the door to on the spot testing, like drug
checks at crime scenes or bedside medical diagnostics without needing large laboratory equipment.
Automation and Artificial Intelligence: Traditional chromatography often required long hours of
manual operation. Now, robotic autosampler can run hundreds of samples overnight while AI-driven
software helps optimize conditions like solvent choice or column type. This shift not only improves
reproducibility but also frees researchers to focus on interpreting results rather than handling repetitive
technical tasks.
Size Exclusion
Chromatography (SEC)
Gentle method that preserves delicate
biomolecules and polymers
Limited capacity for samples
and lower resolution compared
to HPLC.
Supercritical Fluid
Chromatography (SFC)
Combines the efficiency of gas
chromatography with the flexibility of liquid
chromatography, use little solvent and is more
environmentally friendly.
Works best with non-polar or
moderately polar compounds,
equipment is specialized and
complex.
Affinity Chromatography
Extremely specific, produces very pure results,
commonly used in biotechnology and drug
production
Stationary phases such as
antibodies are expensive,
elution can sometimes damage
the molecules being studied

27
Advanced Materials: A lot of progress is also happening in the design of new stationary phases.
Innovations such as core-shell particles allow HPLC to achieve excellent separation at lower pressures,
making the systems more efficient. At the same time new chiral selectors are improving our ability to
separate mirror-image molecules (enantiomers). This is especially important in drug research since the
two “hands” of a molecule can have completely different effects in the body.
Conclusion
Chromatography provides solution for nearly every separation challenge. The choice between gas and
liquid methods usually depends on whether a compound can be vaporized. From simple teaching tools
like TLC to large-scale purification with columns and the precision of HPLC or GC, each technique
serves a unique role. As new technologies develop through the miniaturization, automation, and
innovative materials chromatography will remain a key driver of progress in science and industry.
Moreover, As environmental regulations tighten and laboratory standards evolve the integration of green
practices chromatographic workflows will become not just beneficial, but also essential.

28
References
➢ (https://www.vedantu.com/chemistry/gas-chromatography, n.d.)
➢ (https://www.birchbiotech.com/blogs/hplc-lc-ms-articles/how-to-determine-purity-from-
gas-chromatography, n.d.)
➢ (https://gkscientist.com/chromatographic-separation-methods/, n.d.)
➢ (https://blog.epcland.com/gas-chromatographs, n.d.)
➢ Types of stationary phases in gas chromatography| Phenomenex. (n.d.).
https://www.phenomenex.com/knowledge-center/gc-knowledge-center/stationary-phases-
gc-types-key-insights
➢ JoVE. (2025, July 4). Gas chromatography: types of columns and stationary phases
[Video]. JoVE. https://www.jove.com/science-education/v/14788/gas-chromatography-
types-of-columns-and-stationary-
phases#:~:text=Overview,noted%20for%20their%20high%20polarity.
➢ Admin. (2022, November 16). Adsorption chromatography. BYJUS.
https://byjus.com/chemistry/adsorption-chromatography/
➢ https://lab-training.com/partition-chromatography/
➢ https://en.wikipedia.org/wiki/Size-exclusion_chromatography
➢ https://goldbio.com/articles/article/2-common-types-of-ion-exchange-chromatography-
anion-vs-
cation#:~:text=Ion%20exchange%20chromatography%20includes%20a,of%20interest%
20and%20purification%20purpose.
➢ Rawal, A. (2025, April 3). Affinity Chromatography: principle, parts, steps, uses. Microbe
Notes. https://microbenotes.com/affinity-chromatography/
➢ Ahuja, S. (2003). Chromatography and separation Science. Elsevier.

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