Chromatography is a separation technique used to separate and analyze the components of a mixture. It involves the distribution of the components between two phases: a mobile phase and a stationary phase. The different affinities of the components for these phases lead to their separation based on their physical or chemical properties.

2. L.N Paramedical College
C- SECTOR, SARVADHARAM, KOLAR ROAD, BHOPAL
MADHYA PRADESH Session – 2022 – 2023
Project on
HPLC
Class: BMLT 3rd YEAR
Submitted to :- Submitted by:-
BMLT Department Md Shams tabrez

3. L.N Paramedical college C- SECTOR,
SARVADHARAM, KOLAR ROAD, BHOPAL, MADHYA
PRADESH

4. CERTIFICATE
This is to certify that MR. Md Shams Tabrez is the student of BMLT 3rd
year L.N PARAMEDICAL COLLEGE, BHOPAL. He submitted his assignment
on the title HPLC during the session 2022- 2023 to head of department
of PARAMEDICAL.
DR.Vivek Srivastava
Head of department
Paramedical department

5. ACKNOWLEDGEMENT
My Assignment was only possible in the guidance and supervision of
SHAILESH VERMA and Mr NAREN SARKAR
I am thankful to him for support and discussion on the work done.
I am sincerely thankful to Mrs. DR. SHAILJA KURELE, Mr. Surya Dev
Singh Sir faculty and lab technician,
Mr.SANJEEV AHIRWAR ,Mr.MD MASUD AZAHAR of department of
Paramedical , LN PARAMEDICAL COLLEGE, BHOPAL for their cooperation
and blessings, to complete this work.
I am thankful to my family members for their blessings and support.
Last but not the least I am thankful to almighty.
Md Shams Tabrez
BMLT 3rd year

6. INDEX
• Contents
 WHAT IS CHARAMATOGRAPHY
 WHAT IS HPLC
 TYPES OF HPLC
 IMPORTANCE OF HPLC
 REVERSE PHASE CHROMTOGRAPHY
 NORMAL PHASE CHROMTOGRAPHY
 IRON EXCHANGE CHROMTOGRAPHY

7. Whats is CHRAMATOGRAPHY
Chromatography is a separation technique used to separate and analyze the
components of a mixture. It involves the distribution of the components between
two phases: a mobile phase and a stationary phase. The different affinities of the
components for these phases lead to their separation based on their physical or
chemical properties.
Here are some key points about chromatography:
1. Principle: Chromatography works on the principle of differential partitioning or
distribution of the components between the mobile phase and the stationary phase.
The components with stronger interactions with the stationary phase will move more
slowly, while those with weaker interactions will move more quickly, resulting in
separation.
2. Mobile Phase: The mobile phase is a fluid or gas that carries the sample through
the chromatographic system. It can be a liquid (in liquid chromatography) or a gas (in
gas chromatography). The choice of mobile phase depends on the nature of the
sample and the separation requirements.
3. Stationary Phase: The stationary phase is a solid or liquid phase that is immobile
and interacts with the components of the sample. It can be a solid adsorbent, such as
silica gel or a resin, or a liquid coating on a solid support. The stationary phase
selectively retains or interacts with the components based on their physical or
chemical properties.
4. Types of Chromatography: There are various types of chromatography techniques,
including:
- Gas Chromatography (GC): In GC, the mobile phase is a carrier gas, and the
separation is based on the components' volatility and their interaction with the
stationary phase.

8. - Liquid Chromatography (LC): LC uses a liquid mobile phase and can be further
classified into several techniques, such as High-Performance Liquid Chromatography
(HPLC), Reverse-Phase Chromatography, Ion-Exchange Chromatography, and Size-
Exclusion Chromatography.
- Thin-Layer Chromatography (TLC): TLC involves the separation of components on a
thin layer of adsorbent material coated on a solid support.
- Paper Chromatography: Paper chromatography uses a piece of porous paper as
the stationary phase.
5. Detection: Chromatographic techniques employ various detectors to measure and
quantify the separated components. Common detectors include UV-Vis detectors,
fluorescence detectors, mass spectrometers, refractive index detectors, and
electrochemical detectors. The choice of detector depends on the nature of the
sample and the analytes being analyzed.
6. Applications: Chromatography has widespread applications in various fields,
including pharmaceutical analysis, environmental monitoring, food and beverage
analysis, forensic science, biochemical analysis, and quality control. It allows for the
separation, identification, and quantification of components in complex mixtures.
Chromatography is a versatile technique that offers high resolution, sensitivity, and
selectivity for separating and analyzing complex mixtures. It plays a vital role in
research, quality control, and various industries where the separation and analysis of
components are critical.
WHAT IS HPLC:
HPLC stands for High-Performance Liquid
Chromatography. It is a widely used analytical
technique that separates, identifies, and quantifies

9. components in a liquid mixture. HPLC is based on the
principles of liquid chromatography, where a liquid
mobile phase carries the sample through a stationary
phase, and the components in the sample interact
differently with the stationary phase, leading to their
separation.
Here are some key features and components of an
HPLC system:
1. Pump: HPLC systems include a high-pressure pump
that delivers the mobile phase at a constant flow
rate. The pump ensures a consistent and
reproducible flow of the liquid mobile phase through
the system.
2. Injector: The injector is responsible for introducing
the sample into the mobile phase stream. It typically
consists of a sample loop or a syringe that delivers a
precise volume of the sample into the system.

10. 3. Column: The column is the heart of the HPLC
system. It contains a stationary phase that separates
the components of the sample based on their
interaction with the stationary phase. Columns can
vary in length, diameter, and stationary phase
chemistry, depending on the specific analytical
needs.
4. Detector: HPLC systems employ various types of
detectors to monitor the separated components. The
most commonly used detector is the UV-Vis detector,
which measures the absorbance of the components
at specific wavelengths. Other detectors, such as
fluorescence detectors, refractive index detectors,
and mass spectrometers, can be used for specific
applications or when enhanced sensitivity or
selectivity is required.
5. Chromatographic Data System (CDS): A CDS is a
computer-based system that controls the HPLC
system, collects data from the detector, and
performs data analysis. It allows for real-time

11. monitoring of chromatograms, data processing, and
integration of peaks for quantification.
6. Mobile Phase: The mobile phase is the liquid
solvent or mixture of solvents that carries the sample
through the column. It is typically a mixture of water
and organic solvents, with the composition optimized
based on the sample and the separation
requirements.
7. Stationary Phase: The stationary phase is the solid
or liquid phase that is immobilized in the column and
interacts with the components of the sample. It can
be a porous solid (such as silica or polymer) or a
liquid phase coated onto solid support (such as a
bonded phase). The choice of stationary phase
depends on the analytes being separated and the
desired separation mechanism.
HPLC is used in a wide range of applications,
including pharmaceutical analysis, environmental
monitoring, food and beverage analysis, forensic

12. analysis, and many others. It offers high sensitivity,
selectivity, and reproducibility, making it a versatile
and powerful technique for qualitative and
quantitative analysis of complex mixtures.
Types of HPLC
There are several types of High-Performance Liquid
Chromatography (HPLC) techniques, each offering
unique separation mechanisms and applications.
Here are some commonly used types of HPLC:
1. Reverse-Phase Chromatography (RPC): In reverse-
phase chromatography, the stationary phase is
nonpolar (hydrophobic), typically a hydrocarbon
chain bonded to a solid support, while the mobile
phase is polar (aqueous or organic solvent). This
technique is widely used in pharmaceutical analysis,
drug discovery, and quality control applications.
2. Normal-Phase Chromatography (NPC): Normal-
phase chromatography utilizes a polar stationary

13. phase, such as silica gel, and a nonpolar mobile
phase, such as a mixture of nonpolar organic
solvents. It is often employed for the separation of
polar compounds, such as natural products, organic
acids, and carbohydrates.
3. Ion-Exchange Chromatography (IEC): Ion-exchange
chromatography separates components based on
their ionic interactions with charged stationary
phases. The stationary phase can be either an anion-
exchange resin (positively charged) or a cation-
exchange resin (negatively charged). IEC is used for
the analysis of ions, amino acids, proteins, and other
charged compounds.
4. Size-Exclusion Chromatography (SEC): Size-
exclusion chromatography, also known as gel
filtration chromatography, separates components
based on their size or molecular weight. It uses a
porous stationary phase, and larger molecules elute
first, while smaller molecules are retained longer. SEC

14. is often used for the analysis of polymers, proteins,
and biomolecules.
5. Affinity Chromatography: Affinity chromatography
exploits the specific binding interactions between a
target analyte and a stationary phase containing
immobilized ligands or antibodies. This technique
allows for highly selective separation and purification
of target compounds, such as proteins, enzymes, and
biomolecules.
6. Chiral Chromatography: Chiral chromatography
separates enantiomers, which are stereoisomers that
are mirror images of each other. It utilizes chiral
stationary phases or chiral additives in the mobile
phase to discriminate between enantiomers based
on their unique interactions. Chiral HPLC is essential
in pharmaceutical development, as many drugs exist
as chiral compounds with distinct biological
activities.
7. Hyphenated Techniques: HPLC can be coupled
with other analytical techniques to enhance

15. separation and detection capabilities. For example,
liquid chromatography-mass spectrometry (LC-MS)
combines the separation power of HPLC with the
mass analysis capabilities of a mass spectrometer,
enabling identification and structural elucidation of
analytes.
These are just a few examples of the types of HPLC
techniques available. Each technique has its
advantages and applications, and the choice depends
on the nature of the sample and the specific
separation requirements. Researchers and analysts
select the appropriate HPLC method based on the
target analytes, sample matrix, sensitivity, selectivity,
and resolution needed for their particular analysis.
The Importance of HPLC
High-Performance Liquid Chromatography (HPLC) is
an essential analytical technique with wide-ranging

16. importance in various fields. Here are some key
reasons why HPLC is important:
1. Separation and Analysis of Complex Mixtures:
HPLC allows for the separation and analysis of
complex mixtures containing multiple components. It
enables the identification and quantification of
individual analytes within a sample, even in the
presence of interfering substances. This is
particularly valuable in fields such as pharmaceutical
analysis, environmental monitoring, food and
beverage analysis, forensic science, and quality
control, where accurate and precise determination of
component concentrations is crucial.
2. High Sensitivity: HPLC offers high sensitivity,
enabling the detection and quantification of analytes
at low concentrations. With appropriate sample
preparation techniques and sensitive detectors, HPLC
can detect and analyze trace amounts of substances
in a sample. This is essential in areas such as

17. pharmaceutical research, environmental analysis,
and forensic toxicology, where the presence of
minute quantities of substances can have significant
implications.
3. Selectivity and Specificity: HPLC provides excellent
selectivity and specificity in separating and analyzing
components within a mixture. By using different
stationary phases and mobile phases, specific
separation mechanisms can be employed to target
analytes of interest. This selectivity allows for the
detection and quantification of analytes in the
presence of interfering substances, ensuring accurate
and reliable results.
4. Wide Range of Applications: HPLC has extensive
applications in various industries and research fields.
It is used in pharmaceutical analysis for drug
development, quality control, and pharmacokinetic
studies. Environmental scientists employ HPLC to
monitor and analyze pollutants, pesticides, and

18. contaminants in air, water, and soil. In the food and
beverage industry, HPLC is used to determine
nutritional content, identify additives, and detect
contaminants. HPLC is also widely used in forensic
analysis, biochemical research, clinical diagnostics,
and many other areas.
5. Method Development and Optimization: HPLC
allows for method development and optimization to
achieve the desired separation and analytical
performance. By selecting the appropriate stationary
phase, mobile phase, and detection parameters,
analysts can tailor HPLC methods to specific sample
matrices and separation goals. This flexibility and
versatility enable the development of robust and
efficient analytical methods for different applications.
6. Quantitative Analysis and Quality Control: HPLC
plays a critical role in quantitative analysis and
quality control. It enables the accurate determination
of component concentrations in samples, ensuring

19. the safety, efficacy, and compliance of
pharmaceuticals, food products, and industrial
materials. HPLC methods are often used as standard
analytical techniques in official pharmacopoeias and
quality control laboratories.
7. Method Validation and Regulatory Compliance:
HPLC is an established and widely accepted analytical
technique, making it essential for method validation
and regulatory compliance. HPLC methods undergo
rigorous validation procedures to demonstrate their
accuracy, precision, linearity, specificity, and
robustness. These validated methods are relied upon
in regulated industries to meet quality standards,
comply with regulatory requirements, and ensure
the safety and efficacy of products.
In summary, HPLC is of paramount importance due
to its ability to separate complex mixtures, its high
sensitivity, selectivity, and specificity, and its wide
range of applications in diverse fields. Its quantitative

20. capabilities, method development flexibility, and
adherence to regulatory standards make it an
indispensable analytical tool for research, industry,
and quality control.
Reverse-Phase Chromatography
(RPC)
Reverse-Phase Chromatography (RPC) is a widely
used technique in High-Performance Liquid
Chromatography (HPLC). It is based on the principle
of partitioning analytes between a nonpolar

21. stationary phase and a polar mobile phase. Here are
some key points about Reverse-Phase
Chromatography:
1. Principle: In RPC, the stationary phase is nonpolar
(hydrophobic), typically a hydrocarbon chain bonded
to a solid support, while the mobile phase is polar
(aqueous or organic solvent). The nonpolar
stationary phase retains nonpolar or weakly polar
analytes, while polar analytes are eluted more
quickly.
2. Separation Mechanism: The separation in RPC is
primarily driven by the hydrophobic interactions
between the analytes and the nonpolar stationary
phase. Nonpolar analytes have stronger affinity for
the stationary phase and are retained longer,
resulting in slower elution. On the other hand, polar
analytes have weaker interactions and elute more
quickly.

22. 3. Applications: Reverse-Phase Chromatography is
widely employed in pharmaceutical analysis, drug
discovery, quality control, and other areas where the
separation and analysis of organic compounds are
required. It is particularly useful for analyzing
hydrophobic and lipophilic compounds, such as
drugs, natural products, lipids, and metabolites.
4. Stationary Phase: The stationary phase in RPC
consists of a nonpolar hydrocarbon chain, typically
attached to a silica-based support. Common
stationary phases include C18 (octadecyl) and C8
(octyl) alkyl chains, which provide different degrees
of hydrophobicity and selectivity. The choice of
stationary phase depends on the analytes being
separated and the desired separation characteristics.
5. Mobile Phase: The mobile phase in RPC is typically
a mixture of water and organic solvents, such as
methanol or acetonitrile. The composition and ratio
of the mobile phase can be adjusted to optimize the
separation of analytes. The addition of an acid or a

23. buffer to the mobile phase can also influence the
separation, especially for ionizable compounds.
6. Detector: Various detectors can be used in RPC,
depending on the nature of the analytes and the
specific requirements of the analysis. Common
detectors include UV-Vis detectors, which measure
the absorbance of the analytes at specific
wavelengths, and mass spectrometers, which provide
additional identification and structural information.
7. Method Development: RPC methods require
careful optimization of the mobile phase
composition, stationary phase choice, and operating
conditions to achieve the desired separation and
resolution. Parameters such as the organic solvent
concentration, pH, and flow rate can be adjusted to
improve the separation efficiency and selectivity.
8. Advantages: RPC offers several advantages,
including high resolution, good peak shape, and
compatibility with a wide range of analytes. It is

24. suitable for both analytical and preparative-scale
separations. RPC also provides a simple and efficient
method for the separation and analysis of complex
mixtures.
In summary, Reverse-Phase Chromatography is a
powerful technique in HPLC that relies on the
partitioning of analytes between a nonpolar
stationary phase and a polar mobile phase. It is
widely used for the separation and analysis of
organic compounds in various industries, particularly
in pharmaceutical analysis and drug discovery.
Normal-Phase Chromatography
(NPC)
Normal-Phase Chromatography (NPC) is a type of
chromatographic technique used in High-
Performance Liquid Chromatography (HPLC). It is the
opposite of Reverse-Phase Chromatography (RPC)
and operates on the principle of partitioning analytes
between a polar stationary phase and a nonpolar

25. mobile phase. Here are some key points about
Normal-Phase Chromatography:
1. Principle: In NPC, the stationary phase is polar,
typically consisting of a polar adsorbent material,
such as silica gel or alumina. The mobile phase is
nonpolar, usually a mixture of nonpolar organic
solvents. The polar stationary phase retains polar or
weakly polar analytes, while nonpolar analytes are
eluted more quickly.
2. Separation Mechanism: The separation in NPC is
based on the differential affinity of analytes for the
polar stationary phase and the nonpolar mobile
phase. Polar analytes have stronger interactions with
the polar stationary phase, leading to slower elution,
while nonpolar analytes have weaker interactions
and elute more quickly.
3. Applications: Normal-Phase Chromatography is
commonly used for the separation and analysis of
polar compounds, such as natural products, organic

26. acids, carbohydrates, and some pharmaceuticals. It is
particularly useful for compounds that are too polar
for reverse-phase separation or for resolving closely
related isomers.
4. Stationary Phase: The stationary phase in NPC is
typically a polar adsorbent material, such as silica gel
or alumina, which is packed into a column. Silica gel,
a common stationary phase, consists of porous silica
particles with surface silanol groups that provide
polar interactions. The choice of stationary phase
depends on the analytes being separated and the
desired separation characteristics.
5. Mobile Phase: The mobile phase in NPC is a
nonpolar organic solvent or a mixture of organic
solvents. Common mobile phase solvents include
hexane, heptane, diethyl ether, and chloroform. The
composition and ratio of the mobile phase can be
adjusted to optimize the separation of analytes.

27. 6. Detector: Similar to Reverse-Phase
Chromatography, NPC can utilize various detectors,
such as UV-Vis detectors and mass spectrometers,
depending on the analytes and specific requirements
of the analysis.
7. Method Development: NPC methods require
careful optimization of the mobile phase
composition, stationary phase choice, and operating
conditions to achieve the desired separation and
resolution. Parameters such as the organic solvent
composition, flow rate, and temperature can be
adjusted to improve the separation efficiency and
selectivity.
8. Advantages: Normal-Phase Chromatography offers
advantages for the separation of polar compounds
and is particularly useful for compounds that are too
hydrophilic or polar for reverse-phase separation. It
allows for the resolution of closely related isomers
and can provide different selectivity compared to
other chromatographic techniques.

28. In summary, Normal-Phase Chromatography is a
chromatographic technique in HPLC that utilizes a
polar stationary phase and a nonpolar mobile phase.
It is effective for the separation and analysis of polar
compounds and can provide different selectivity
compared to Reverse-Phase Chromatography. NPC is
commonly used in various applications, including
natural product analysis, organic acid analysis, and
carbohydrate analysis.
Ion-Exchange Chromatography
(IEC)
Ion-Exchange Chromatography (IEC) is a powerful
chromatographic technique used in High-
Performance Liquid Chromatography (HPLC) for the
separation and analysis of charged molecules based
on their ionic interactions with a stationary phase.
Here are some key points about Ion-Exchange
Chromatography:

29. 1. Principle: Ion-Exchange Chromatography relies on
the reversible exchange of ions between the sample
analytes and the charged functional groups on the
stationary phase. The stationary phase can be either
an anion-exchange resin (positively charged) or a
cation-exchange resin (negatively charged). Analytes
with opposite charges to the functional groups on
the resin will interact and be retained, while analytes
with similar charges will elute more quickly.
2. Separation Mechanism: In IEC, the separation is
based on the differences in the ionic interactions
between the analytes and the stationary phase.
Analytes with stronger ionic interactions with the
resin will be retained longer, resulting in slower
elution, while analytes with weaker interactions will
elute more quickly.
3. Applications: Ion-Exchange Chromatography is
commonly used for the separation and analysis of
ions, charged molecules, and biomolecules such as
proteins, peptides, amino acids, nucleic acids, and

30. carbohydrates. It is also used for the purification and
isolation of target compounds based on their charge
properties.
4. Stationary Phase: The stationary phase in IEC
consists of a resin or solid support with charged
functional groups. Anion-exchange resins have
positively charged functional groups, such as
quaternary ammonium, while cation-exchange resins
have negatively charged functional groups, such as
sulfonate or carboxylate. The choice of stationary
phase depends on the target analytes and their
charges.
5. Mobile Phase: The mobile phase in IEC is typically
an aqueous buffer solution containing ions of
opposite charge to the stationary phase. The pH and
ionic strength of the mobile phase can be adjusted to
control the interactions between the analytes and
the stationary phase. Changes in pH or ionic strength
can affect the degree of binding and elution of
analytes.

31. 6. Detector: Various detectors can be used in IEC,
depending on the nature of the analytes and the
specific requirements of the analysis. Common
detectors include UV-Vis detectors for measuring
absorbance at specific wavelengths, conductivity
detectors for monitoring changes in ionic strength,
and mass spectrometers for identification and
structural analysis.
7. Method Development: IEC methods require
careful optimization of the mobile phase
composition, pH, ionic strength, and operating
conditions to achieve the desired separation and
resolution. The choice of buffer, pH, and ionic
strength can influence the selectivity and efficiency
of the separation.
8. Advantages: Ion-Exchange Chromatography offers
several advantages, including high selectivity for
charged molecules, the ability to separate
compounds with similar molecular weights but

32. different charges, and the potential for large-scale
purification of biomolecules. It is widely used in
biochemistry, biotechnology, pharmaceutical
analysis, and environmental analysis.
In summary, Ion-Exchange Chromatography is a
valuable technique in HPLC that utilizes the ionic
interactions between analytes and a charged
stationary phase. It allows for the separation and
analysis of charged molecules and biomolecules
based on their charges and is widely applied in
various fields for research, analysis, and purification
purposes.

33. THANK YOU