Abstract

Chromatography encompasses a group of techniques designed

to separate the components of a mixture. It operates using two
distinct phases: the stationary phase and the mobile phase.
The separation process relies on the variation in the partition
coefficients between these phases. The term
"chromatography" has its roots in the Greek words Chroma
(colour) and graphene (to write).
Highly regarded for its analytical capabilities,
chromatography is widely employed in various fields.
Different methods under this umbrella include paper
chromatography, gas chromatography, liquid
chromatography, thin-layer chromatography, ion-exchange
chromatography, and high-performance liquid
chromatography (HPLC).
This discussion places particular emphasis on HPLC,
exploring its principles, classifications, instrumentation, and
applications. Recognized as an advanced chromatographic
technique, HPLC plays a crucial role in both scientific
research and industrial processes, making it a cornerstone in
modern analytical practices.
KEY WORDS: Chromatography, Mobile phase, Stationary
phase, Analytic
 INTRODUCTION
DIFFERENT METHODS OF ANALYSIS
▪ STEPS IN QUANTITATIVE ANALYSIS
▪ INTRODUCTION:

Analytical Chemistry focuses on developing methods to

determine the chemical composition of various materials. It
plays a crucial role in breaking down compounds into their
basic components, identifying elements, and detecting any
foreign substances present. This field is divided into two main
branches: qualitative and quantitative analysis.
Qualitative analysis aims to identify the atomic or molecular
species or functional groups present in a sample. On the other
hand, quantitative analysis provides numerical data on the
relative amounts of one or more components within the
sample. When faced with an unknown sample, the primary
objective for analysts is often to identify the substances it
contains. This can extend to determining impurities or
confirming their absence, tasks that fall under the scope of
qualitative analysis. Once the identity of the constituents is
established, analysts are frequently required to quantify them,
which is the domain of quantitative analysis.
Both branches are vital in industries to ensure that raw
materials meet specific standards and that final products
adhere to quality benchmarks. Furthermore, analytical
chemists are instrumental in the development of new
products, including mixtures, by identifying the optimal
composition for their intended use.
When an appropriate sample is obtained, selecting the most
effective method for analysis becomes essential. Analysts
must consider several factors, such as:
 The type of information needed.
 The sample size and the proportion of the constituent to
be analysed.
 The purpose for which the analytical data will be used.
Precision and accuracy are paramount, and the choice of
methodology significantly impacts the reliability of results.
By evaluating these considerations, analysts can ensure that
their methods are suitable for achieving the desired outcomes.

AIMS OF ANALYTICAL CHEMISTRY:-

1. Advancing the theoretical framework of analytical

methodologies across diverse domains.
2. Enhancing and providing scientific validation for current
analytical techniques to strengthen their reliability and
application.
3. Innovating and developing new analytical approaches
tailored to meet the evolving demands of modern science
and industrial practices.
4. Conducting comprehensive analysis of natural
substances, environmental samples, and industrial
materials to derive meaningful insights and ensure
quality standards.
 HISTORY

Before the advent of High-Performance Liquid

Chromatography (HPLC), researchers relied on conventional
liquid chromatographic techniques, which were notably
inefficient due to the solvent flow rate being dependent on
gravity. These methods often required several hours or even
days to complete separations. While gas chromatography
(GC) was more efficient than liquid chromatography (LC) at
the time, it was believed that separating. The thermal
instability of solutes made GC unsuitable for many organic
chemists, prompting the exploration of alternative approaches,
which eventually led to the development of HPLC.
Building on the pioneering work of Martin and Synge in
1941, scientists like Cal Giddings, Josef Huber, and others in
the 1960s predicted that liquid chromatography could achieve
higher efficiency by significantly reducing the particle size of
the packing material (to well below the typical LC and GC
size of 150 µm) and using pressure to increase the mobile
phase velocity. This vision underwent extensive
experimentation and refinement throughout the 1960s and
1970s.
Early research in this period focused on improving LC
particles, and the development of Zipax, a particle with
external permeability, showed promise for advancing HPLC
technology. The 1970s marked significant progress in HPLC
instrumentation. Researchers began using pumps and injectors
to simplify system configurations. Gas amplifier pumps were
particularly favoured for their ability to maintain consistent
pressure, eliminating the need for leak-proof seals or check
valves, thereby ensuring steady flow and reliable quantitation.
Although these instrumental advancements were crucial, the
evolution of HPLC is largely intertwined with the
development of particle technology. After introducing
permeable layer particles, there was a consistent move
towards reducing particle size to improve efficiency.
However, reducing particle size introduced challenges such as
excessive pressure drops needed to drive the mobile phase
through the column and difficulties in achieving uniform
packing with extremely fine materials. Each reduction in
particle size necessitated a corresponding round of
instrumental innovations to address these issues and manage
the increased pressures.
 HPLC Method Development Purpose
The primary aim of developing analytical methods in High-
Performance Liquid Chromatography (HPLC) is to create a
dependable and efficient approach for the separation,
identification, and quantification of components within a
sample. This is crucial for applications spanning various
fields, such as pharmaceuticals, environmental monitoring,
food safety, and more. The key goals of method development
include:
1. Selectivity: Formulating a process that efficiently
distinguishes target compounds from any interfering
substances to ensure precise and specific outcomes.
2. Sensitivity: Fine-tuning the method to detect and
quantify substances at the required concentration levels,
which can vary depending on the application.
3. Accuracy and Precision: Creating a procedure that
yields consistent and accurate results, minimizing
variations in measurements.
4. Robustness: Ensuring the method remains reliable
despite changes in environmental conditions, equipment
settings, or sample properties.
5. Efficiency: Reducing the time needed for analysis, as
well as minimizing the use of solvents and the generation
of waste, all while maintaining high analytical standards.
6. Validation: Confirming through validation that the
method is suitable for its intended purpose, particularly
when regulatory standards must be met.
7. Cost-effectiveness: Striking a balance between accuracy
and cost by considering factors such as the required
equipment, consumables, and the time involved in the
process.
 High-Performance Liquid Chromatography (HPLC)
has several modes or types, each designed for specific
purposes. Some common modes of HPLC include:
1. Normal Phase HPLC (NP-HPLC): In this method, a
polar stationary phase is paired with a nonpolar mobile
phase. It is commonly utilized for the separation of
nonpolar compounds.
2. Reverse Phase HPLC (RP-HPLC): This approach uses
a nonpolar stationary phase and a polar mobile phase.
RP-HPLC is highly effective for separating polar
substances such as pharmaceuticals and biomolecules.
3. Ion-Exchange Chromatography: This technique
separates substances based on their ionic properties. It is
further divided into:
o Cation-Exchange Chromatography: Targets

positively charged ions.

o Anion-Exchange Chromatography: Focuses on

negatively charged ions.

4. Size-Exclusion Chromatography (SEC): Also known
as Gel Filtration Chromatography, this method relies on
molecular size for separation. Larger molecules elute
first as they are unable to penetrate the porous stationary
phase.
5. Affinity Chromatography: This technique leverages the
specific interaction between a ligand attached to the
stationary phase and the target molecule, making it ideal
for purifying biomolecules such as proteins.
6. Chiral Chromatography: Designed for the separation
of enantiomers (mirror-image isomers), this mode
 utilizes chiral stationary phases to distinguish compounds
with differing optical isomerism.
7. Hydrophilic Interaction Chromatography (HILIC):
HILIC integrates features of both normal and reverse-
phase chromatography and is frequently employed to
separate polar compounds.
8. Hydrophobic Interaction Chromatography (HIC):
This method sorts biomolecules based on their
hydrophobic characteristics, making it effective for
specialized biochemical analyses.

PRINCIPLE
The separation process in High-Performance Liquid
Chromatography (HPLC) occurs within a separation column,
involving two primary phases: the stationary phase and the
mobile phase. The stationary phase consists of granular
material with fine porous particles packed within the column.
In contrast, the mobile phase is a solvent or a combination of
solvents that is pumped at high pressure through the column.
Samples are introduced into the mobile phase flow via a valve
equipped with a sample loop—a small stainless steel tube or
capillary. Using a syringe, the sample is injected into the
system, where it is carried by the mobile phase towards the
separation column.
Within the column, the components of the sample migrate at
varying speeds due to differences in their interactions with the
stationary phase. These differing retention levels result in the
separation of individual compounds. Once the compounds exit
the column, they are detected by an appropriate detector,
which converts the signals into data that can be processed by
HPLC software on a computer.
At the end of this process, a chromatogram is generated by the
software. This chromatogram serves as a graphical
representation, enabling the identification and quantification
of the various substances present in the sample.

INSTRUMENTATION
Main components of HPLC are as following:
1. Solvent reservoirs
2. Degassing system
3. Filters 15
4. Mixing chambers
5. Pumps
6. Sample injection system
7. Pre/Guard column
8. Analytical column
9. Detectors
10. Recorders
1. Solvent Reservoirs: These containers, made of stainless
steel or glass, are designed to hold up to one litre (1 L) of
the mobile phase. The solvents used can be pure organic,
inorganic, or buffer solutions, and the reservoirs must be
resistant to corrosion. The number of reservoirs depends
 on the types and quantities of solvents used in the HPLC
system.
a. Isocratic Elution: In this method, a single,
unchanging mobile phase is used throughout the
process. While simple, it is less effective for
separating complex mixtures and often requires
more time, resulting in lower resolution.
b. Gradient Elution: This technique involves altering
the composition of the mobile phase either
incrementally or continuously during the analysis.
By using a gradient controller, a specific proportion
of the mobile phase can be delivered to the column.
Gradient elution is more effective for separating
complex mixtures, offering better resolution in a
shorter time and producing higher-quality
chromatograms compared to isocratic elution.
2. Degassing System:
Degassing involves removing unwanted gases and air
bubbles dissolved in the mobile phase. The presence of
air bubbles in the mobile phase of an HPLC system can
lead to significant challenges. These issues arise from
dissolved gases, such as oxygen and nitrogen, which can
be transported into the column and interfere with the
operation of the pump and detector.
Air bubbles, due to their compressibility, can reduce the
volume of the mobile phase delivered by the pump, which in
turn impacts the reproducibility of results. Additionally,
variations in flow caused by these bubbles can generate noise
in the detector's readings, compromising the accuracy of the
analysis. In some cases, large air bubbles can completely
disrupt the functioning of the pump, leading to a halt in
operations.
 3. Filters:
In HPLC, the filtration of the mobile phase is essential
before transferring the solvent to the reservoir. This
process typically involves using micro bore filters with
pore sizes ranging from 0.45 μm to 0.20 μm to achieve
effective purification. The filtration step removes UV-
absorbing impurities, particulate matter, and microbial
growth that could increase the baseline noise during
analysis.
For this purpose, membrane filters with diameters between 25
mm and 90 mm and pore sizes from 0.2 μm to 0.45 μm are
commonly used. One example of such filters is wwPTFE
(water-wet table polytetrafluoroethylene), which is
specifically designed for efficient filtration in HPLC systems.
4. Mixing Chamber:
In HPLC systems, solvents often have varying viscosities
and miscibility levels. To ensure they are thoroughly
blended before reaching the column, a mixing chamber is
employed. This chamber is filled with inert fibres that
generate the necessary turbulence for effective mixing,
enabling a uniform and consistent solvent composition to
be delivered to the column.

5. Pumping System:
The pumping system in HPLC is responsible for
propelling the liquid mobile phase through the
chromatographic column. The three primary types of
pumps utilized in HPLC are:
  Reciprocating Pumps: These pumps operate by using a
reciprocating piston to deliver the mobile phase in a
steady flow.
 Displacement Pumps (Syringe Type): These pumps are
designed to displace liquid in a controlled manner,
providing consistent flow rates.
 Pneumatic Pumps (Constant Pressure Pumps): These
pumps utilize gas pressure to maintain a constant flow of
the mobile phase.
1. Reciprocating Pump:
This pump consists of a small chamber where the mobile
phase is moved back and forth using a motor-driven
piston. Alternatively, pressure can be transmitted through
a diaphragm that is hydraulically pumped by the
reciprocating piston.
Advantages:
 Can generate high pressures, up to 40,000 poise.
 Provides consistent flow rates that are unaffected by
column back pressure or solvent viscosity.
 Offers unlimited volume capacity.
 Supports gradient elution.
Disadvantage:
 Produces a pulsed flow, requiring the use of a pulse
damper for smooth operation.

2. Displacement Pump:
This pump operates on the principle of positive
displacement. It features a large syringe-like chamber
 with a capacity of 250–500 ml, equipped with a plunger
activated by a motor-driven screw.
Advantages:
 Delivers a pulse-free flow.
 Can generate high pressures ranging from 200 to 478
atm.
 Flow rate is unaffected by column back pressure and
solvent viscosity.
 Easy to operate, allowing solvent flushing with a simple
push-button plunger action.
Disadvantages:
 Has limited solvent capacity.
 Not suitable for gradient elution.

3. Pneumatic Pump (Constant Pressure Pump):

Widely used in HPLC, this pump operates by
pressurizing a collapsible container of mobile phase
placed inside a vessel using compressed gas.
Advantages:
 Delivers a pulse-free flow.
 Capable of generating high pressure.
Disadvantages:
 Offers limited volume capacity.
 Flow rate and pressure output are influenced by solvent
viscosity and column back pressure.
  Gradient elution cannot be performed with this type of
pump.
6. Sample Injection System:
In HPLC, it is crucial to introduce a minimum amount of
sample as a single, concentrated slug to avoid
overlapping, which can lead to band broadening. To
ensure optimal results, the sample is typically injected
directly at the column head, minimizing any disturbances
to the column material.
The sample injection process can be carried out using two
primary methods:
 Syringe Injection: The sample is manually introduced
into the system using a syringe.
 Sampling Valve or Loop Method: This approach uses a
valve equipped with a loop to precisely inject the sample
into the mobile phase flow.
7. Column Selection:
The choice of a suitable column in HPLC involves
considering various factors, such as the type of
adsorbent, particle size, surface area, geometry, and
column packing procedures. The selection is also
influenced by the following criteria:
a. Attainable Resolution: High resolution is achieved by
using the smallest possible sample size and allowing for
a longer analysis time. The stationary phase should
consist of particles with the smallest size to ensure a
sufficient number of theoretical plates.
b. Speed of Analysis: A shorter column should be used to
minimize the time required for analysis.
 c. Load Capacity: To handle an adequate sample load, the
column’s cross-sectional area must be increased to
accommodate a larger stationary phase volume.

Guard Column or Pre-Column:

Guard columns are short (2–10 cm) and do not interfere with
the chromatographic separation process. Their stationary
phase is similar to that of the main column but contains larger
particles. These columns are cost-effective, easy to clean, and
can be conveniently replaced. They are known as guard
columns because they prolong the life of the main analytical
column by removing particulate matter and contaminants
from the solvent. Additionally, they pre-saturate the mobile
phase with the stationary phase, thereby minimizing losses
from the main column.

Main Columns:
In HPLC, main columns are typically constructed from
stainless steel or durable heavy glass to withstand the high
pressure of the system. The internal surface of the column
must be smooth and free from longitudinal striations, as these
markings can disrupt the solvent’s flow and significantly
reduce the efficiency of the chromatographic process.
8. Detectors:
Detectors are electronic instruments responsible for
processing data and providing results in the form of
signals, such as peaks or bands. They represent one of
the most critical components in an HPLC system. The
primary role of a detector is to identify and track the
presence of individual components as they exit the
column along with the mobile phase, ensuring accurate
monitoring and analysis.
Types of detectors
A) Specific HPLC detectors
1. Mass spectrometric HPLC detectors
2. UV/ vis detectors
3, Photo diode array detectors: 21
4. Fluorescence HPLC detectors
B) Bulk property HPLC detectors

1. Electrical conductivity HPLC detectors

2. Refractive index HPLC detectors
3. Electrochemical HPLC detectors
4. Light scattering HPLC detectors
 The advantages of HLPC
 HPLC provides a fast, automated, and highly accurate
technique for identifying specific chemical components
within a sample.
 High-Performance Liquid Chromatography ensures
precise and efficient quantitative analysis in various
applications.
 Certain methods allow for the implementation of a
gradient solvent system to improve results.
 This technique is highly reproducible, ensuring
consistent outcomes.
 HPLC systems can be integrated with mass spectrometry
for advanced analytical capabilities.
 Compared to other chromatographic methods, HPLC is
significantly faster, more efficient, and delivers superior
resolution.
 It optimizes every aspect of analysis to enhance overall
productivity.
The disadvantages of HPLC
 HPLC can be costly, as it requires numerous expensive
organic solvents, a consistent power supply, and routine
maintenance to ensure proper functioning.
 One limitation of HPLC is the absence of a universal
detector. For example, the commonly used UV-Vis
detector can only identify chromophore compounds.
 The efficiency of separation in High-Performance Liquid
Chromatography is comparatively lower than that
achieved in Gas Chromatography.
 Operating HPLC systems can pose challenges for
beginners due to its complex processes and technical
nuances.
  The reliability of HPLC pump operation is highly
dependent on maintaining the cleanliness of the sample,
mobile phase, and the overall system's correct usage.

APPLICATIONS OF HPLC
High-Performance Liquid Chromatography (HPLC) is a
versatile and essential analytical technique used for both
qualitative and quantitative analysis of pharmaceutical
compounds. It is widely applied in the identification,
characterization, quantification, and separation of biological
substances. Additionally, HPLC plays a crucial role in the
isolation and purification of chemical compounds.
Its extensive applicability, rapid analysis, and high sensitivity
have made HPLC one of the most widely used
chromatographic techniques. Nearly all types of biological
molecules have been successfully purified using Reverse
Phase Partition HPLC, which is particularly effective for
separating polar compounds such as drugs, their metabolites,
peptides, vitamins, polyphenols, and steroids.
Before the introduction of this chromatography method,
isolating polar compounds was challenging and often required
pre-derivatization to convert them into less polar forms for
better separation. The technique is extensively employed in
clinical and pharmaceutical research, as biological fluids like
serum and urine can be directly injected into the column,
ideally with the aid of a guard column to enhance analytical
accuracy.
Owing to its broad range of applications, HPLC remains an
indispensable tool in various scientific and industrial fields.
The following sections explore its diverse applications in
greater detail.
1. General Applications of HPLC
High-Performance Liquid Chromatography (HPLC) continues
to be widely utilized in routine analytical processes. With
ongoing advancements in support materials and the
development of more sensitive detectors, HPLC is expected to
become increasingly indispensable. It has already replaced
numerous gas chromatographic methods due to its superior
speed, resolution, and sensitivity. Additionally, the reusability
of HPLC columns adds to its efficiency.
This technique is particularly beneficial for separating
compounds with high molecular weights. Unlike gas
chromatography, HPLC can effectively analyse substances
that have low vapor pressure or are prone to thermal
degradation at elevated temperatures.
HPLC has been successfully employed in the analysis of
various natural and manufactured substances, including:
 Natural compounds such as nucleic acids, urine, serum,
carbohydrates, lipids, amino acids, and bile acids.
 Industrial and pharmaceutical products such as drugs,
pesticides, herbicides, surfactants, and antioxidants.
Furthermore, Reverse Phase HPLC (RP-HPLC) is particularly
useful for improving the separation of highly polar
compounds such as amino acids, organic acids, and
catecholamine, which are challenging to resolve using
conventional reverse-phase chromatography.
 2. Applications of HPLC in
Pharmaceuticals
High-Performance Liquid Chromatography (HPLC) is
extensively utilized in the pharmaceutical industry and is one
of the most reliable analytical techniques for drug
development and manufacturing. It plays a vital role in
conducting dissolution studies to assess the release profiles
of pharmaceutical products across various dosage forms.
HPLC is fundamental for quality control in pharmaceuticals,
ensuring the identification, purification, and quantification of
active ingredients extracted from various sources.
Additionally, it is widely employed in determining the shelf
life of pharmaceutical compounds, helping monitor drug
stability over time. Routine analysis of drug formulations—
both in bulk and in final dosage forms—is a key function of
HPLC in pharmaceutical industries.
Many pharmaceutical lead compounds originate from plants,
animals, and microorganisms. HPLC facilitates the separation,
purification, and characterization of these bioactive
molecules. Furthermore, it is instrumental in
differentiating enantiomers (mirror-image
isomers) by utilizing a chiral stationary phase,
ensuring precision in pharmaceutical research and
development.
 3. HPLC in Clinical Diagnosis and
Healthcare
High-Performance Liquid Chromatography (HPLC) plays a
crucial role in the medical field, particularly in the diagnosis
of various disorders. It is widely used to detect metabolic
abnormalities and conditions related to endocrine and
exocrine gland secretions. Additionally, HPLC aids in
identifying alterations in body fluids, allowing for accurate
medical assessments.
HPLC enables the precise estimation of metabolites such as
purines and pyrimidines, along with other essential biological
compounds, from plasma, cerebrospinal fluid, and urine
samples. This technique is also valuable for determining
corticoid levels in plasma, which is essential for diagnosing
adrenal gland disorders, as these glands regulate endocrine
hormone secretion.
Its reliability and sensitivity make HPLC indispensable in
clinical and pharmaceutical research, contributing
significantly to modern healthcare advancements.

4. HPLC in Food and Flavour Analysis

High-Performance Liquid Chromatography (HPLC) plays a
crucial role in food and beverage testing by enabling rapid
screening and analysis of various components in non-
alcoholic drinks. It is widely used for assessing the quality
of food products, soft drinks, and water, ensuring
compliance with industry standards.
One significant application of HPLC is the detection of
polycyclic compounds in vegetables, which helps monitor
their safety and nutritional quality. Additionally, HPLC is
employed to analyse the sugar composition in fruit juices,
ensuring consistency and quality in production.
Many food and flavoured beverages contain preservatives
that help maintain their freshness and shelf life. HPLC
facilitates the identification and quantification of these
additives across various food products. Another advanced
application is multiresidue analysis of pesticides in food
samples, which is effectively carried out using LC-MS
(Liquid Chromatography-Mass Spectrometry), ensuring
food safety and regulatory compliance.

5. HPLC in Forensic Science Applications

High-Performance Liquid Chromatography (HPLC) plays a
significant role in forensic investigations, aiding in the
analysis of biological and chemical samples submitted for
examination. The results of such analyses are crucial in
assisting legal authorities with judicial proceedings.
One of the primary forensic applications of HPLC is the
quantitative detection of drugs in biological samples such as
blood, urine, sweat, and hair. Additionally, it is widely used to
identify anabolic steroids, which may be present in these
bodily fluids.
Another important forensic use of HPLC is in textile dye
analysis, helping investigators trace specific dyes back to
their sources. Moreover, HPLC is instrumental in detecting
illegal substances, including cocaine, morphine, and charas,
ensuring regulatory compliance and law enforcement.
Advanced techniques, such as LC-MS (Liquid
Chromatography-Mass Spectrometry), are employed for
analysing specialized drug classes like benzodiazepines,
particularly in oral fluid samples, offering high precision in
forensic toxicology.

6. Environmental Applications of HPLC

With rapid urbanization, air pollution has become a major
global concern, posing severe risks to human health. Toxic
pollutants released from vehicles and industrial activities have
been linked to serious diseases, including cancer.
High-Performance Liquid Chromatography (HPLC) serves as
a powerful tool in monitoring and analysing air pollutants,
helping researchers and environmental experts develop
preventive strategies to mitigate pollution. This technique
enables the rapid separation and identification of carbonyl
compounds, which are key contributors to atmospheric
contamination.
HPLC is also utilized in the analysis of sediment samples,
particularly for detecting diphenhydramine, a substance
known for its environmental toxicity. Additionally, it plays a
crucial role in quantifying phenols and related compounds
in water sources, ensuring the assessment of water quality
and safety.
By providing precise and reliable data, HPLC supports efforts
to control pollution, safeguard ecosystems, and promote
sustainable environmental practices.

7. Impurity Profiling of Pharmaceuticals

Using HPLC
Impurities in pharmaceutical products can originate from
various sources, potentially compromising the quality and
purity of drug molecules. Such contamination may lead to
regulatory concerns, forcing the withdrawal of products from
the market. To address this issue, High-Performance Liquid
Chromatography (HPLC) has become an essential tool in
impurity profiling studies, ensuring drug safety and
compliance with quality standards.
HPLC plays a critical role in identifying and characterizing
unknown impurities at different stages of the manufacturing
process. By systematically analysing each step, this technique
helps detect contaminants that may affect drug stability and
efficacy.
For deeper insights into impurity structures, Liquid
Chromatography-Mass Spectrometry (LC-MS) is
employed. This advanced technique allows for structural
elucidation, facilitating precise identification of impurities to
ensure pharmaceutical formulations meet safety and
regulatory requirements.

8. Additional Applications of HPLC

 Inorganic Chemistry: HPLC is used for estimating
various anions (F⁻, Cl⁻, Br⁻, HPO₄²⁻, HPO₃²⁻, NO₂⁻,
NO₃⁻, SO₄²⁻) and cations (NH₄⁺, Na⁺, Cu²⁺, Fe³⁺).
 Separation of Non-Ionic Inorganic Solutes: HPLC
effectively isolates non-ionic inorganic substances for
further analysis.
 Detection of Poisons and Intoxicants: The technique
plays a vital role in forensic investigations by identifying
harmful substances.
 Separation of Alkaloids: HPLC is instrumental in
analysing Ergot alkaloids and phenyl alkyl amines
using adsorption or ion-exchange chromatography.
 Forensic Identification: It assists in lipstick smear
analysis, enabling forensic comparisons for crime
investigations.
 Food and Lipid Analysis: The method is employed in
lipid separation from food products and ergot alkaloid
purification.
 Steroidal Compound Analysis: HPLC enables precise
separation of steroids and steroidal hormones for
medical and research applications.
 Hormone Monitoring: It aids in detecting oestrogen
levels in female urine, which is significant for
pregnancy monitoring.
 Nucleic Acid Separation: The technique is crucial for
isolating nucleic acids in physiological fluids, including
their metabolites.
 Drug and Narcotic Analysis: HPLC is widely used in
separating opiates, cocaine, hallucinogens, and
addictive drugs, making it a valuable tool in toxicology
and drug
 Separation of biomolecules such as amino acids,
proteins, carbohydrates, and various essential compounds
found in coal and oil products.
 Analysis of nutrients in food including amino acids,
proteins, preservatives, antioxidants, and vitamins in
human diets.
 Detection of narcotics and illicit substances like
opiates, cocaine, and hallucinogens for forensic and
pharmaceutical investigations.
 Analysis of pharmaceutical compounds such as
barbiturates and benzodiazepine derivatives, ensuring
quality control and safety.
 Isolation of plant-based compounds such as saponins,
sapogenins, and biogenic amines used in medicinal
research.
 Screening of psychotropic substances present in body
fluids, including antidepressants and phenothiazine
derivatives.
 Extraction and characterization of natural products
including active components from digitalis, cinchona,
liquorice, and ergot extracts.
 Drug stability testing to assess degradation products
and ensure pharmaceutical formulations remain effective
over time.
 Clinical diagnostics for identifying diseases and
disorders by analysing biological fluids and biomarkers.
 Scientific research applications facilitating the
discovery of new molecules and the advancement of
analytical methodologies.
 Food industry applications supporting quality control
of food products, preservatives, and additives for safety
assessments.