Assignment on

High Performance Liquid Chromatography (HPLC)

Course Title: Advanced Pharmaceutical Analysis

Course Code: PHARM 6201

Submitted to:
Bidduth Kumar Sarkar
Lecturer
Department of Pharmacy
Comilla University

Submitted by:
Md. Akil Mahmud
ID: 22015020
Session: 2019-20
Department of Pharmacy
Comilla University

Date of Submission: 13/10/22

Table of Contents
Chromatography ........................................................................................................................... 3
Types of Chromatography ........................................................................................................... 5
1. Affinity chromatography ...................................................................................................... 5
2. Anion exchange chromatography ........................................................................................ 6
3. Cation exchange chromatography ....................................................................................... 8
4. Column chromatography ..................................................................................................... 9
5. Gas chromatography........................................................................................................... 10
6. High-Performance Liquid Chromatography (HPLC) ..................................................... 12
7. Ion exchange chromatography ........................................................................................... 14
10. Thin-layer chromatography (TLC) ................................................................................. 18
Basics of HPLC ........................................................................................................................... 19
Basic Principles of HPLC ........................................................................................................... 20
How HPLC works ....................................................................................................................... 24
Factors Affecting HPLC Separations........................................................................................ 25
Isocratic versus Gradient Separations ...................................................................................... 26
Applications of High-Performance Liquid Chromatography (HPLC).................................. 27
Advantages of High-Performance Liquid Chromatography (HPLC) ................................... 27
 High Performance Liquid Chromatography (HPLC)

Chromatography

Chromatography is a technique used to separate the different chemical compounds of a mixed

sample. It uses two phases: a nonmoving "stationary" phase and a mobile phase which moves
through the stationary phase, carrying along the chemical compounds. As per dictionary definition
Chromatography is a method for separating the constituents of a solution by exploiting the
different properties of different molecules.

The technique employs a mobile phase to transport the solution to be analyzed through the
stationary phase which absorbs or impedes different components of the solution to different
degrees and thus causes their separation as different layers. It is an invaluable tool in the hands of
analytical scientists for the separation and quantification of components in a mixture of organic
compounds Take a look at this setup below.
The paper acts as the stationary phase, and a solvent is used as the mobile phase. This is the basic
principle behind all chromatographic methods,chromatography, technique for separating the
components, or solutes, of a mixture on the basis of the relative amounts of each solute distributed
between a moving fluid stream, called the mobile phase, and a contiguous stationary phase. The
mobile phase may be either a liquid or a gas, while the stationary phase is either a solid or a liquid.
If, for a particular solute, the distribution favours the moving fluid, the molecules will spend most
of their time migrating with the stream and will be transported away from other species whose
molecules are retained longer by the stationary phase.

For a given species, the ratio of the times spent in the moving and stationary regions is equal to
the ratio of its concentrations in these regions, known as the partition coefficient.(The term
adsorption isotherm is often used when a solid phase is involved). A mixture of solutes is
introduced into the system in a confined region or narrow zone , whereupon the different species
are transported at different rates in the direction of fluid flow.

The driving force for solute migration is the moving fluid, and the resistive force is the solute
affinity for the stationary phase; the combination of these forces, as manipulated by the analyst,
produces the separation. In this case, the driving force is an electric field, which exerts different
forces on solutes of different ionic charge. The resistive force is the viscosity of the nonflowing
solvent. It is widely used in biochemical research for the separation and identification of chemical
compounds of biological origin. In the petroleum industry the technique is employed to analyze
complex mixtures of hydrocarbons.

It is capable of separating all the components of a multicomponent chemical mixture without

requiring an extensive foreknowledge of the identity, number, or relative amounts of the
substances present.
Types of Chromatography

1. Affinity chromatography
Affinity chromatography is a separation technique where the components of a mixture are
separated based on their affinity towards the stationary phase of the system.
Principle of Affinity chromatography

1. This chromatography technique is based on the principle that components of a mixture are
separated when the element having an affinity towards the stationary phase binds to the
stationary phase. In contrast, other components are eluted with the mobile phase.
2. The substrate/ ligand is bound to the stationary phase so that the reactive sites for the binding
of components are exposed.
3. Now, the mixture is passed through the mobile phase where the components with binding
sites for the substrate bind to the substrate on the stationary phase while the rest of the
components are eluted out with the mobile phase.
4. The components attached to the stationary phase are then eluted by changing the pH, ionic
strength, or other conditions.

Figure: Affinity chromatography. Image Source: Creative Biostructure.

Steps of Affinity chromatography

1. The column is prepared by loading it with solid support like agarose or cellulose, onto which
the substrate/ ligand with the spacer arm, is attached.
2. The mobile phase containing the mixture is poured into the column at a constant rate.
 3. Once the process is complete, the ligand-molecule complex is eluted from the stationary
phase by changing the conditions that favor the separation of ligand and components of the
mixture.
Uses of Affinity chromatography

1. Affinity chromatography is used as a staple separation technique from enzymes and other
proteins.
2. This principle is also applied in the in vitro antigen-antibody reactions.
3. This technique is used for the separation of components as well as the removal of impurities
from a mixture.
4. Affinity chromatography can be used in the detection of mutation and nucleotide
polymorphisms in nucleic acids.

2. Anion exchange chromatography

Anion exchange chromatography is the separation technique for negatively charged molecules by
their interaction with the positively charged stationary phase in the form of ion-exchange resin.
Principle of Anion exchange chromatography

1. This technique is based on the principle of attraction of positively charged resin and the
negatively charged analyte. Here the exchange of positively charged ions takes place to
remove the negatively charged molecules.
2. The stationary phase is first coated with positive charges where the components of the
mixture with negative charges will bind.
3. An anion exchange resin with a higher affinity to the negatively charged components then
binds the components, displacing the positively charged resin.
4. The anion exchange resin-component complex then is removed by using different buffers.
 Figure: Anion exchange chromatography. Image

Steps of Anion exchange chromatography

1. A column packed with positively charged resin is taken as the stationary phase.
2. The mixture with the charged particles is then passed down the column where the negatively
charged molecules bind to the positively charged resins.
3. The anion exchange resin is then passed through the column where the negatively charged
molecules now bind to the anion exchange resin displacing the positively charged resin.
4. Now an appropriate buffer is applied to the column to separate the complex of anion
exchange resins and the charged molecules.
Uses of Anion exchange chromatography

1. Anion exchange chromatography is used to separate proteins and amino acids from their
mixtures.
2. Negatively charged nucleic acids can be separated, which helps in further analysis of the
nucleic acids.
3. This method can also be used for water purification where the anions are exchanged for
hydroxyl ions.
4. Anion exchange resins can be used for the separation of metals as they usually have
negatively charged complexes that are bound to the anion exchangers.
3. Cation exchange chromatography

Anion exchange chromatography is the separation technique for positively charged molecules by
their interaction with negatively charged stationary phase in the form of ion-exchange resin.
Principle of Cation exchange chromatography

1. This technique is based on the principle of attraction of negatively charged resin and the
positively charged analyte. Here the exchange of negatively charged ions takes place to
remove the positively charged molecules.
2. The stationary phase is first coated with negative charges where the components of the
mixture with positive charges will bind.
3. A cation exchange resin with a higher affinity to the positively charged components then
binds the components, displacing the negatively charged resin.
4. The cation exchange resin-component complex then is removed by using different buffers.
Steps of Cation exchange chromatography

1. A column packed with negatively charged resin is taken as the stationary phase.
2. The mixture with the charged particles is then passed down the column where the positively
charged molecules bind to the negatively charged resins.
3. The cation exchange resin is then passed through the column where the positively charged
molecules now bind to the cation exchange resin displacing the negatively charged resin.
4. Now an appropriate buffer is applied to the column to separate the complex of cation
exchange resins and the charged molecules.
Uses of Cation exchange chromatography

1. Cation exchange chromatography is used for the analysis of the products obtained after the
hydrolysis of nucleic acids.
2. This can also be used for the separation of metals where the metal ions themselves bind to
the negatively charged resins to remove the negatively charged complexes.
3. Cation exchange chromatography helps in purification of water by exchanging the positively
charged ion by the hydrogen ions.
4. It is also used to analyze the rocks and other inorganic molecules.
4. Column chromatography

Column chromatography is the separation technique where the components in a mixture are
separated on the basis of their differential adsorption with the stationary phase, resulting in them
moving at different speeds when passed through a column.
It is a solid-liquid chromatography technique in which the stationary phase is a solid & mobile
phase is a liquid or gas.
Principle of Column chromatography

1. This technique is based on the principle of differential adsorption where different molecules
in a mixture have different affinities with the absorbent present on the stationary phase.
2. The molecules having higher affinity remain adsorbed for a longer time decreasing their
speed of movement through the column.
3. However, the molecules with lower affinity move with a faster movement, thus allowing the
molecules to be separated in different fractions.
4. Here, the stationary phase in the column chromatography also termed the absorbent, is a solid
(mostly silica) and the mobile phase is a liquid that allows the molecules to move through
the column smoothly.

Figure: Column chromatography. Image Source: PrepGenie.

Steps of Column chromatography

1. The column is prepared by taking a glass tube that is dried and coated with a thin, uniform
layer of stationary phase (cellulose, silica).
2. Then the sample is prepared by adding the mixture to the mobile phase. The sample is
introduced into the column from the top and is allowed to pass the sample under the influence
of gravity.
 3. The molecules bound to the column are separated by elution technique where either solution
of the same polarity is used (isocratic technique), or different samples with different
polarities are used (gradient technique).
4. The separated molecules can further be analyzed for various purposes.
Uses of Column chromatography

1. Column chromatography is routinely used for the separation of impurities and purification
of various biological mixtures.
2. This technique can also be used for the isolation of active molecules and metabolites from
various samples.
3. Column chromatography is increasingly used for the detection of drugs in crude extracts.

5. Gas chromatography

Gas chromatography is a separation technique in which the molecules are separated on the basis
of their retention time depending on the affinity of the molecules to the stationary phase.
The sample is either liquid or gas that is vaporized in the injection point.
Principle of Gas chromatography

1. Gas chromatography is based on the principle that components having a higher affinity to
the stationary phase have a higher retention time as they take a longer time to come out of
the column.
2. However, the components having a higher affinity to the stationary phase have less retention
time as they move along with the mobile phase.
3. The mobile phase is a gas, mostly helium, that carries the sample through the column.
4. The sample once injected in converted into the vapor stage is then passed through a detector
to determine the retention time.
5. The components are collected separately as they come out of the stationary phase at different
times.
 Figure: Gas chromatography. Image Source: Bitesize Bio.
Steps of Gas chromatography

1. The sample is injected into the column where it is vaporized into a gaseous state. The
vapourised component than mixes with the mobile phase to be carried through the rest of the
column.
2. The column is set with the stationary phase where the molecules are separated on the basis
of their affinity to the stationary phase.
3. The components of the mixture reach the detector at different times due to differences in the
time they are retained in the column.

Uses of Gas chromatography

1. This technique is used to calculate the concentration of different chemicals in various

samples.
2. This is used in the analysis of air pollutants, oil spills, and other samples.
3. Gas chromatography can also be used in forensic science to identify and quantify various
biological samples found in the crime scene.
4. Molecules is appropriate enough to enter the pores, they remain in the pores partly or wholly.
5. However, molecules with a larger size are retained from entering the pores, causing them to
be moved with the mobile phase, out of the column.
6. If the mobile phase used in an aqueous solution, the process is termed gel filtration
chromatography.
7. If the mobile phase used is an organic solvent, it is termed as gel permeation chromatography.
 Figure: Gel-filtration chromatography. Image Source: MBL Life Science.
Steps

1. The column is filled with semi-permeable, porous polymer gel beads with a well-defined
range of pore sizes.
2. The sample, mixed with the mobile phase, is then injected into the column from the top of
the column.
3. The molecules bound to the column are separated by elution solution where either solution
of the same polarity is used (isocratic technique), or different samples with different
polarities are used (gradient technique).
4. Elution conditions (pH, essential ions, cofactors, protease inhibitors, etc.) can be selected,
which will complement the requirements of the molecule of interest.

6. High-Performance Liquid Chromatography (HPLC)

High-performance liquid chromatography is a modified form of column chromatography where

the components of a mixture are separated on the basis of their affinity with the stationary phase.
Principle of HPLC

1. This technique is based on the principle of differential adsorption where different molecules
in a mixture have a varying degree of interactions with the absorbent present on the stationary
phase.
2. The molecules having higher affinity remain adsorbed for a longer time decreasing their
speed of movement through the column.
3. However, the molecules with lower affinity move with a faster movement, thus allowing the
molecules to be separated in different fractions.
 4. This process is slightly different from the column chromatography as in this case; the solvent
is forced under high pressures of up to 400 atmospheres instead of allowing it to drip down
under gravity.
5.

Figure: High-performance liquid chromatography (HPLC). Image Source: Toppr.

Steps of HPLC

1. The column is prepared by taking a glass tube that is dried and coated with a thin, uniform
layer of stationary phase (cellulose, silica).
2. Then the sample is prepared by adding the mixture to the mobile phase. The sample is
introduced into the column from the top, and a high-pressure pump is used to pass the sample
at a constant rate.
3. The mobile phase then moves down to a detector that detects molecules at a certain
absorbance wavelength.
4. The separated molecules can further be analyzed for various purposes.
Uses of HPLC
1. High-performance liquid chromatography is used in the analysis of pollutants present in
environmental samples.
2. It is performed to maintain product purity and quality control of various industrial
productions.
3. This technique can also be used to separate different biological molecules like proteins and
nucleic acids.
4. The increased speed of this technique makes the process faster and more effective.
7. Ion exchange chromatography

Ion exchange chromatography is the separation technique for charged molecules by their
interaction with the oppositely charged stationary phase in the form of ion-exchange resin.
Principle of Ion exchange chromatography

1. This technique is based on the principle of attraction of charged resin and the oppositely
charged analyte. Here the exchange of negatively/ positively charged ions takes place to
remove the charged molecules.
2. The stationary phase is first coated with particular charges where the components of the
mixture with opposite charges will bind.
3. A cation or anion exchange resin with a higher affinity to the charged components then binds
the components, displacing the oppositely charged resin.
4. The cation or anion exchange resin-component complex then is removed by using different
buffers.

Figure: Ion exchange chromatography.

Steps of Ion exchange chromatography

1. A column packed with charged resin that can either be positively charged or negatively
charged is taken as the stationary phase.
2. The mixture with the charged particles is then passed down the column where the charged
molecules bind to the oppositely charged resins.
 3. If a cation exchange resin is used, the positively charged molecules now bind to the cation
exchange resin displacing the negatively charged resin.
4. Similarly, if an anion exchange resin is used, the negatively charged molecules bind to the
anion exchange resin displacing the positively charged resin.
5. Now an appropriate buffer is applied to the column to separate the complex of charged
exchange resins and the charged molecules.
Uses of Ion exchange chromatography

1. Ion exchange chromatography is used in the purification of water where the positively
charged ions are replaced by hydrogen ions, and the negatively charged ions are replaced by
hydroxyl ions.
2. This method also works as an effective method for the analysis of the products formed after
hydrolysis of nucleic acids.
3. The separation of metals and other inorganic compounds is also facilitated by the ion-
exchange chromatography.

8. Liquid chromatography

Liquid chromatography is a separation technique where the mobile phase used is liquid, and the
separation can take place either in a column or a plain surface.

Principle of Liquid chromatography

1. The process of liquid chromatography is based on the principle for the affinity of the
molecules to the mobile phase.
2. If the components to be separated have a higher affinity to the mobile phase, the molecules
move along with the mobile phase and come out of the column faster.
3. However, if the components have a lower degree of interaction with the mobile phase, the
molecules move slowly and thus come out of the column later.
4. Thus, if two molecules in a mixture have different polarities and the mobile phase is of a
distinct polarity, the two molecules will move at different speeds through the stationary
phase.
 Figure: Liquid chromatography. Image Source: Vânia Margaret Flosi Paschoalin
(Researchgate).

Steps of Liquid chromatography

1. The column or paper is prepared where the stationary phase (cellulose or silica) is applied on
the solid support.
2. The sample is added to the liquid mobile phase, which is then injected into the
chromatographic system.
3. The mobile phase moves through the stationary phase before coming out of the column or
the edge of the paper.
4. An elution solution is applied to the system to separate the molecules from the stationary
phase.
Uses of Liquid chromatography

1. Liquid chromatography is an effective method for the separation of a colored solution as they
form two separate bands after separation.
2. This method can also be used over other techniques as it is quite simple and less expensive.
3. It can be used for the separation of solid molecules that are insoluble in water.

Figure: Paper chromatography. Image Source: Enyoh Christian Ebere (Researchgate).

9. Reverse-phase chromatography

Reverse-phase chromatography is a liquid chromatography technique where the separation of

molecules is achieved through hydrophobic interaction between the liquid mobile phase and the
stationary phase.
Principle of Reverse-phase chromatography

1. The principle of reverse phase chromatography is based on the interaction between two
molecules with hydrophobic groups.
2. Here, the stationary phase is solid support applied with both hydrophobic and hydrophilic
groups.
3. The solvent molecules containing hydrophobic regions interact with the hydrophobic groups,
thus separating them from the molecules with hydrophilic groups.
4. The interaction is then reversed by applying an elution solution with decreasing salt gradient,
which causes the molecules with hydrophobic groups to be separated from the stationary
phase.

Figure: Steps of a reversed-phase chromatography separation. Image Source: Annette C

Moser (Researchgate).

Steps of Reverse-phase chromatography

1. The column is prepared with a glass tube applied with solid support like silica gel, upon
which hydrophobic groups like phenyl, octyl butyl, are attached.
2. The sample is prepared by adding the mixture to the mobile phase of organic and inorganic
solvents.
 3. The sample is then injected into the column from the top of the column.
4. The molecules with hydrophobic groups form an interaction with the hydrophobic groups of
the stationary phase. In contrast, the molecules without such groups move out of the column
with the mobile phase.
5. Then a particular elution solution with decreasing salt gradient is then passed into the column
that removes the bound molecules from the stationary phase.
Uses of Reverse-phase chromatography

1. Reverse chromatography, in combination with high-performance liquid chromatography, is

increasingly used for the separation of biomolecules.
2. This is also used in the study of the analysis of drugs, metabolites, and active molecules.
3. It can also be used to remove impurities from various environmental samples.

10. Thin-layer chromatography (TLC)

Thin-layer chromatography is a separation technique where the stationary phase is applied as a

thin layer on a solid support plate with a liquid mobile phase.
Principle of Thin-layer chromatography (TLC)

1. This chromatography technique is based on the principle that components of a mixture are
separated when the component having an affinity towards the stationary phase binds to the
stationary phase. In contrast, other components are eluted with the mobile phase.
2. The substrate/ ligand is bound to the stationary phase so that the reactive sites for the binding
of components are exposed.
3. Now, the mixture is passed through the mobile phase where the components with binding
sites for the substrate bind to the substrate on the stationary phase while the rest of the
components are eluted out with the mobile phase.
4. After separation, the molecules are seen as spots at a different location throughout the
stationary phase.
5. The detection of molecules is performed by various techniques.
 Figure: Thin-layer chromatography (TLC). Image Source: MZ-Analysentechnik GmbH.

Steps of Thin-layer chromatography (TLC)

1. The stationary phase is uniformly applied on the solid support (glass, thin plate or aluminum
foil) and dried.
2. The sample is injected as spots on the stationary phase about 1 cm above the edge of the
plate.
3. The sample loaded plate is then carefully dipped into the mobile phase not more than the
height of 1 cm.
4. After the mobile phase reaches near the edge of the plate, the plate is taken out.
5. The retention factor is calculated as in paper chromatography, and the separated components
are detected by different techniques.
Uses of Thin-layer chromatography (TLC)

1. Thin-layer chromatography is routinely performed in laboratories to identify different

substances present in a mixture.
2. This technique help s in the analysis of fibers in forensics.
3. TLC also allows the assay of various pharmaceutical products.
4. It aids in the identification of medicinal plants and their composition.

Basics of HPLC

High-performance liquid chromatography (HPLC) is a broad analytical chemistry technique used

to separate compounds in a chemical mixture. These separations utilize the pressure-driven flow
of a mobile phase through a column packed with a stationary phase.
The mobile phase carries a liquid sample through the column to the detector, and compounds or
analytes separate due to varying degrees of interaction with the stationary phase.

A detector measures the analytes after elution from the column, and a chromatography data system
(CDS) translates the detected signal.

The translated data output of an HPLC analysis is called a chromatogram, where the x-axis is a
measure of time and the y-axis measures a specific signal generated by the detector.

Basic Principles of HPLC

Analyte – target compound(s) of interest for detection in an HPLC analysis

Mobile phase – phase in motion and composed of solvent or eluents flowing from injection to
detection

Stationary phase – the still phase where physical separation of analytes occurs

Flow rate – how fast the mobile phase flows with respect to time

Retention time – time between sample injection and the maximum peak signal of the analyte in a
chromatogram
 Efficiency – given as the number of theoretical plates, a key metric for quantifying performance
of a separation

Resolution – ability to distinguish between peaks and the primary concern for any separation

Selectivity – ability to separate two analytes

Void volume – all volume within a column not occupied by the stationary phase.

Limit of detection – the smallest quantity of an analyte which can be reliably detected

Limit of quantitation – the lower or upper quantity of an analyte which can be reliably quantified

 The purification takes place in a separation column between a stationary and a mobile phase.
 The stationary phase is a granular material with very small porous particles in a separation
column.
 The mobile phase, on the other hand, is a solvent or solvent mixture which is forced at high
pressure through the separation column.
 Via a valve with a connected sample loop, i.e. a small tube or a capillary made of stainless steel,
the sample is injected into the mobile phase flow from the pump to the separation column using
a syringe.
 Subsequently, the individual components of the sample migrate through the column at different
rates because they are retained to a varying degree by interactions with the stationary phase.
 After leaving the column, the individual substances are detected by a suitable detector and passed
on as a signal to the HPLC software on the computer.
 At the end of this operation/run, a chromatogram in the HPLC software on the computer is
obtained.
 The chromatogram allows the identification and quantification of the different substances.

Instrumentation of High-Performance Liquid Chromatography (HPLC)

The Pump

 The development of HPLC led to the development of the pump system.

 The pump is positioned in the most upper stream of the liquid chromatography system and
generates a flow of eluent from the solvent reservoir into the system.
 High-pressure generation is a “standard” requirement of pumps besides which, it should also
to be able to provide a consistent pressure at any condition and a controllable and
reproducible flow rate.
 Most pumps used in current LC systems generate the flow by back-and-forth motion of a
motor-driven piston (reciprocating pumps). Because of this piston motion, it produces
“pulses”.

Injector

 An injector is placed next to the pump.

 The simplest method is to use a syringe, and the sample is introduced to the flow of eluent.
 The most widely used injection method is based on sampling loops.
 The use of the autosampler (auto-injector) system is also widely used that allows repeated
injections in a set scheduled-timing.
Column

 The separation is performed inside the column.

 The recent columns are often prepared in a stainless steel housing, instead of glass columns.
 The packing material generally used is silica or polymer gels compared to calcium carbonate.
The eluent used for LC varies from acidic to basic solvents.
 Most column housing is made of stainless steel since stainless is tolerant towards a large
variety of solvents.

Detector

 Separation of analytes is performed inside the column, whereas a detector is used to observe
the obtained separation.
 The composition of the eluent is consistent when no analyte is present. While the presence
of analyte changes the composition of the eluent. What detector does is to measure these
differences.
 This difference is monitored as a form of an electronic signal. There are different types of
detectors available.

Recorder

 The change in eluent detected by a detector is in the form of an electronic signal, and thus it
is still not visible to our eyes.
 In older days, the pen (paper)-chart recorder was popularly used. Nowadays, a computer-
based data processor (integrator) is more common.

Degasser

The eluent used for LC analysis may contain gases such as oxygen that are non-visible to our eyes.
  When gas is present in the eluent, this is detected as noise and causes an unstable
baseline.
 Degasser uses special polymer membrane tubing to remove gases.
 The numerous very small pores on the surface of the polymer tube allow the air to go
through while preventing any liquid to go through the pore.

Column Heater

The LC separation is often largely influenced by the column temperature.

 In order to obtain repeatable results, it is important to keep consistent temperature conditions.
 Also for some analysis, such as sugar and organic acid, better resolutions can be obtained at
elevated temperatures (50 to 80°C).
 Thus columns are generally kept inside the column oven (column heater).

How HPLC works

An HPLC instrument has four major components: a pump to deliver the mobile phase, an
autosampler to inject the sample, a stationary phase column to separate the sample compounds,
and a detector to measure the compounds. Additional elements include connective capillaries and
tubing to allow the continuous flow of the mobile phase and sample through the system and a CDS
package to control the HPLC instrument, separation, detection, and result evaluation.
Every HPLC analysis includes the following steps:

1. Mobile phase begins to flow. The pump pushes the eluents or solvents through the system at
a specified flow rate.

2. Sample injection. Once injected into the mobile phase flow path, the sample travels with the
mobile phase from the injection point to the head of the column.

3. Compound separation. Physical separation of the compounds happens on the column

stationary phase. After elution from the column, the separated sample components travel to
the detector.

4. Analyte detection. Detection of target analytes based on an electrical signal generated by

specific properties.

5. Chromatogram generation. Translation of the detected analyte signal by the CDS into a
chromatogram of analyte signal versus time

Factors Affecting HPLC Separations

Many factors, including mobile phase composition, stationary phase chemistry, and temperature
influence HPLC separations. Successful separation only occurs if the analytes have differing
affinities for the stationary phase, so selecting the appropriate stationary phase for your compounds
is crucial. The main factors influencing the overall separation process are:

 Physiochemical properties of the analyte, such as size, charge, polarity, and volatility
 Physiochemical properties of the stationary phase, such as polarity, charge, and viscosity
 Physiochemical properties of the mobile phase used and interaction with the analyte and
stationary phases
Isocratic versus Gradient Separations

All HPLC separations are carried out in one of two modes, isocratic or gradient.

Isocratic methods separate by using a consistent eluent composition during analysis, like 100%
acetonitrile or a 50:50 mixture of acetonitrile to water.

On the other hand, gradient methods include a change in the mobile phase composition across a
separation. These methods often employ two solvents, called A and B. The run will begin with a
certain percentage of A to B, like 60% water to 40% acetonitrile, for instance, followed by a
percentage change throughout a separation.

Gradient separations usually provide superior performance over isocratic modes but are more
complex and require advanced pump hardware.
Applications of High-Performance Liquid Chromatography (HPLC)

The HPLC has developed into a universally applicable method so that it finds its use in almost all
areas of chemistry, biochemistry, and pharmacy.
 Analysis of drugs
 Analysis of synthetic polymers
 Analysis of pollutants in environmental analytics
 Determination of drugs in biological matrices
 Isolation of valuable products
 Product purity and quality control of industrial products and fine chemicals
 Separation and purification of biopolymers such as enzymes or nucleic acids
 Water purification
 Pre-concentration of trace components
 Ligand-exchange chromatography
 Ion-exchange chromatography of proteins
 High-pH anion-exchange chromatography of carbohydrates and oligosaccharides

Advantages of High-Performance Liquid Chromatography (HPLC)

 Speed
 Efficiency
 Accuracy
 Versatile and extremely precise when it comes to identifying and quantifying chemical
components.
Reference

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2. Anupama Sapkota (2022). 14 Types of Chromatography (Definition, Principle, Steps, Uses).

[online] Microbe Notes. Available at: https://microbenotes.com/types-of-chromatography/
[Accessed 13 Oct. 2022].

3. Thermofisher.com. (2015). Introduction to UHPLC Whiteboard Video. [online] Available at:

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