GURU NANAK INSTITUTE OF PHARMACEUTICAL SCIENCE AND

TECHNOLOGY
(An Autonomous Institute)
Affiliated to Maulana Abul Kalam Azad University of Technology
PRESENTATION ON : HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
PRESENTED BY :
ATANU KUNDU (186122401004)
RINI DAS (186122401010)
SUBJECT NAME : MODERN PHARMACEUTICAL ANALYTICAL
TECHNIQUES
SUBJECT CODE : MPT_1061T

M. PHARM (PHARMACEUTICS), 1ST YEAR, 1ST SEMESTER

ACADEMIC SESSION : 2024-2025
 CONTENTS
SERIAL NO. TOPIC

1. INTRODUCTION

2. PRINCIPLE OF HPLC

3. TYPES OF HPLC

4. INSTRUMENTATION

5 CHROMATOGRAPHIC PARAMETERS

6. APPLICATION OF HPLC

7. ADVANTAGES OF HPLC

8. DISADVANTAGES OF HPLC

9. REFERENCES
 INTRODUCTION
The word Chromatography comes from the greek words “chroma” (color) and “graphein” (to write).
Chromatography is a technique that separates a mixture into its components by passing it over a stationary
phase while it's flowing with a mobile phase.
HPLC stands for High-performance liquid chromatography (also named high-pressure liquid
chromatography).
HPLC is a chromatographic technique used to separate a mixture of compounds in analytical chemistry and
biochemistry with the purpose of identifying, quantifying or purifying the individual components of the
mixture.
Some terms that are related to HPLC:
Mobile phase: A solvent that flows through the supporting medium.
Stationary phase: A layer or coating on the supporting medium that interacts with the analytes.
Supporting medium: A solid surface on which the stationary phase is bound or coated.
Elution time: The time between when a solute enters the column and when it elutes.
 PRINCIPLE OF HPLC
The main principle of separation is adsorption.
HPLC is a separation technique that involves:
Small volume of liquid sample is injected into a tube packed with tiny particles (3 to 5 micron in diameter and is called the
stationary phase) where individual components of the sample are moved down the packed tube (column) with a liquid (mobile
phase) forced through the column by high pressure delivered by a pump. [1]

These components are separated from one another by the column packing that involves various chemical and/or physical
interactions between their molecules and the packing particles. [1]

These separated components are detected at the exit of this tube by a detector that measures their amount and the output from
the detector is called a liquid chromatogram. [1]
 TYPES OF HPLC
1. Normal Phase HPLC:[2] [4]
This method separates analytes on the basis of polarity.
It uses polar stationary phase and non- polar mobile phase.
The stationary phase is usually silica and mobile phases are
hexane, methylene chloride, chloroform, diethyl ether.
Polar samples are thus retained on the polar surface of the column
packing longer than less polar materials.

Fig: Normal Phase HPLC

2. Reverse Phase HPLC: [2] [4]

The stationary phase is non-polar in nature (C18 Silica).
The mobile phase aqueous, moderately polar in nature (such
as mixtures of water and methanol or acetonitrile).
It works on the principle of hydrophobic interactions hence
the more non-polar the material is, the longer it will be
retained.

Fig: Reserve Phase HPLC

3. Size-exclusion HPLC:[2] [4]
The column is filled with material having precisely
controlled pore sizes.
The particles are separated according to their
molecular size.
Larger molecules are rapidly washed through the
column.
Smaller molecules penetrate inside the pores of the
packing particles and elute later.
This method is highly useful to determine molecular
weight of polysaccharides.

Fig: Size-exclusion HPLC

[2] [4]
4. Ion-Exchange HPLC:
The stationary phase has an ionically charged surface
of opposite charge to the sample ions.
This technique is used almost exclusively with ionic or
ionizable samples.
The stronger the charge on the sample, the stronger it
will be attracted to the ionic surface and thus, the
longer it will take to elute.
The mobile phase is an aqueous buffer, where both pH
and ionic strength are used to control elution time.
Fig: Ion-Exchange HPLC
5. Bio-affinity chromatography: [2]
[4]

This method involves separation according

to the specific reversible interaction of
proteins with ligands that are attached to
the solid support on a bio-affinity matrix
through covalent bonds.

Fig: Bio-affinity chromatography

 INSTRUMENTATION
I. Solvent Reservoir
II. Mixing system
III. Degassing system
IV. High Pressure Pump
V. Sample Injector
VI. Column
VII. Detectors
VIII. Data Recording System
Fig: HPLC instrumentation over-view
1. Solvent Reservoir:[4]
The appropriate solvents [mobile phase] from the reservoirs are allowed to enter the mixing chamber where a
homogenous mixture is obtained.

2. Mixing unit:[4]
Mixing unit is used to mix solvents in different proportions and pass through the column.
There are two types of mixing units. They are:
- Low pressure mixing chamber
- High pressure mixing chamber
Mixing of solvent is done either with a static mixer or a dynamic mixer.

3. Solvent degassing:[4]
Several gases are soluble in organic solvents.
When solvents are pumped under high pressure, gas bubbles are formed which will interfere with the separation process,
steady baseline and the shape of the peak. Hence degassing is necessary.
This can be done by using following techniques:
-Vacuum filtration
-Helium purging
-Ultrasonification
4. Pump:[2] [4]
The role of the pump is to force a liquid through the liquid chromatograph at a specific flow rate, expressed in
mL/min.
Normal flow rates in HPLC are in the 1 to 2 mL/min range.
Pumps can reach pressures in the range of 6000- 9000 psi.
Most commonly used pumps are: Reciprocating piston pump, Screw driven syringe pump, Constant pressure
pump.

(i) Reciprocating piston pump: [2] [4]

The piston is moved in and out of a solvent chamber by an eccentric
cam or gear.
The forward-stroke closes the inlet-check value while the outlet
valve opens and the respective mobile phase is duly pumped into the
column.
Consequently, the return-stroke-closes the outlet valve and it refills
the chamber.
They have unlimited capacity.
The flow rate can be altered by varying the length of piston stroke
or the motor speed. Fig: Reciprocating piston pump
(ii) Screw driven syringe pump:[2] [4]
Variable speed stepper motor turns a screw driven piston, which
displaces the mobile phase from chamber.
The flow is pulse free and varies with motor speed.
Mobile phase capacity depends on solvent chamber volume.
Much solvent is wasted while flushing out.

Fig: Screw driven syringe pump

(iii) Constant Pressure Pump or Pneumatic pump:[2] [4]

It consist of collapsible solvent container inside a vessel
pressurized by a compressed gas.
These are inexpensive and pulseless.
They suffer from limited capacity and pressure output.
Their pump rates rely on solvent viscosity.

Fig: Pneumatic Pump

 5. Injector:[2] [4]
The injector serves to introduce the liquid sample into the mobile phase.
Typical sample volumes are 5-20 microliters.
The injector must also be able to withstand the high pressure of the liquid system. An auto sampler is the
automatic version for when the user has many samples to analyze or when manual injection is not practical. There
are 3 types of injectors:
(i) Septum injectors:[2] [4]
The sample is introduced through a high pressure syringe via self
sealing septum or elastometer.
It give rise to a leaching effect that results in pseudo peaks.

(ii) Stop flow injector: [2] [4] Fig: Septum injector

The flow of mobile phase is stopped for a while.
The column top is opened and the sample is introduced at the top of
the packing.

(iii) Rheodyne injector:[2] [4]

It is the most popular injector.
This has a fixed volume loop like 20µL or 50µL or more. It has 2
modes:
-Load position
-Inject mode Fig: Rheodyne injector
6. Column: [1]
The column [stationary phase] separates the sample components of interest using various
physical and chemical parameters.
Column length: varies from 5cm to 30cm
Column diameter: ranges from 2mm to 50mm
Particle size: from 1 to 20µ
Particle nature: spherical, uniform sized, porous materials are used.

Types of columns in HPLC: [1] [2]

(i) Pre column or Guard column:

It is set between the injector and an analytical
column, are used to protect analytical columns
from chemical impurities in samples.
Guard columns are suitably used with samples
containing non-volatile residues that could
contaminate a column.
These residues get deposited in the guard
column, and thus the interaction between the
residues and the sample is reduced since the
solute may not adhere in the guard column.
Guard column requires periodic cutting or
trimming upon a build-up of residues. Fig: Guard column and analytical column
(ii) Analytical column:
It is responsible for separating and analyzing sample components.
The column is filled with a stationary phase material that interacts with the sample as it
passes through.

Materials of construction for the tubing:

Stainless steel (the most popular, gives high pressure capabilities)
Glass (mostly for biomolecules)
PEEK (poly ether ether ketone) and polymer (biocompatibility and chemically inert to most
solvents) [3]

Packing material:
The packing material is prepared from silica particle, alumina particle.
Porous plug of stainless steel or teflon are used in the end of the columns to retain the packing material.
[3]
 7. Detector:[4]
It monitors the mobile phase passing out of the column.
It releases electrical signals that are directly proportional to the characteristics of the solute or
the mobile phase. The commonly used detectors in HPLC:
▪ Bulk-Property Detectors: Refractive Index detector
▪ Solute-Property Detectors: UV Detector and Fluorescence Detector
▪ Multipurpose Detectors: Perkin-Elmer 3D system that combines UV absorption, fluorescence
and conductometric detection altogether
▪ Electrochemical Detectors: Amperometric or coulometric detector

(a) Ultraviolet (UV) Detector:

An UV-detector works on the principle of
absorption of UV visible light from the
effluent emerging out of the column and
passing through a photocell positioned in the
radiation beam.
They are cost-effective and popular and are
widely used in industry.
Wavelength ranging from 210-800 nm are
used for selective detection. Fig: UV Detector
(b) Fluorescence Detector:
Radiation emitted from a xenon or
deuterium source is concentrated on the
flow cell using a filter.
Usually at 90o to the incident beam, the
fluorescent radiation emitted by the
sample is measured.

Fig: Fluorescence Detector

(c) Refractive Index (RI) Detector:

Light emitted from the source(s) is
concentrated into the cell containing the
sample and reference sample.
Both the chambers of cells are separated
by a diagonal glass sheet.
The light passes through the cell and
reaches the beam splitter (B) that diverts
the light towards two photocells (P₁ and
P₂). Fig: Refractive Index Detector
8. Data recording system: [1]
Frequently called the data system,
It takes the signal from the detector and uses it to determine the time of elution
(retention time) of the sample components (qualitative analysis) and the amount of
sample (quantitative analysis).
The concentration of each detected component is calculated from the area or
height of the corresponding peak and reported.

Fig: Data recording system

 CHROMATOGRAPHIC PARAMETERS
1. Retention Time (tR): Time between the sample injection point & the analyte reaching detector [2]
2. Void Time (tM): Retention time of unretained component (or) an first baseline disturbance due to elution of the
sample solvent [2]
3. Capacity factor (k): A measurement of solute retention obtained by dividing the net retention time by the void time
[2]

4. Net Retention time: Retention Time (tR)- void time (tM) [2]

Fig: The above diagram shows retention time, void time and
capacity factor
 CHROMATOGRAPHIC PARAMETERS
5. Separation factor/Selectivity (α): [2]
The ratio of retention of two adjacent peaks(K1
and K2 )is called selectivity.
α> 1 indicates good selectivity.

Fig: Selectivity or separation factor

6. Resolution(RS): [2]
The degree of separation between two peaks is
called Resolution.
The difference in their retention time (tR1 and tR2)
divided by the average peak width (Wb1 and Wb2)
is the resolution.
Fig: Resolution
 APPLICATIONS OF HPLC
1)Stability Studies: [4]
Stability of various pharmaceutical compounds, degradation products and other chemical substances can be studied
using the technique of HPLC.

2) Bioassays and its Complementation:[4]

HPLC is commonly used for the bioassay and analysis of peptide hormones, and some antibiotics (e.g.. cotrimoxazole).

3)Design of Dosage Form : [4]

Biopharmaceutics of the dosage form and the pharmacokinetic properties of the drugs are studied with the help of
HPLC.

4)In Cosmetic Industry : [4]

HPLC Is used for analyzing the quality of various cosmetic products such as lipsticks, gels, creams, etc.

5) Isolation of Pharmaceutically Active Compounds: [4]

HPLC is the most specific and sensitive method used for the separation of different therapeutically active components
present in plant extracts.
HPLC method is used in the isolation of different types of alkaloids and glycosides.
 ADVANTAGES OF HPLC: [4]

1) It is a simple, rapid, and reproducible technique.

2) It is highly sensitive.
3) It shows a better performance.
4) It's resolution and separation capacity is high.
5) It provides accurate and precise result.
6) It needs a small amount of mobile phase for developing chamber.
7) It enables easy visualization of separated components.
8) It is useful in qualitative and quantitative analysis.

DISADVANTAGES OF HPLC: [4]

1) It is difficult to detect co elution (two compounds escaping from the tubing at once) with HPLC,
which may lead to inaccurate compound categorization.
2) There is a high cost for equipment needed to conduct HPLC.
3) It’s operation can be complex, requiring a trained technician to operate.
 REFERENCES
1. CHATWAL G.R., Instrumental methods of chemical Analysis. FIFTH EDITION. India:
Himalaya Pub.

2. Ravisankar, P., Anusha, S., Supriya, K., Kumar, U. A., & Department of Pharmaceutical
Analysis and Quality Assurance, Vignan Pharmacy College, Vadlamudi, Guntur, A.P, India.
(2020). Fundamental chromatographic parameters. International Journal of Pharmaceutical
Sciences Review and Research, 46–50.

3. Salvato, F., Costa Da Cruz De Carvalho, M., De Lima Leite, A., University of Campinas,
Universidade Estadual do Norte do Paraná, & University of São Paulo. (2012). Strategies for
protein separation. In Integrative Proteomics.

4. Malathi S. , Patil P.M. , Kumar S. , Instrumental Methods of Analysis. FIFTH EDITION.

India: Thakur Publication PVT Limited.
Thank you