CALCUTTA INSTITUTE OF PHARMACEUTICAL

TECHNOLOGY & AHS

PRESENTED BY
UJJAL MANNA & SOUVIK MAITI
COURSE – M.PHARM (PHARMACEUTICS)
Year – 1st , SEMESTER – 1st
TOPIC- HPLC

SESSION – 2024-2026
 contents

❑ Introduction
❑definition
❑ principle
❑ types of hplc techniques
❑ instrumentation
❑ application
❑ advantages
❑ disadvantages
❑ new amendments of hplc
INTRODUCTION

High Performance Liquid

Chromatography (HPLC) is a form of
column chromatography that pumps a
sample mixture or analyte in a solvent
(known as the mobile phase) at high
pressure through a column with
chromatographic packing material
(stationary phase).
 DEFINITION

➢ HPLC or High Performance Liquid Chromatography is a

form of liquid chromatography and analytical technique
used to separate , identify, quantify each component that are
dissolved in solution.
➢ HPLC has the ability to separate , and identify compounds
that are present in any samples that can be dissolved in a
liquid in trace concentrations as low as parts per trillion.
Sample retention time will vary depending on the interaction
between the stationary phase, the molecules being analyzed
and the solvent or solvents used.
 PRINCIPLE

❑ The basic principle of HPLC in normal phase and reverse phase mode is adsorption. The sample is
introduced into HPLC column , different components of the sample move according to their
affinities towards the stationary phase .
❑ Components having high affinity get absorb on stationary phase while components having less
affinity towards stationary phase travel faster down the column
❑ As no two components have the same adsorption and affinity towards stationary phase, the
components get separated by this method.
 INSTRUMENTATION
❑ SOLVENT RESERVOIRS
❑ SOLVENT DEGASSING
❑ PUMP
❑ SAMPLE INJECTOR
❑ COLUMN
❑ DETECTOR
❑ RECORDER
❖ SOLVENT RESERVOIRS a modern HPLC apparatus is equipped with one or more glass or stainless

steel reservoirs, containing 500 ml or more of solvent.

❖ THE SOLVENT DEGASSING is used to de-gasify the air bubbles that are formed during the test.

❖ THE PUMP is used to generate a specific flow of mobile phase through the column.

Three types of pump

1. Pneumatic Pump
2. Syringe Pump
3. Reciprocating Pump
Pumps are designed in order to maintain a stable flow rate, avoiding pulsation even when the
composition of the mobile phase varies.
 1. Pneumatic Pump
❑ Working:
• The driving air is applied, piston moves, inlet closes & outlet open pushing
mobile phase to the column.
• Pressure on solvent is proportional to the ration of piston usually 50:1.
• A lower pressure gas source of 1-10 atm can be used to generate high liquid
pressure. ( 1-400atm)
• About 70 ml of the mobile phase is pumped from every stroke.
❑ Advantages: Pulse free flow & Generates high pressure.
❑ Disadvantages: 1) Limited volume capacity(70 ml)
2) Pressure outpit and flow rate depends on the viscosity &
column back pressure.
3) Gradient elusion is not possible.
 2. Syringe Pump
• Working:
• Works on the principle of positive solvent pressure.
• Rotatory motion provides continuous movement of the mobile phase which is
propelled by the revolving screw at greater speed and pushes solvent through small
needle like outlet.
• Consist of large syringe like chamber of capacity 250-500 ml.
• Advantages:
• Flow is pulse free
• Provide high pressure upto 200-475 atm
• Simple operation.
• Disadvantages:
• Limited solvent capacity
• Gradient elution is not easy.
3. Reciprocating Pump
• Working:
• Contains reciprocating piston that moves back and forth in hydraulic chamber.
• By the movement of piston solvent flow into the column under high pressure.
• The reduction in volume in main chamber due to forward motion of piston
result in mobile phase moving out of the exit valve under high pressure.
• Advantages:
• Generate high output pressure ( upto 10000 poise)
• Ready adaptability to gradient elusion.
• Provide constant flow rate.
• Disadvantages:
• Pulsed flow which must be damped as they produce a base line noise on the
chromatogram.
 Pneumatic Pump: Syringe Pump Reciprocating Pump

1. About 70 ml of the 1.capacity 250-500 ml. 1. 70 ml of the mobile phase

mobile phase is pumped is pumped from every
from every stroke. stroke. Provide constant
flow rate.
2. generate high liquid 2. Provide high pressure 2. Generate high output
pressure. upto 200-475 atm. pressure ( upto 10000 psi)
(1-400atm).
❖ THE INJECTOR or auto sampler introduces the solvent into a phase stream.
Three types of injector
1. Micro liter injector
2. Rotary valve injector
3. Loop injector
❖ THE COLUMN contains specific packing material needed to effect separation.
❑ Types of column based on mode of operation
1. Normal phase chromatography columns
2. Reverse phase chromatography column
❑ Types of column based on scale of preparation
1. Analytical Column
2. Preparative column
 Microlitre Injection

Sample Inject Sample Load

Types of column based on scale of preparation
Property Guard Column Analytical Column
Particle Size Large Small( 5-10 micron)

Length 2-10 cm 25-100 cm

Use Remove the Effective separation
impurities from the of compounds
solvent, it can protect
the analytical column

Analytical Column
 Types of column based on mode of operation
Normal Phase Reversed Phase
Stationary Phase Polar( Silica Gel) Non- Polar(C18 group
bonded - ODS)
Mobile Phase Non-Polar (Organic Polar( Aqueous/
Solvent) Organic)
Sample Movement Non polar fastest Polar fastest
Use Long Time Analysis Drug analysis in QC

Normal Phase Reverse Phase

 ❑ Normal phase mode
❑ Reverse phase mode
❑ Reverse Phase- HPLC C18 column:
• C18 means octa decyl silane
• In partition HPLC uses liquid bonded phase column, where the liquid stationary phase is
chemically bonded to the packing material.
• The packing material usually hydrolysed silica.
❑ In Reverse Phase HPLC:
• Strongly hydrophobic substances are strongly retained by the stationary phase.
• Thus have relatively long retention times.
❑ In Normal Phase HPLC:
• Hydrophilic substance are strongly retained by the stationary phase.
• Thus have relatively long retention times.
• Based on elution Technique:

▪ Isocratic Elution:
• In this technique the mobile phase composition is fixed constant throughout the chromatographic
procedure.
• For example, if a method consisting of mobile phase as methanol and water in the ratio of 70:30, the
same ratio is maintained for the entire chromatographic procedure in isocratic method.
▪ Gradient Elution:
• In this technique, the composition of mobile phase is changed either stepwise or continuously as
elution proceeds.
• For example, Initially a composition (methanol :water, 70:30) for some time period (10 min), is
maintained then the polarity is modified by changing the ratio to (80:20) for next 5 min and then to
90:10 for another 5 min.
❖ THE DETECTOR is needed to see the separated compound bands as they elute from the high pressure
column.

1) UV Detector:

2) Refractive Index Detector or RI Detector

3) Fluorescent Detector

4) Conductometric Detector

❖ THE RECORDER is used to record all the valuable data that are obtained after the experiments. The
recorder helps analyze and interpret the data.
 APPLICATIONS OF HPLC

The applications of HPLC are as follows:

1) Checking the impurity of a compound.
2) Quantitative analysis.
3) Qualitative analysis.
4) Isolation and detection / identification of drugs.
5) Used in the ion exchange chromatography of proteins.
6) Used in the purification of water.
• Identification of Drug(Paracetamol):
➢ A series of standard solution containing 10-100 µg/mL of paracetamol and the sample solution of
pharmaceutical preparations were applied respectively. 10 µL aliquot of each solution was injected into
the column in a duplicate and the chromatograms were recorded. Calibration graph was constructed by
plotting the mean peak area versus concentration of paracetamol. The concentration of the unknown
was calculated from the regression equation derived from the concentration and peak area data, or was
read from calibration graph.
➢ Tablets 20 tablets were weighed, powdered and transfered an accurately weighed portion of the powder
equivalent to 5 mg paracetamol into 100 mL volumetric flasks and diluted with mobile phase to the
volume, and the amount of paracetamol was determined by comparing the peak area of the assay
preparation with the standard preparation at the same concentration.
 ADVANTAGES

1. Separation of volatile and non volatile components.

2. Quick analysis.
3. High resolution is often found.
4. More reproducibility due to close control of the parameters affecting the
efficiency of separation.
5. Adaptability to large scale, preparative procedures.
6. Easy automation of instruments operation and data analysis.
 DISADVANTAGES

1. HPLC costs high in market.

2. HPLC is very much complex to operate.
3. Volatile substances are better separated by gas chromatography.
4. Low peak resolution.
5. Clogging of columns.
6. HPLC has low sensitivity for some certain compounds.
 NEW AMENDMENTS IN HPLC TECHNIQUE

HPLC is compared with the classical techniques are characterized by:

• Rapid Resolution Liquid Chromatography (RRLC)
• Ultra Performance Liquid Chromatography (UPLC)
• Ultra-fast Liquid Chromatography (UFLC)
• Nano Liquid Chromatography (NANO LC)
• For Better Understand:
https://www.youtube.com/watch?v=Vr5t-cgHHG4