HPLC is an advanced form of liquid chromatography that uses pumps to force a pressurized liquid solvent containing a sample mixture through a column filled with adsorbent material. Each component interacts slightly differently with the adsorbent, leading to separation as components flow out the column at different rates. Key components of an HPLC system include solvent reservoirs, pumps that maintain high pressure, an injector port for samples, a column packed with porous silica or other material, and various detectors to monitor the separated components emerging from the column. HPLC is used to separate, identify, and quantify individual components in a mixture through differential partitioning between a mobile and stationary phase under high pressure.

1. GOVT. HOLKAR SCIENCE COLLEGE
SESSION-2016
DEPARTMENT OF PHARMACEUTICAL CHEMESTRY
TOPIC- HIGHER PERFOMANCE LIQUID CHROMATOGRAPHY
SUBMITTED BY-
AMAN KAKNE
M.Sc Previous 2sem

2. Chromatography
In Chromatography the mixture is dissolve in a fluid called
mobile phase which carries it through a structure holding
another material called stationary phase.The various
constituent of mixture travelled at different speed causing
them to separate.The separation is based on a differential
partition between mobile phase and stationary phase.
Chroma – colour
Graphy - writing

3. Steps in chromatography
 Adsorption of mixture
 Developing solvent
 Elution ( running of solvent)
 Quantitative and qualitative analysis
 Calculation by retention factor :-
= Distance travelled by solute from original line/
Distance travelled by Solvent from original line

4. H
P
igh
erformance
Liquid
Chromatography

5. Introduction of HPLC
HPLC (Higher Pressure Liquid Chromatography) is an
advance form of a liquid chromatography used to separate
compounds of a mixture (analyzes).
In HPLC chromatography the mixture is dissolved in a
solvent (mobile phase) and then force to flow through a
chromatographic column under a higher pressure (pump).
In a column the mixture is resolved into its components.

6. Principal of HPLC
HPLC is a technique in analytical chemistry used to
separate, identify, and quantify each component
in a mixture. It relies on pumps to pass a
pressurized liquid solvent containing the sample
mixture through a column filled with a
solid adsorbent material. Each component in the
sample interacts slightly differently with the
adsorbent material, causing different flow rates
for the different components and leading to the
separation of the components as they flow out
the column.

8. Instrumentation

9. Solvent reservoir
 Are used to store mobile phase.
 Made up of inert material like glass.
 It can be transparent or amber colored.
 The mobile phase is pumped under pressure
from reservoirs.
 Filtration is needed to eliminate suspended
particles and organic impurities.

10. The Function:
The pump provide a flow of the mobile-phase
through the HPLC injector, column, and detector.
.
The requirements of standard HPLC pump include:
 Generation of pressures up to 400-1500 psi.
 Pulse-free output.
 Flow rate ranging from 0.1 to 10 ml/min.
 Made of corrosion-resistant materials (stainless steel).
Pump

11.  Advantages:
Simple, inexpensive, easy to operate, and free from
pulsations, resulting in smooth baselines.
 Disadvantages:
Flow rate must be monitored carefully and constantly.
Types of HPLC Pumps:
 Constant-Pressure Pump.
 Constant-Flow Pump.

12. Sample Injector port
 The sample is introduced
using a syringe into the
sample loop via a port on the
injection valve.
 The samples introduce
with high precision, without
interruption of the mobile
phase flow.

13. COLUMN
The dimensions:
10-300 cm long
4-5 mm internal diameter.
Packing material:
Porous silica particles gives better column efficiency,
sample capacity, and speed of analysis
It is made out of stainless steel tubes
Silica is inert to most compounds

15. Detectors
 The detector from HPLC consist of a photometric detector
fitted with a low volume flow cell.
function:
Monitoring the mobile phase
as it emerges from the column..

16. Some detector in HPLC
 Refractive index detector
 Ultraviolet detector
 Fluorescence detector
 Electrochemical detector

17. Working
17
Pump
Sample injection unit
(injector)
Column
Column Oven
(thermostatic colum
n chamber)
Detector
solvent
(mobile phase)
Drain
Data processor
Degasser

18. Block diagram of HPLC

20. GRAPH

21. THANKING YOU