1. MEHRAN UNIVERSITY Of ENGINEEING
AND TECHNOLOGY JAMSHORO.
PRESENTATION TOPIC:
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
Group Leader:
Hameer Khan Khuhro

2. INTRODUCTION1
CONTENTS
PRINCIPLE3
INSTRUMENTATION4
ADVANTAGES5
FIELDS OF APPLICATION6
DEFINITION2

3. INTRODUCTION
 In 1964 J.Calvin Giddings predicted improved liquid
chromatography.
 Few years later C.Horvath and S.R Lipsky built the first HPLC and
called it High pressure liquid chromatography.
 Today HPLC has become known as High performance liquid
chromatography.

4. WHAT IS HPLC
 HPLC, is a chromatographic technique used to
separate the components in a mixture, to identify
each component, and to quantify each
component.

5. PRINCIPLE:
The sample mixture to be separated and analyzed is
introduced, in a discrete small volume (typically microliters),
into the stream of mobile phase percolating through the
column. The components of the sample move through the
column at different velocities, which are function of specific
physical interactions with the sorbent (also called stationary
phase). The velocity of each component depends on its
chemical nature, on the nature of the stationary phase
(column) and on the composition of the mobile phase. The
time at which a specific analyte elutes (emerges from the
column) is called its retention time. The retention time
measured under particular conditions is considered an
identifying characteristic of a given analyte.

6.  A flow scheme for HPLC

7. Instrumentation
 Solvent Reservoirs
 Pump
 Sample Injector
 Column(s)
 Detector
 Data System

8. 1. Solvent Reservoir
 It stores Mobile Phase.
 The liquid used as Mobile Phase is called Eluent.
 Mobile Phase may be a single solvent or solvent
mixture of constant composition and is called
Isocratic elution.
 Mobile Phase Reservoir can be filled with a wide
range of solvents with different polarities and is
called Gradient elution. Example Acetonitrile or
Methanol with water.
 The solvents must be pure and degassed to avoid
formation of gas bubbles.

9. 2.Solvent Delivery System (Pump)
 The pump is the most critical piece of equipment for a
successfully operating HPLC.
Functions :
HPLC Pump has three basic functions:
1. Provide accurate and constant flow.
2. Provide accurate mobile phase compositions.
3. Provide the force necessary to push the mobile
phase through the tightly packed column.

10. HPLC Pump Criteria
 Constructed of materials inert toward solvents to be used
 Deliver high volumes (flow rates) of solvent (to 10 mL/min)
 Deliver precise and accurate flow (<0.5% variation)
 Deliver high pressure (to 6000 psi)
 Deliver pulse free flow
 Have low pump-head volume
 Be reliable
HPLC Pumps: Types
 Reciprocating pumps
 Syringe pumps
 Constant pressure pumps

11. 3. Sample Injector
 The function of the injector is to place the sample
into the high-pressure flow in as narrow volume
as possible so that the sample enters the column
as a homogeneous
 To minimize spreading of the injected volume
during transport to the column, the shortest
possible length of tubing should be used from the
injector to the column.

12.  Performance Requirements
 No sample remaining in unit
 Minimal broadening of sample band
 Free adjustment of injection volume
 Minimal loss
 Superior durability and pressure resistance

13. Manual Injectors
Front View Inject

14. Automatic Injectors

15. 4. HPLC columns
 The column is one of the most important components of
the HPLC chromatograph because the separation of the
sample components is achieved when those components
pass through the column. The High performance liquid
chromatography apparatus is made out of stainless steel
tubes with a diameter of 3 to 5mm and a length ranging
from 10 to 30cm.
 Normally, columns are filled with silica gel because its
particle shape, surface properties, and pore structure
help to get a good separation.
 Its chromatographic behavior is generally predictable
and reproducible.

16.  PARAMETERS
 Length (5-15 cm); much shorter than GC column
 Diameter (4 mm down to 50mm)
 Particle size (3, 5, or 10 mm)
 Typically detection limit is decreased by
decreasing the column diameter

17. 5.Detector
 There are several ways of detecting when a substance has passed
through the column.

18. 6.Data System
 Data is processed using specific softwares that are
connected to HPLC machine.
 Receive the information and present it as a graph.
 It automatically compare the graph with standard graph
and gives results.

19. Advantages of High Performance Liquid
Chromatography
 High separation capacity, enabling the batch analysis of
multiple components
 Superior quantitative capability and reproducibility
 Moderate analytical conditions
 Unlike GC, the sample does not need to be vaporized.
 Generally high sensitivity
 Low sample consumption
 Easy preparative separation and purification of samples

20. Fields in Which High Performance
Liquid Chromatography Is Used
 Biogenic substances
 Sugars, lipids, nucleic acids, amino acids, proteins, peptides, steroids,
amines, etc.
 Medical products
 Drugs, antibiotics, etc.
 Food products
 Vitamins, food additives, sugars, organic acids, amino acids, etc.
 Environmental samples
 Inorganic ions
 Hazardous organic substances, etc.
 Organic industrial products
 Synthetic polymers, additives, surfactants, etc.