MEHRAN UNIVERSITY Of ENGINEEINGAND TECHNOLOGY JAMSHORO.

PRESENTATION TOPIC: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Group Leader: Hameer Khan Khuhro

INTRODUCTION1

CONTENTS

PRINCIPLE 3

INSTRUMENTATION4

ADVANTAGES5

FIELDS OF APPLICATION6

DEFINITION2

INTRODUCTIONIn 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.

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.

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.

 A flow scheme for HPLC 

InstrumentationSolvent Reservoirs

Pump

Sample Injector

Column(s)

Detector

Data System  

1.  Solvent  ReservoirIt 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.

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.

HPLC Pump CriteriaConstructed 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

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.

Performance RequirementsNo sample remaining in unitMinimal broadening of sample bandFree adjustment of injection volumeMinimal lossSuperior durability and pressure resistance

Manual Injectors  

Front View Inject

Automatic Injectors

4. HPLC columnsThe 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.

PARAMETERSLength (5-15 cm); much shorter than GC

columnDiameter (4 mm down to 50mm)Particle size (3, 5, or 10 mm)Typically detection limit is decreased by

decreasing the column diameter

5.Detector There are several ways of detecting when a substance

has passed through the column.

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.

Advantages of High Performance Liquid Chromatography

High separation capacity, enabling the batch analysis of multiple components

Superior quantitative capability and reproducibility

Moderate analytical conditionsUnlike GC, the sample does not need to be vaporized.

Generally high sensitivityLow sample consumptionEasy preparative separation and purification of

samples

Fields in Which High Performance Liquid Chromatography Is Used

Biogenic substancesSugars, lipids, nucleic acids, amino acids, proteins, peptides,

steroids, amines, etc.

Medical productsDrugs, antibiotics, etc.

Food productsVitamins, food additives, sugars, organic acids, amino acids,

etc.Environmental samples

Inorganic ionsHazardous organic substances, etc.

Organic industrial productsSynthetic polymers, additives, surfactants, etc.