HPLC.pptx principle, instrumentation , theory, columns

High performance liquid chromatography
( HPLC)
Pune District Education Association ,
Shankarrao Ursal College of
Pharmaceutical Sciences & Research
Centre, Kharadi, Pune
Presented by :- Dr. Vijaya Barge
(Vice Principal & Professor)

LEARNING OBJECTIVES :
After completing this sub-unit the students should be able:
 To study principle, advantages and disadvantages of HPLC.
 To learn different types of HPLC.
 To study instrumentation and different components used in HPLC.
 To study derivatization techniques under HPLC.
 To study different applications of HPLC.

Contents
1. Background
2. Different types of chromatography
3. Introduction
4. Latest instrument
5. Principle
6. Types of HPLC
7. Application of HPLC
8. Reference

Background
 Chromatography :-
Chromatography is technique which is used to separate component mixture due to the different
time take for each component travel through stationary phase carry to mobile phase.
Basically, All chromatography systems consists of two phases.
 Mobile phase :- liquid or gaseous flows over through the stationary phase .
 Stationary phase :- solid , liquid mixture which is immobilized.
 Analyte :- Substance that is separate during chromatography.
 Effluent :- Mobile phase leaving the columns.

Different types of chromatography:-
1. Paper chromatography
2. Liquid chromatography
3. Gas chromatography
4. High performance liquid chromatography

Introduction
 HPLC stands for “High-performance liquid chromatography” ( sometimes referred to as
High-pressure liquid chromatography).
 HPLC was developed in 1960 .
 HPLC is a form of liquid chromatography used to Separate compounds that are dissolved
in solution.
 HPLC instruments consist of a reservoir of mobile phase, pump, an injector, a separation
column, and a detector.
 Compounds are separated by injecting a sample mixture into the column.

 The mobile phase must be degassed to eliminate the formation of air bubbles.
 This technique base on the same mode of separation as column chromatography.
 HPLC is most powerful tool of Analytical techniques.
 In HPLC coloumns are tightly packed and the eluent is forced through the column
under high pressure up to 5,000 psi by a pump.
 HPLC allows to used a very smaller particle size for the column packing material which
gives a much greater surface area for interactions between the stationary phase and
the molecules flowing through it.
 HPLC allows a much better separation of the components of the mixture.

Principle
 To understand the principle of HPLC, we must first look at the principle behind
liquid chromatography.
 Liquid chromatography is a separation technique that involves:-
1. The injection of a small volume of liquid sample
2. Into a tube packed with porous particles ( stationary phase )
3. Where individual components of the sample are transported along the packed
tube ( column) by a liquid moved by gravity.
 The main principle of separation is adsorption.

 They travel according to their relative affinities towards the stationary phase. The component
which has more affinity towards the adsorbent, travels slower.
 The component which has less affinity towards the stationary phase travels faster.
 Since no two components have the same affinity towards the stationary phase, the
components are separated.

 HPLC is a separation technique that involves:-
 The injection of a small volume of liquid sample into a tube packed with tiny
particles ( 3 to 5 micron in diameter called the stationary phase).
 Where individual comments of the sample are moved down the packed tube (
column) with a liquid ( mobile phase ) force through the column by high pressure
delivered by a pump.
 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.

 These separated components are detected at the exit of this tube (
column) by a flow-through device ( detector) that measures their amount. The
output from the detector is called a liquid chromatogram.
 In principle, LC and HPLC work the same way except the speed, efficiency,
sensitivity and ease of operation of HPLC is vastly superior.

Types of HPLC
• Based on modes of chromatography
1. Normal phase mode
2. Reverse phase mode
• Based on principle of chromatography
1. Absorption chromatography
2. Ion exchanging chromatography
• Based on elution technique
1. Isocratic Separation
2. Gradient separation
• Based on the type of Analysis
1. Qualitative analysis
2. Quantitative Analysis

 Based on modes of chromatography there are two modes such as Normal phase mode
and Reverse phase mode.
 Before explaining the modes, It is important to know the interaction which occurs
between solute, stationary and mobile phase.
 Polar-polar :- Interaction or affinity is more .
 Non-polar – Non-polar :- Interaction or affinity is more .
 Polar - Non-polar :- interaction or affinity is less
Based on modes of chromatography

In Normal phase mode , the Stationary phase (e.g. silica gel) is polar in nature and mobile
phase is Non-polar.
In this technique Non-polar compound travels faster and are eluted first.
This is because of less affinity between solute and stationary phase.
Normal phase mode

Reverse phase mode
In reverse phase technique , a Non-polar Stationary phase is used. The Mobile phase is polar in nature.
Hence polar components get eluted first and Non-polar compound are retained for longer time.

Absorption Chromatography :-
The principle of absorption Chromatography separation of components takes place because of
the difference in affinity of compound towards stationary phase.
Based on principle of separation

Ion exchange chromatography
The principle of separation is ion exchange, which is reversible exchange of functional
groups.
In ion exchange chromatography, an ion exchange resin is used to separate a mixture of
similar charged ions.

Based on elution Technique
Isocratic separation :-
In this technique the same mobile phase combination is used throughout the process of
separation.
The same polarity or elution strength is maintained throughout the process .
Gradient separation :-
In this technique a mobile phase combination of lower polarity or elution strength is used
followed by gradually increasing the polarity or elution strength.

Based on the type of analysis
Qualitative Analysis :-
Which is used to identify compounds, detect the presence of impurities, to find out the
number of compound etc. This is done by using retention time values.
Quantitative Analysis :-
This is done to determine the quantity of the individual or several components in a mixture.
This is done by comparing the peak area of the standard and sample.

1. HPLC which is used in Chemistry and Biochemistry research analysing complex mixtures.
2. HPLC which is used to purifying chemical compounds.
3. HPLC which is used quality control to ensure the purity of raw material.
4. HPLC which is used to analysing air and water pollutants.
5. HPLC which is used to purification of some compounds of natural or synthetic origin on
preparative scale.
6. HPLC which used in stability studies.
7. HPLC which used in Biopharmaceutical and pharmacokinetic studies.
8. HPLC which is used in isolation and identification of drugs or metabolites in urine ,
plasma, serum etc. Can be carried out.
9. HPLC which is used in isolation and identification of mixtures of components of natural or
synthetic origin.

References
1. Harold Variety practical clinical chemistry.
2. http ://scimedia.com
3. http ://www.forumsci.co.il/HPLC.
4. Dr.Walode S.G, Dr.Chandan R.S., Instrumental method of Analysis, Nirali Prakashan,
first edition sep2020, page no 13.1, 13.36

HPLC.pptx principle, instrumentation , theory, columns

HPLC.pptx principle, instrumentation , theory, columns

More Related Content

What's hot

Similar to HPLC.pptx principle, instrumentation , theory, columns

More from Dr. Vijaya Barge

Recently uploaded

HPLC.pptx principle, instrumentation , theory, columns