High Performance Liquid Cromatography

1. High-performance Liquid Chromatography
HPLC
Presented By:
All An Shariah Brishti
Sec:13th (B)
Daffodil International
University

2. High-performance liquid chromatography (HPLC) is an analytical technique
to separate, identify, and quantify components in a mixture. It is the single biggest
chromatography technique essential to most laboratories worldwide.
INTRODUCTION

3. • Biogenic substances
Sugars, lipids, nucleic acids, amino acids,
proteins , peptides, steroids, amines, etc.
• Medical products
Drugs, antibiotics, etc.
• Clinical Tests
Urine analysis, antibiotics analysis in
blood. bilirubin, biliverdin analysis in
hepatic disorders.
APPLICATION OF HPLC

4.  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.
APPLICATION OF HPLC….

5. • High Performance Liquid Chromatography (HPLC) is a form of
column chromatography that pumps a sample mixture in a solvent
at high pressure through a column with chromatographic packing
material.
• All chromatographic separations, including HPLC operate under
the same basic principle; separation of a sample into its
constituent parts because of the difference in the relative affinities
of different molecules for the mobile phase and the stationary
phase used in the separation.
PRINCIPLE OF HPLC

6. • HPLC has the ability to separate, and identify compounds that
are present in any sample that can be dissolved in a liquid in
trace concentrations as low as parts per trillion.
• As the sample passes through the column it interacts between
the two phases at different rate, primarily due to different
polarities in the analytes.
• Analytes that have the least amount of interaction with the
stationary phase or the most amount of interaction with the
mobile phase will exit the column faster.
PRINCIPLS OF HPLC….

7. INSTRUMENTATION

8. Solvent Reservoir: A reservoir holds the solvent called the mobile phase.
Pump : A high-pressure pump aspirates the mobile phase from the solvent reservoir
and forces it through the system’s column and detector.
Sample Injector : An injector is able to introduce the sample into the continuously
flowing mobile phase stream that carries the sample into the HPLC column.
How does it work…..

9. Columns : The column contains the chromatographic packing material needed to
effect the separation.
Detector : A detector is needed to see the separated compound bands as they
elute from the HPLC column.
Data Collection Devices :The computer integrates the response of the detector
to each component and places it into a chromatograph that is easy to read and
interpret.
How does it work…..

10. • It is a Powerful , adaptable and automated
process.
• results obtained quickly and easily.
• High resolution results are obtained.
• Affordable.
• Accurate results.
• Quick method for analysis of sample.
ADVANTAGES OF HPLC