High Performance Liquid Chromatography (HPLC)

1. High Performance Liquid
Chromatography
(HPLC)
By:
LinkedIn: Shumookh AlSufyani

2. • Introduction .
• High Performance Liquid
Chromatography
• Principle Of HPLC
• Components of HPLC
• Applications.
Contents:

3. Introduction
Introduction
IUPAC definition of chromatography (1993):
Chromatography is a physical method of separation in which the components to be
separated are distributed between two phases.
Phase Of chromatography
Mobile Phase
It’s the phase used to move the sample
Either it’s gas or liquid.
Stationary Phase
The phase doesn’t move Either it’s
solid or liquid.

4. Principle of chromatography :
Introduction

5. Introduction
Classification of chromatographic Methods:
Adsorption
Gas Mobile phase,
column (GC GSC)
Liquid Mobile phase
Column (LC) & (HPLC)
Liquid Mobile phase
Planer (TLC) , (PC)
Partition
Liquid Mobile
phase, Colum
(LC) , (HPLC)
Gas Mobile phase,
Column
(GCGLC)
Ion Exchange
Liquid Mobile
phase, Column
(IEC) ,
(IC HPIC)
Permeation
Liquid Mobile
phase, Column
(GPC)

6. HPLC
igh erformance iquid hromatography:
➢ Its separated methods using the basic principle of column chromatography and then
identified and quantified by spectroscopy.
➢ In the 1960s the column chromatography LC with its low-pressure suitable glass columns
was further developed to the HPLC with its high-pressure adapted metal columns.
➢ HPLC is thus basically a highly improved form of column liquid chromatography. Instead of
a solvent being allowed to drip through a column under gravity, it is forced through under
high pressures of up to 400 atmospheres.

7. Principle Of HPLC
➢ The purification takes place in a separation column between a stationary and a
mobile phase.
➢ The stationary phase is a granular material with very small porous particles in a
separation column.
➢ The mobile phase, on the other hand, is a solvent or solvent mixture which is
forced at high pressure through the separation column.
➢ Via a valve with a connected sample loop, i.e., a small tube or a capillary made
of stainless steel, the sample is injected into the mobile phase flow from the
pump to the separation column using a syringe
Principle Of HPLC:

8. Components of HPLC :
Components Of HPLC
➢ Injector.
➢ pump
➢ Column
➢ Detector

9. 1- Injector
Components Of HPLC
Manual injector Auto-sampler injector

10. 2- Pump
Components Of HPLC
➢ Solvent delivery system.
o Displacement pumps (Syringe pump)
Syringe pumps push the solvent at a
constant speed using a large syringe
o Reciprocating pump (Plunger pump)
Its strengths are compactness, low-cost,
and continuous delivery

11. 3- Column
Size Exclusion
Ion-exchange
phase
Reverse phase
Normal phase
Pore sizes Particles
Ion-exchange resin
Non-Polar
particles
Hydrophobic ,
Alkyl group
Polar Particles
Alumni, Silica
Stationary
Phase
Aqueous Solution
Aqueous buffer
Polar solvent
Non-Polar solvent
Mobile phase
Base on size
Ionic sample
Polar
Non-polar
Separating
Physical
Chemical
Chemical
Chemical
Property
Components Of HPLC

12. 4- Detectors
Components Of HPLC
comments
Analytes
Detector
Useful for many HPLC
Any chromophores
UV-vis
Used to analyzed derivatized
compound
Fluorescent compound
Fluorescence
Virtually a universal detector
limited sensitivity
Compound with different RI to
mobile phase
Refractive index (Ri)
Used for Ion Exchange method
Charged or polar compound
Conductivity
Very selective & sensitive
Readily oxidized (Biological sample)
Electrochemical
Virtually a universal detector high
sensitivity, Not selective
Virtually all compound
Evaporate light Scattering
(ELSD)
High sensitivity & powerful
2𝑛𝑑
diamention Analytical tool
Broad range of compound
Mass spectrometer (MS)

13. Application Of HPLC
Application Of HPLC
Clinical Test
• Urine &
antibiotic
analysis in blood
• Analysis of
bilirubin,
biliverdin in
hepatic
disorders.
Environmental
• Detection of
phenolic
compounds in
drinking water.
• Bio-monitoring
of pollutants.
Forensics
• Quantification of
drugs in samples
• Forensic of
textile dyes.
Food
• Analysis of
polycyclic
compound in
vegetables.
• Measurement of
quality Of water
& soft drink.