high pressure liquid chromatography

1. HPLC
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
BY.
Ayush sharma (QA)
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2. What is HPLC ?
It stands for the “High performance liquid
chromatography” & sometimes also referred
as the High pressure liquid chromatography.
HPLC is a chromatographic technique that can
separate a mixture of compounds .
It is also used in the biochemistry, separation
of combination drugs and in analytical
chemistry to identify , quantify and purify the
individual components of a mixture.
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3. HPLC Apparatus
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4. • Chromatography
Physical method in which separation of
components takes place between two phases i.e..
A stationary phase and a mobile phase.
• Stationary phase
The substance on which adsorption of the analyte
(the substance to be separated during chromatography )
takes place. It can be a solid, a gel, or a solid liquid
combination.
• Mobile phase
Solvent which carries the analyte (a liquid or a gas).
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5. PRINCIPLE
• The main principle of separation is adsorption.
• The components which has a lesser affinity
towards the stationary phase travels faster,
where as the components which has the greater
affinity towards the stationary , travels slower.
• No two components have the same affinity
towards the stationary phase on the basis of
that the components are separated .
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6. • HPLC is a separation technique that
involves : The injection of the small volume
of liquid sample into a tube packed with tiny
particles (3 to 5 micron) in diameter called
the stationary phase .
where the individual components of the
sample are moved down the packed tube
(column) with a liquid (mobile phase) forced
through the column by high pressure delivered
by a pump.
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7. • These separated components are detected
at the exit of this tube (column) by a flow
through device (detector) that measure
their amount. The output from the detector
is called a liquid chromatogram .
LC & HPLC works in the same way except
the speed ,efficiency , sensitivity and ease
of operation of HPLC is vastly superior.
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8. Working process of HPLC
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9. HPLC 9

10. TYPES OF HPLC
 Based on mode of Separation
 Based on principle of Separation
 Based on Elution technique
 Based on scale of operation
 Based on type of analysis
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11. Based on mode of separation
1- Normal based chromatography
Stationary phase is polar (hydrophilic) & mobile phase
if non-polar (hydrophobic).
2-Reverse phase chromatography
Stationary phase is non-polar (hydrophobic) and mobile
phase is polar (hydrophilic).
Polar-Polar bonds and non polar-non polar bonds have
more affinity that Polar –Non polar bonds.
Reverse phase chromatography is used for the drugs
which are usually hydrophilic.
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12. Based on principle of
Separation
1- Adsorption Chromatography
2- Ion exchange chromatography
3- Ion pair Chromatography
4- Gel permeation Chromatography
5- Affinity Chromatography
6- Chiral Chromatography
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13. Adsorption Chromatography
In the adsorption chromatography solute
molecules bond directly to the surface of the
stationary phase.
The components which has the more affinity
towards the mobile phase elutes first & the
components which the less affinity towards
the stationary phase elutes later.
No two components have the same affinity
towards mobile phase & stationary phase.
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14. Based on Scale of Operation
1- Analytical HPLC
No recovery of individual components of
substance
2- Preparative HPLC
Individual components of substance can be
recovered
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15. Based on type of Analysis
1- Qualitative analysis
Analysis of a substance in order to ascertain the nature
of its chemical constituents , here separation is possible
but cannot assess the quantity in this analysis .
2- Quantitative analysis
Determining the amounts and proportions of its
chemical constituents .
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16. Based on Elution Technique
The process of the extracting one material from another
by washing with a solvent, as in washing loaded ion
exchange resins to remove captured ions.
1- Isocratic elution
A separation in which the mobile phase composition
remains constant throughout the procedure is termed as
isocratic elution.
2- Gradient elution
A separation in which the mobile phase composition is
changed during the separation process is described as a
gradient elution.
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17. APPLICATIONS
1- Pharmaceutical
2- Environmental
3- Forensics
4- Clinical
5- Food & Flavour
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18. APPLICATIONS
HPLC is one of the most widely applied analytical
separation techniques .
1) Pharmaceutical
• Pharmaceutical quality control.
• Shelf life determination of pharmaceutical products.
• Tablet dissolution of pharmaceutical dosages.
• Identification of counterfeit drug products.
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19. Environmental
• Phenols in Drinking water.
• Estrogens in coastal waters- The sewage
source.
• Biomonitering of PAH (polycyclic aromatic
hydrocarbons) pollution in high altitude
lakes.
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20. Forensics
• Identification of anabolic steroids in serum ,
urine , sweat and hair.
• Simultaneous quantification of
psychotherapeutic drugs in human plasma.
• A mobile HPLC apparatus at dance parties on
site identification and quantification of the drug
Ecstasy.
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21. Clinical
• Analysis of antibiotics.
• Quantification of DEET (N,N-diethyl-meta-
toluamide) in human urine.
Food & Flavor
• Ensuring soft drink consistency and quality
• Sugar analysis in fruit juices.
• Analysis of vicinal di-ketones in beer.
• Polycyclic aromatic hydrocarbons in Brazillien
vegetables and fruits .
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22. ADVATAGES OF HPLC
• Separation is fast & Efficient.
• Accurate quantitative measurement.
• Repetitive & reproducible analysis using the same
column.
• It can be applied for the separation & analysis of the
very complex mixtures.
• Both aqueous & non-aqueous samples can be
analyzed.
• It provides a means for determining of multiple
components in a single analysis.
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23. Disadvantages
• Expensive
• Complex to operate
• Tedious to detect co-elution
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24. Thank You
Note .. Questions puchana mana ha.
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