What is HPLC?

2. Presented by: Gauri
Maske
Volunteer of AFRS
HIGH
PERFORMANCE
LIQUID
CHROMATOGRAPHY

3. CONTENT
 Introduction
 Types of HPLC
 Instrumentation
 Application
 Advantages
 Conclusion

4.  Introduction
It is a chromatographic technique used to separate components
of mixture for the purpose to identify, quantify or purify the
individualcomponentsof themixture.
High performance liquid chromatography is a specific form of
column chromatography generally used in biochemistry and
analysistoseparate,identifyandquantifytheactivecompounds.

5.  Types of HPLC
 Normal Phase :
Separation of polar analyte by partitioning onto a polar bonded
stationary phase.
Normal phase chromatography also known as Normal phase HPLC
(NP-HPLC), thismethodseparatesanalytebasedonpolarity.

6.  Reversed phase
Separation of non-polar analyte by partitioning onto a
non-polar bonded stationary phase and an aqueous,
moderately polarmobilephase.

7.  Size exclusionChromatography
(SEC) separates molecules based on their size by filtration
through a gel. Molecules separates based on their size as
they pass through the column and are eluted in order of
decreasingmolecular weight.

8.  Ion exchange chromatography
Separation of organic and inorganic ions by their
partitioning onto ionic stationary phases bonded to a solid
support.
Ion-exchange chromatographyof carbohydrates and
oligosaccharidesetc.

9.  Bio-Affinitychromatography
Bio-affinity chromatography are separation based on
specificreversible interaction of proteinwithligands.
 Bio-specific elution
 A-specific elution

10. INSTRUMENTATION
The HPLC instrumentation involves :
o Solvent Reservoir
o Pump
o Injector
o Columns
o Detector
o Recorder
o Degasser
o Data Collection and output

12. • Solvent Reservoir
The contents of mobile phase are present in
glass container.
• Pump
The pump is positioned in the most upper stream of the
liquid chromatography system and generates a flow of
eluent from the solvent reservoir into the system.
• Injector
The simplest method is to use a syringe and the sample
is introduced to the flow of eluent.
• Columns
The separation is performed inside the column. The
recent columns are often prepared in stainless steel
housing, instead of glass columns.

13. • Detector
Separation of analytes is performed inside the
column, whereas a detector is used to observe
the obtained separation.
• Recorder
The change in eluent detected by a detector is in
the form of an electronic signal and thus it is still
not visible to our eyes.
• Degasser
The eluent used for LC analyasis may contain
gases such as oxygen that are non-visible to
eyes.
• Data Collection

14. Application of HPLC
The HPLC has several application in the fields of pharmacy,
forensic, environment and clinical. It also helps in the separation
andpurificationofcompounds.
Application in Forensic
HPLC is one of the separation techniques most frequently used in
forensictoxicology.
HPLC can also be used to detect certain drugs and has been used
in investigations into terrorism, drug cartels, murders and
organizedcrimesyndicates.

15. Advantages
• Speed
• Efficiency
• Accuracy
• Versatile and extremely precise when it comes to
identifyingandquantifyingchemicalcomponents.

16. CONCLUSION
TheHPLCismostusedanalyticaltechnique.
It is having several advantages such as with the
use of HPLC one can produce extremely pure
compounds.