SlideShare a Scribd company logo
1 of 21
Download to read offline
…
HPLC
Presented by;
Salman Shehzada
CONTENTS
Introduction
History
Instrumentation
Operation
Types
Advantages
Disadvantages
Introduction
 Abbreviated for;
High-performance liquid chromatography
OR
High-pressure liquid chromatography
 Defined as;
“A chromatographic technique used to separate components of
mixture for the purpose to identify, quantify or purify the
individual components of the mixture “.
 Widely used in field of biochemistry and analytical chemistry.
History
 Invented by Martin and Synge as extension of gas
chromatography.
 The main idea was that;
“Small sorbent particles and pressure could produce fast
liquid chromatography techniques”.
 The method was used practically in late 1960s for separation
amino acids.
Instrumentation
 HPLC involves following functional instruments.
1. Mobile phase reservoir
2. Pump
3. Injector
4. Stationary phase ( Column)
5. Detector
6. Computer
1. Mobile phase reservoir:
• Commonly glass bottles with caps are used.
• Teflon tubings and filters are connected to purge gas (helium) for
degassing.
• Vaccum for 5-10 min is also used for degassing.
2. Pump:
• It forces the mobile phase to pass through column.
• Flow rate is 1-2 ml/ min.
• Trypical pressure is 6000 – 9000psi.
3. Injector:
• Can be manually (syringe) or automated.
• Sample volume 5-20µl.
• Ideal to stand pressure of mobile phase.
• Autosampler is used for analysis of many samples automatically.
4. Stationary phase (Column):
• Heart of HPLC.
• Separate sample components on basis of physical and chemical
parameters.
• Lenght 10-30cm.
• Diameter 4-10nm.
• Packing material 5-10nm thick.
5. Detector:
• Detection of elutes from column.
• Quantitative analysis of sample components.
• Output transferred to recorder/ computer.
6. Computer:
• Data system that controls modules of HPLC.
• Signals from detector are interpreted to determine elution time,
quantitative and qualitative analysis of sample.
Figure displaying instrumentation of HPLC
Figure displaying complex instrumentation of HPLC
Operation
• Operation strategies are explained as follows;
1. Sample:
 The sample to be analyzed is introduced into stream of mobile
phase in minute quantity, mostly in microliters.
 The components of sample moves through different velocities
reaching the column where it physically interacts with the
sorbent.
 Velocity of sample components depends on;
• Chemical nature of sample
• Nature of column
• Composition of mobile phase
2. Column:
• Also known as stationary phase.
• Composed of sorbent material varying in particle size, and surface
chemistry.
• Size of particles can be smaller which improves chromatographic
resolution.
• In terms of surface chemistry, the column can be hydrophobic and
polar in nature.
A B
A. Plant extracts separated by HPLC by passing through stationary phase (column)
B. Columns used in HPLC device
3. Eluent:
• Also known as mobile phase.
 Type of solvent used:
• Commonly it is composed of water miscible with organic solvents. For
example methanol.
• Some HPLC utilizes water free mobile phase and uses acids such as
formic acid or phosphoric acid.
• Many mobile phase works well by using salts such as sodium
phosphate.
 Composition of eluent:
• It depends on;
• Intensity of interactions between analytes and stationary phase. For
example;
i. Hydrophobic interactions in reversed-phase HPLC
ii. Polar interactions in normal phase HPLC
iii. Ionic interaction in ion exchange HPLC
• Often a series of trial runs are performed with the sample in
order to find the HPLC method which gives adequate separation.
Figure showing mobile phase reserviors
Figure showing operation of HPLC
Figure showing operation of HPLC and analysis of
analyte present in the sample
Types
1. Partition chromatography
2. Normal phase chromatography
3. Displacement chromatography
4. Reversed phase chromatography
5. Size-exclusion chromatography
6. Ion-exchange chromatography
7. Bioaffinity chromatography
8. Aqueous normal-phase chromatography
Advantages
• Advantages of HPLC is;
• Seapartion of voltile and non- voltile components.
• Thermally unstable compounds isolated.
• Quick analysis
• High resolution
• Less cumbersome
• More reproducibility
Disadvantages
• Disadvantages of HPLC are;
• Tedious to detect co-elution.
• High cost.
• Complex to operate.
Thank you …

More Related Content

What's hot

Adsorption chromatography
Adsorption chromatographyAdsorption chromatography
Adsorption chromatographySharmi143
 
Theory of high performance liquid chromatography ppt
Theory of high performance liquid chromatography pptTheory of high performance liquid chromatography ppt
Theory of high performance liquid chromatography pptshweta more
 
HPTLC- Principle, Instrumentation and Software (Abhishek Gupta)
HPTLC- Principle, Instrumentation and Software (Abhishek Gupta)HPTLC- Principle, Instrumentation and Software (Abhishek Gupta)
HPTLC- Principle, Instrumentation and Software (Abhishek Gupta)Abhishek Gupta
 
instrumentation of HPLC
instrumentation of HPLCinstrumentation of HPLC
instrumentation of HPLCsamiya shaik
 
Adsorption Chromatography Assignment.pdf
Adsorption Chromatography Assignment.pdfAdsorption Chromatography Assignment.pdf
Adsorption Chromatography Assignment.pdfMuhammadFaizan389
 
atomic absorption spectroscopy
atomic absorption spectroscopyatomic absorption spectroscopy
atomic absorption spectroscopyJyotiMhoprekar
 
HPLC Principle,Instrumentation and Application
HPLC Principle,Instrumentation and ApplicationHPLC Principle,Instrumentation and Application
HPLC Principle,Instrumentation and ApplicationAlakesh Pradhan
 

What's hot (20)

Adsorption chromatography
Adsorption chromatographyAdsorption chromatography
Adsorption chromatography
 
Paper chromatography
Paper chromatographyPaper chromatography
Paper chromatography
 
HPTLC
HPTLCHPTLC
HPTLC
 
Column chromatography
Column chromatographyColumn chromatography
Column chromatography
 
Theory of high performance liquid chromatography ppt
Theory of high performance liquid chromatography pptTheory of high performance liquid chromatography ppt
Theory of high performance liquid chromatography ppt
 
HPTLC- Principle, Instrumentation and Software (Abhishek Gupta)
HPTLC- Principle, Instrumentation and Software (Abhishek Gupta)HPTLC- Principle, Instrumentation and Software (Abhishek Gupta)
HPTLC- Principle, Instrumentation and Software (Abhishek Gupta)
 
Paper chromatography
Paper chromatography Paper chromatography
Paper chromatography
 
HPLC
HPLCHPLC
HPLC
 
Lc ms
Lc msLc ms
Lc ms
 
instrumentation of HPLC
instrumentation of HPLCinstrumentation of HPLC
instrumentation of HPLC
 
Adsorption Chromatography Assignment.pdf
Adsorption Chromatography Assignment.pdfAdsorption Chromatography Assignment.pdf
Adsorption Chromatography Assignment.pdf
 
Gas Chromatography
Gas ChromatographyGas Chromatography
Gas Chromatography
 
solid phase extraction method.pptx
solid phase extraction method.pptxsolid phase extraction method.pptx
solid phase extraction method.pptx
 
Partition column-chromatography
Partition column-chromatographyPartition column-chromatography
Partition column-chromatography
 
Gas chromatography
Gas chromatographyGas chromatography
Gas chromatography
 
Columns in HPLC
Columns in HPLCColumns in HPLC
Columns in HPLC
 
AFFINITY CHROMATOGRAPHY
AFFINITY CHROMATOGRAPHYAFFINITY CHROMATOGRAPHY
AFFINITY CHROMATOGRAPHY
 
atomic absorption spectroscopy
atomic absorption spectroscopyatomic absorption spectroscopy
atomic absorption spectroscopy
 
GEL CHROMATOGRAPHY.pptx
GEL CHROMATOGRAPHY.pptxGEL CHROMATOGRAPHY.pptx
GEL CHROMATOGRAPHY.pptx
 
HPLC Principle,Instrumentation and Application
HPLC Principle,Instrumentation and ApplicationHPLC Principle,Instrumentation and Application
HPLC Principle,Instrumentation and Application
 

Viewers also liked

HPLC - High Performance Liquid Chromatography
HPLC - High Performance Liquid ChromatographyHPLC - High Performance Liquid Chromatography
HPLC - High Performance Liquid ChromatographyDivya Basuti
 
Hplc (basic principles, operation and maintenance)
Hplc (basic principles, operation and maintenance)Hplc (basic principles, operation and maintenance)
Hplc (basic principles, operation and maintenance)alim125135
 
Instrumentation Of HPLC
Instrumentation Of HPLCInstrumentation Of HPLC
Instrumentation Of HPLCmddwivedi
 
Theory and application of High Performance Liquid Chromatography (HPLC)
Theory and application of High Performance Liquid Chromatography (HPLC)Theory and application of High Performance Liquid Chromatography (HPLC)
Theory and application of High Performance Liquid Chromatography (HPLC)Protik Biswas
 
Introduction to High Performance Liquid Chromatography (HPLC)
Introduction to High Performance Liquid Chromatography (HPLC)Introduction to High Performance Liquid Chromatography (HPLC)
Introduction to High Performance Liquid Chromatography (HPLC)Saurabh Arora
 
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR HIGH PRESSURE LIQUID CHROMAT...
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMAT...HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMAT...
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR HIGH PRESSURE LIQUID CHROMAT...Dr. Ravi Sankar
 
uv -visible spectroscopy
 uv -visible spectroscopy uv -visible spectroscopy
uv -visible spectroscopykeshav pai
 
UV Visible Spectroscopy
UV Visible SpectroscopyUV Visible Spectroscopy
UV Visible Spectroscopykrishslide
 
Introduction To Spectroscopy
Introduction To SpectroscopyIntroduction To Spectroscopy
Introduction To Spectroscopyguest824336
 
UV-Visible Spectroscopy
UV-Visible SpectroscopyUV-Visible Spectroscopy
UV-Visible SpectroscopySudha Reddy
 

Viewers also liked (20)

HPLC - High Performance Liquid Chromatography
HPLC - High Performance Liquid ChromatographyHPLC - High Performance Liquid Chromatography
HPLC - High Performance Liquid Chromatography
 
HPLC
HPLCHPLC
HPLC
 
Hplc (basic principles, operation and maintenance)
Hplc (basic principles, operation and maintenance)Hplc (basic principles, operation and maintenance)
Hplc (basic principles, operation and maintenance)
 
Hplc
HplcHplc
Hplc
 
HPLC
HPLCHPLC
HPLC
 
HPLC
HPLCHPLC
HPLC
 
Introduction to hplc
Introduction to hplcIntroduction to hplc
Introduction to hplc
 
Hplc instrumentation
Hplc instrumentationHplc instrumentation
Hplc instrumentation
 
HPLC instrument
HPLC instrumentHPLC instrument
HPLC instrument
 
Instrumentation Of HPLC
Instrumentation Of HPLCInstrumentation Of HPLC
Instrumentation Of HPLC
 
Hplc
Hplc Hplc
Hplc
 
Theory and application of High Performance Liquid Chromatography (HPLC)
Theory and application of High Performance Liquid Chromatography (HPLC)Theory and application of High Performance Liquid Chromatography (HPLC)
Theory and application of High Performance Liquid Chromatography (HPLC)
 
Akshay ppt
Akshay pptAkshay ppt
Akshay ppt
 
Electrophoresis
ElectrophoresisElectrophoresis
Electrophoresis
 
Introduction to High Performance Liquid Chromatography (HPLC)
Introduction to High Performance Liquid Chromatography (HPLC)Introduction to High Performance Liquid Chromatography (HPLC)
Introduction to High Performance Liquid Chromatography (HPLC)
 
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR HIGH PRESSURE LIQUID CHROMAT...
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMAT...HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR  HIGH PRESSURE LIQUID CHROMAT...
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR HIGH PRESSURE LIQUID CHROMAT...
 
uv -visible spectroscopy
 uv -visible spectroscopy uv -visible spectroscopy
uv -visible spectroscopy
 
UV Visible Spectroscopy
UV Visible SpectroscopyUV Visible Spectroscopy
UV Visible Spectroscopy
 
Introduction To Spectroscopy
Introduction To SpectroscopyIntroduction To Spectroscopy
Introduction To Spectroscopy
 
UV-Visible Spectroscopy
UV-Visible SpectroscopyUV-Visible Spectroscopy
UV-Visible Spectroscopy
 

Similar to Hplc

HPLC BY RAJ JAISWAL
HPLC BY RAJ JAISWAL HPLC BY RAJ JAISWAL
HPLC BY RAJ JAISWAL RAJJAISWAL68
 
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY/SAGAR/SHARMA/DEPARTMENT OF PHARMACEUTI...
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY/SAGAR/SHARMA/DEPARTMENT OF PHARMACEUTI...HIGH PERFORMANCE LIQUID CHROMATOGRAPHY/SAGAR/SHARMA/DEPARTMENT OF PHARMACEUTI...
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY/SAGAR/SHARMA/DEPARTMENT OF PHARMACEUTI...SagarMudgil1
 
HPLC Instrumentation, Pharmaceutical analysis, HPLC detectorsInstmn sem sharu(4)
HPLC Instrumentation, Pharmaceutical analysis, HPLC detectorsInstmn sem sharu(4)HPLC Instrumentation, Pharmaceutical analysis, HPLC detectorsInstmn sem sharu(4)
HPLC Instrumentation, Pharmaceutical analysis, HPLC detectorsInstmn sem sharu(4)Sharath Hns
 
High Performance Liquid Chromatography(HPLC).pptx
High Performance Liquid Chromatography(HPLC).pptxHigh Performance Liquid Chromatography(HPLC).pptx
High Performance Liquid Chromatography(HPLC).pptxOMJHA20
 
High performance liquid chromatography.pptx
High performance liquid chromatography.pptxHigh performance liquid chromatography.pptx
High performance liquid chromatography.pptxDhanashreeKavhale
 
High Performance Liquid Chromatography
High Performance Liquid ChromatographyHigh Performance Liquid Chromatography
High Performance Liquid ChromatographyMd. Kader
 
High performance liquid chromatography (hplc)
High performance liquid chromatography (hplc)High performance liquid chromatography (hplc)
High performance liquid chromatography (hplc)Pharm Ajahson
 
Basics of RP-HPLC.pptx
Basics of RP-HPLC.pptxBasics of RP-HPLC.pptx
Basics of RP-HPLC.pptxDeveshPelagade
 
High Performance Liquid Chromatography
High Performance Liquid ChromatographyHigh Performance Liquid Chromatography
High Performance Liquid ChromatographyPayal Vaswani
 
Demostration of HPLC Chromatography
Demostration of HPLC ChromatographyDemostration of HPLC Chromatography
Demostration of HPLC ChromatographyShwetaYadav150
 
Separation technology
Separation technologySeparation technology
Separation technologySarla Rao
 
Hplc by Bachaspati jana
Hplc by  Bachaspati janaHplc by  Bachaspati jana
Hplc by Bachaspati janaBachaspatiJana
 
high performance liquid chromatography 22
high performance liquid chromatography 22high performance liquid chromatography 22
high performance liquid chromatography 22alishapep2020
 

Similar to Hplc (20)

HPLC BY RAJ JAISWAL
HPLC BY RAJ JAISWAL HPLC BY RAJ JAISWAL
HPLC BY RAJ JAISWAL
 
Hplc
HplcHplc
Hplc
 
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY/SAGAR/SHARMA/DEPARTMENT OF PHARMACEUTI...
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY/SAGAR/SHARMA/DEPARTMENT OF PHARMACEUTI...HIGH PERFORMANCE LIQUID CHROMATOGRAPHY/SAGAR/SHARMA/DEPARTMENT OF PHARMACEUTI...
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY/SAGAR/SHARMA/DEPARTMENT OF PHARMACEUTI...
 
HPLC Instrumentation, Pharmaceutical analysis, HPLC detectorsInstmn sem sharu(4)
HPLC Instrumentation, Pharmaceutical analysis, HPLC detectorsInstmn sem sharu(4)HPLC Instrumentation, Pharmaceutical analysis, HPLC detectorsInstmn sem sharu(4)
HPLC Instrumentation, Pharmaceutical analysis, HPLC detectorsInstmn sem sharu(4)
 
High Performance Liquid Chromatography(HPLC).pptx
High Performance Liquid Chromatography(HPLC).pptxHigh Performance Liquid Chromatography(HPLC).pptx
High Performance Liquid Chromatography(HPLC).pptx
 
High performance liquid chromatography.pptx
High performance liquid chromatography.pptxHigh performance liquid chromatography.pptx
High performance liquid chromatography.pptx
 
High Performance Liquid Chromatography
High Performance Liquid ChromatographyHigh Performance Liquid Chromatography
High Performance Liquid Chromatography
 
High performance liquid chromatography (hplc)
High performance liquid chromatography (hplc)High performance liquid chromatography (hplc)
High performance liquid chromatography (hplc)
 
Basics of RP-HPLC.pptx
Basics of RP-HPLC.pptxBasics of RP-HPLC.pptx
Basics of RP-HPLC.pptx
 
Hplc
Hplc Hplc
Hplc
 
High Performance Liquid Chromatography
High Performance Liquid ChromatographyHigh Performance Liquid Chromatography
High Performance Liquid Chromatography
 
Hplc and gc ppt
Hplc and gc pptHplc and gc ppt
Hplc and gc ppt
 
Liquid chromatography
Liquid chromatographyLiquid chromatography
Liquid chromatography
 
Demostration of HPLC Chromatography
Demostration of HPLC ChromatographyDemostration of HPLC Chromatography
Demostration of HPLC Chromatography
 
Hplc
HplcHplc
Hplc
 
Separation technology
Separation technologySeparation technology
Separation technology
 
Chromatography & PCR
Chromatography & PCRChromatography & PCR
Chromatography & PCR
 
Hplc by Bachaspati jana
Hplc by  Bachaspati janaHplc by  Bachaspati jana
Hplc by Bachaspati jana
 
high performance liquid chromatography 22
high performance liquid chromatography 22high performance liquid chromatography 22
high performance liquid chromatography 22
 
HPLC
HPLC HPLC
HPLC
 

More from Musa Khan

Water and food culture
Water and food cultureWater and food culture
Water and food cultureMusa Khan
 
Serum protein electrophpresis
Serum protein electrophpresisSerum protein electrophpresis
Serum protein electrophpresisMusa Khan
 
Blood grouping
Blood groupingBlood grouping
Blood groupingMusa Khan
 
Instruments used in microbiology
 Instruments used in microbiology Instruments used in microbiology
Instruments used in microbiologyMusa Khan
 
Frozen section
Frozen sectionFrozen section
Frozen sectionMusa Khan
 
Media in microbiology
Media in microbiologyMedia in microbiology
Media in microbiologyMusa Khan
 
cytology of body fluid
 cytology of body fluid cytology of body fluid
cytology of body fluidMusa Khan
 
Approach To A Patient With Anemia
Approach To A Patient With Anemia  Approach To A Patient With Anemia
Approach To A Patient With Anemia Musa Khan
 
ATOMIC ABSORPTION SPECTROSCOPY
ATOMIC ABSORPTION SPECTROSCOPYATOMIC ABSORPTION SPECTROSCOPY
ATOMIC ABSORPTION SPECTROSCOPYMusa Khan
 
Mycobacterium.ppt
Mycobacterium.pptMycobacterium.ppt
Mycobacterium.pptMusa Khan
 
Modified elisa
Modified elisaModified elisa
Modified elisaMusa Khan
 
DNA SEQUENCING METHOD
DNA SEQUENCING METHODDNA SEQUENCING METHOD
DNA SEQUENCING METHODMusa Khan
 
CYTOLOGY OF CSF
CYTOLOGY OF CSFCYTOLOGY OF CSF
CYTOLOGY OF CSFMusa Khan
 
CULTURE MEDIA USED IN MICROBIOLOGY
CULTURE  MEDIA USED IN MICROBIOLOGYCULTURE  MEDIA USED IN MICROBIOLOGY
CULTURE MEDIA USED IN MICROBIOLOGYMusa Khan
 
HistoPATHOLOGY
HistoPATHOLOGYHistoPATHOLOGY
HistoPATHOLOGYMusa Khan
 

More from Musa Khan (20)

Water and food culture
Water and food cultureWater and food culture
Water and food culture
 
Serum protein electrophpresis
Serum protein electrophpresisSerum protein electrophpresis
Serum protein electrophpresis
 
Blood grouping
Blood groupingBlood grouping
Blood grouping
 
Instruments used in microbiology
 Instruments used in microbiology Instruments used in microbiology
Instruments used in microbiology
 
Frozen section
Frozen sectionFrozen section
Frozen section
 
Media in microbiology
Media in microbiologyMedia in microbiology
Media in microbiology
 
cytology of body fluid
 cytology of body fluid cytology of body fluid
cytology of body fluid
 
Approach To A Patient With Anemia
Approach To A Patient With Anemia  Approach To A Patient With Anemia
Approach To A Patient With Anemia
 
ATOMIC ABSORPTION SPECTROSCOPY
ATOMIC ABSORPTION SPECTROSCOPYATOMIC ABSORPTION SPECTROSCOPY
ATOMIC ABSORPTION SPECTROSCOPY
 
Porphyria
PorphyriaPorphyria
Porphyria
 
Porphyria
PorphyriaPorphyria
Porphyria
 
KARYOTYPING
KARYOTYPINGKARYOTYPING
KARYOTYPING
 
Mycobacterium.ppt
Mycobacterium.pptMycobacterium.ppt
Mycobacterium.ppt
 
Modified elisa
Modified elisaModified elisa
Modified elisa
 
DNA SEQUENCING METHOD
DNA SEQUENCING METHODDNA SEQUENCING METHOD
DNA SEQUENCING METHOD
 
CYTOLOGY OF CSF
CYTOLOGY OF CSFCYTOLOGY OF CSF
CYTOLOGY OF CSF
 
CULTURE MEDIA USED IN MICROBIOLOGY
CULTURE  MEDIA USED IN MICROBIOLOGYCULTURE  MEDIA USED IN MICROBIOLOGY
CULTURE MEDIA USED IN MICROBIOLOGY
 
ANEMIA
ANEMIAANEMIA
ANEMIA
 
CSF
CSFCSF
CSF
 
HistoPATHOLOGY
HistoPATHOLOGYHistoPATHOLOGY
HistoPATHOLOGY
 

Recently uploaded

Metabolism , Metabolic Fate& disorders of cholesterol.pptx
Metabolism , Metabolic Fate& disorders of cholesterol.pptxMetabolism , Metabolic Fate& disorders of cholesterol.pptx
Metabolism , Metabolic Fate& disorders of cholesterol.pptxDr. Santhosh Kumar. N
 
LEAD5623 The Economics of Community Coll
LEAD5623 The Economics of Community CollLEAD5623 The Economics of Community Coll
LEAD5623 The Economics of Community CollDr. Bruce A. Johnson
 
Dhavni Theory by Anandvardhana Indian Poetics
Dhavni Theory by Anandvardhana Indian PoeticsDhavni Theory by Anandvardhana Indian Poetics
Dhavni Theory by Anandvardhana Indian PoeticsDhatriParmar
 
Plant Tissue culture., Plasticity, Totipotency, pptx
Plant Tissue culture., Plasticity, Totipotency, pptxPlant Tissue culture., Plasticity, Totipotency, pptx
Plant Tissue culture., Plasticity, Totipotency, pptxHimansu10
 
LEAD6001 - Introduction to Advanced Stud
LEAD6001 - Introduction to Advanced StudLEAD6001 - Introduction to Advanced Stud
LEAD6001 - Introduction to Advanced StudDr. Bruce A. Johnson
 
DNA and RNA , Structure, Functions, Types, difference, Similarities, Protein ...
DNA and RNA , Structure, Functions, Types, difference, Similarities, Protein ...DNA and RNA , Structure, Functions, Types, difference, Similarities, Protein ...
DNA and RNA , Structure, Functions, Types, difference, Similarities, Protein ...AKSHAYMAGAR17
 
POST ENCEPHALITIS case study Jitendra bhargav
POST ENCEPHALITIS case study  Jitendra bhargavPOST ENCEPHALITIS case study  Jitendra bhargav
POST ENCEPHALITIS case study Jitendra bhargavJitendra Bhargav
 
UNIT I Design Thinking and Explore.pptx
UNIT I  Design Thinking and Explore.pptxUNIT I  Design Thinking and Explore.pptx
UNIT I Design Thinking and Explore.pptxGOWSIKRAJA PALANISAMY
 
EDD8524 The Future of Educational Leader
EDD8524 The Future of Educational LeaderEDD8524 The Future of Educational Leader
EDD8524 The Future of Educational LeaderDr. Bruce A. Johnson
 
AUDIENCE THEORY - PARTICIPATORY - JENKINS.pptx
AUDIENCE THEORY - PARTICIPATORY - JENKINS.pptxAUDIENCE THEORY - PARTICIPATORY - JENKINS.pptx
AUDIENCE THEORY - PARTICIPATORY - JENKINS.pptxiammrhaywood
 
LEAD6001 - Introduction to Advanced Stud
LEAD6001 - Introduction to Advanced StudLEAD6001 - Introduction to Advanced Stud
LEAD6001 - Introduction to Advanced StudDr. Bruce A. Johnson
 
2024.03.16 How to write better quality materials for your learners ELTABB San...
2024.03.16 How to write better quality materials for your learners ELTABB San...2024.03.16 How to write better quality materials for your learners ELTABB San...
2024.03.16 How to write better quality materials for your learners ELTABB San...Sandy Millin
 
The OERs: Transforming Education for Sustainable Future by Dr. Sarita Anand
The OERs: Transforming Education for Sustainable Future by Dr. Sarita AnandThe OERs: Transforming Education for Sustainable Future by Dr. Sarita Anand
The OERs: Transforming Education for Sustainable Future by Dr. Sarita AnandDr. Sarita Anand
 
Pharmacology chapter No 7 full notes.pdf
Pharmacology chapter No 7 full notes.pdfPharmacology chapter No 7 full notes.pdf
Pharmacology chapter No 7 full notes.pdfSumit Tiwari
 
Riti theory by Vamana Indian poetics.pptx
Riti theory by Vamana Indian poetics.pptxRiti theory by Vamana Indian poetics.pptx
Riti theory by Vamana Indian poetics.pptxDhatriParmar
 
BÀI TẬP BỔ TRỢ TIẾNG ANH 11 THEO ĐƠN VỊ BÀI HỌC - CẢ NĂM - CÓ FILE NGHE (FRIE...
BÀI TẬP BỔ TRỢ TIẾNG ANH 11 THEO ĐƠN VỊ BÀI HỌC - CẢ NĂM - CÓ FILE NGHE (FRIE...BÀI TẬP BỔ TRỢ TIẾNG ANH 11 THEO ĐƠN VỊ BÀI HỌC - CẢ NĂM - CÓ FILE NGHE (FRIE...
BÀI TẬP BỔ TRỢ TIẾNG ANH 11 THEO ĐƠN VỊ BÀI HỌC - CẢ NĂM - CÓ FILE NGHE (FRIE...Nguyen Thanh Tu Collection
 
Alamkara theory by Bhamaha Indian Poetics (1).pptx
Alamkara theory by Bhamaha Indian Poetics (1).pptxAlamkara theory by Bhamaha Indian Poetics (1).pptx
Alamkara theory by Bhamaha Indian Poetics (1).pptxDhatriParmar
 
PHARMACOGNOSY CHAPTER NO 5 CARMINATIVES AND G.pdf
PHARMACOGNOSY CHAPTER NO 5 CARMINATIVES AND G.pdfPHARMACOGNOSY CHAPTER NO 5 CARMINATIVES AND G.pdf
PHARMACOGNOSY CHAPTER NO 5 CARMINATIVES AND G.pdfSumit Tiwari
 
DLL Catch Up Friday March 22.docx CATCH UP FRIDAYS
DLL Catch Up Friday March 22.docx CATCH UP FRIDAYSDLL Catch Up Friday March 22.docx CATCH UP FRIDAYS
DLL Catch Up Friday March 22.docx CATCH UP FRIDAYSTeacherNicaPrintable
 
BBA 205 BE UNIT 2 economic systems prof dr kanchan.pptx
BBA 205 BE UNIT 2 economic systems prof dr kanchan.pptxBBA 205 BE UNIT 2 economic systems prof dr kanchan.pptx
BBA 205 BE UNIT 2 economic systems prof dr kanchan.pptxProf. Kanchan Kumari
 

Recently uploaded (20)

Metabolism , Metabolic Fate& disorders of cholesterol.pptx
Metabolism , Metabolic Fate& disorders of cholesterol.pptxMetabolism , Metabolic Fate& disorders of cholesterol.pptx
Metabolism , Metabolic Fate& disorders of cholesterol.pptx
 
LEAD5623 The Economics of Community Coll
LEAD5623 The Economics of Community CollLEAD5623 The Economics of Community Coll
LEAD5623 The Economics of Community Coll
 
Dhavni Theory by Anandvardhana Indian Poetics
Dhavni Theory by Anandvardhana Indian PoeticsDhavni Theory by Anandvardhana Indian Poetics
Dhavni Theory by Anandvardhana Indian Poetics
 
Plant Tissue culture., Plasticity, Totipotency, pptx
Plant Tissue culture., Plasticity, Totipotency, pptxPlant Tissue culture., Plasticity, Totipotency, pptx
Plant Tissue culture., Plasticity, Totipotency, pptx
 
LEAD6001 - Introduction to Advanced Stud
LEAD6001 - Introduction to Advanced StudLEAD6001 - Introduction to Advanced Stud
LEAD6001 - Introduction to Advanced Stud
 
DNA and RNA , Structure, Functions, Types, difference, Similarities, Protein ...
DNA and RNA , Structure, Functions, Types, difference, Similarities, Protein ...DNA and RNA , Structure, Functions, Types, difference, Similarities, Protein ...
DNA and RNA , Structure, Functions, Types, difference, Similarities, Protein ...
 
POST ENCEPHALITIS case study Jitendra bhargav
POST ENCEPHALITIS case study  Jitendra bhargavPOST ENCEPHALITIS case study  Jitendra bhargav
POST ENCEPHALITIS case study Jitendra bhargav
 
UNIT I Design Thinking and Explore.pptx
UNIT I  Design Thinking and Explore.pptxUNIT I  Design Thinking and Explore.pptx
UNIT I Design Thinking and Explore.pptx
 
EDD8524 The Future of Educational Leader
EDD8524 The Future of Educational LeaderEDD8524 The Future of Educational Leader
EDD8524 The Future of Educational Leader
 
AUDIENCE THEORY - PARTICIPATORY - JENKINS.pptx
AUDIENCE THEORY - PARTICIPATORY - JENKINS.pptxAUDIENCE THEORY - PARTICIPATORY - JENKINS.pptx
AUDIENCE THEORY - PARTICIPATORY - JENKINS.pptx
 
LEAD6001 - Introduction to Advanced Stud
LEAD6001 - Introduction to Advanced StudLEAD6001 - Introduction to Advanced Stud
LEAD6001 - Introduction to Advanced Stud
 
2024.03.16 How to write better quality materials for your learners ELTABB San...
2024.03.16 How to write better quality materials for your learners ELTABB San...2024.03.16 How to write better quality materials for your learners ELTABB San...
2024.03.16 How to write better quality materials for your learners ELTABB San...
 
The OERs: Transforming Education for Sustainable Future by Dr. Sarita Anand
The OERs: Transforming Education for Sustainable Future by Dr. Sarita AnandThe OERs: Transforming Education for Sustainable Future by Dr. Sarita Anand
The OERs: Transforming Education for Sustainable Future by Dr. Sarita Anand
 
Pharmacology chapter No 7 full notes.pdf
Pharmacology chapter No 7 full notes.pdfPharmacology chapter No 7 full notes.pdf
Pharmacology chapter No 7 full notes.pdf
 
Riti theory by Vamana Indian poetics.pptx
Riti theory by Vamana Indian poetics.pptxRiti theory by Vamana Indian poetics.pptx
Riti theory by Vamana Indian poetics.pptx
 
BÀI TẬP BỔ TRỢ TIẾNG ANH 11 THEO ĐƠN VỊ BÀI HỌC - CẢ NĂM - CÓ FILE NGHE (FRIE...
BÀI TẬP BỔ TRỢ TIẾNG ANH 11 THEO ĐƠN VỊ BÀI HỌC - CẢ NĂM - CÓ FILE NGHE (FRIE...BÀI TẬP BỔ TRỢ TIẾNG ANH 11 THEO ĐƠN VỊ BÀI HỌC - CẢ NĂM - CÓ FILE NGHE (FRIE...
BÀI TẬP BỔ TRỢ TIẾNG ANH 11 THEO ĐƠN VỊ BÀI HỌC - CẢ NĂM - CÓ FILE NGHE (FRIE...
 
Alamkara theory by Bhamaha Indian Poetics (1).pptx
Alamkara theory by Bhamaha Indian Poetics (1).pptxAlamkara theory by Bhamaha Indian Poetics (1).pptx
Alamkara theory by Bhamaha Indian Poetics (1).pptx
 
PHARMACOGNOSY CHAPTER NO 5 CARMINATIVES AND G.pdf
PHARMACOGNOSY CHAPTER NO 5 CARMINATIVES AND G.pdfPHARMACOGNOSY CHAPTER NO 5 CARMINATIVES AND G.pdf
PHARMACOGNOSY CHAPTER NO 5 CARMINATIVES AND G.pdf
 
DLL Catch Up Friday March 22.docx CATCH UP FRIDAYS
DLL Catch Up Friday March 22.docx CATCH UP FRIDAYSDLL Catch Up Friday March 22.docx CATCH UP FRIDAYS
DLL Catch Up Friday March 22.docx CATCH UP FRIDAYS
 
BBA 205 BE UNIT 2 economic systems prof dr kanchan.pptx
BBA 205 BE UNIT 2 economic systems prof dr kanchan.pptxBBA 205 BE UNIT 2 economic systems prof dr kanchan.pptx
BBA 205 BE UNIT 2 economic systems prof dr kanchan.pptx
 

Hplc

  • 3. Introduction  Abbreviated for; High-performance liquid chromatography OR High-pressure liquid chromatography  Defined as; “A chromatographic technique used to separate components of mixture for the purpose to identify, quantify or purify the individual components of the mixture “.  Widely used in field of biochemistry and analytical chemistry.
  • 4. History  Invented by Martin and Synge as extension of gas chromatography.  The main idea was that; “Small sorbent particles and pressure could produce fast liquid chromatography techniques”.  The method was used practically in late 1960s for separation amino acids.
  • 5. Instrumentation  HPLC involves following functional instruments. 1. Mobile phase reservoir 2. Pump 3. Injector 4. Stationary phase ( Column) 5. Detector 6. Computer
  • 6. 1. Mobile phase reservoir: • Commonly glass bottles with caps are used. • Teflon tubings and filters are connected to purge gas (helium) for degassing. • Vaccum for 5-10 min is also used for degassing. 2. Pump: • It forces the mobile phase to pass through column. • Flow rate is 1-2 ml/ min. • Trypical pressure is 6000 – 9000psi. 3. Injector: • Can be manually (syringe) or automated. • Sample volume 5-20µl. • Ideal to stand pressure of mobile phase. • Autosampler is used for analysis of many samples automatically.
  • 7. 4. Stationary phase (Column): • Heart of HPLC. • Separate sample components on basis of physical and chemical parameters. • Lenght 10-30cm. • Diameter 4-10nm. • Packing material 5-10nm thick. 5. Detector: • Detection of elutes from column. • Quantitative analysis of sample components. • Output transferred to recorder/ computer. 6. Computer: • Data system that controls modules of HPLC. • Signals from detector are interpreted to determine elution time, quantitative and qualitative analysis of sample.
  • 9. Figure displaying complex instrumentation of HPLC
  • 10. Operation • Operation strategies are explained as follows; 1. Sample:  The sample to be analyzed is introduced into stream of mobile phase in minute quantity, mostly in microliters.  The components of sample moves through different velocities reaching the column where it physically interacts with the sorbent.  Velocity of sample components depends on; • Chemical nature of sample • Nature of column • Composition of mobile phase
  • 11. 2. Column: • Also known as stationary phase. • Composed of sorbent material varying in particle size, and surface chemistry. • Size of particles can be smaller which improves chromatographic resolution. • In terms of surface chemistry, the column can be hydrophobic and polar in nature.
  • 12. A B A. Plant extracts separated by HPLC by passing through stationary phase (column) B. Columns used in HPLC device
  • 13. 3. Eluent: • Also known as mobile phase.  Type of solvent used: • Commonly it is composed of water miscible with organic solvents. For example methanol. • Some HPLC utilizes water free mobile phase and uses acids such as formic acid or phosphoric acid. • Many mobile phase works well by using salts such as sodium phosphate.
  • 14.  Composition of eluent: • It depends on; • Intensity of interactions between analytes and stationary phase. For example; i. Hydrophobic interactions in reversed-phase HPLC ii. Polar interactions in normal phase HPLC iii. Ionic interaction in ion exchange HPLC • Often a series of trial runs are performed with the sample in order to find the HPLC method which gives adequate separation.
  • 15. Figure showing mobile phase reserviors
  • 17. Figure showing operation of HPLC and analysis of analyte present in the sample
  • 18. Types 1. Partition chromatography 2. Normal phase chromatography 3. Displacement chromatography 4. Reversed phase chromatography 5. Size-exclusion chromatography 6. Ion-exchange chromatography 7. Bioaffinity chromatography 8. Aqueous normal-phase chromatography
  • 19. Advantages • Advantages of HPLC is; • Seapartion of voltile and non- voltile components. • Thermally unstable compounds isolated. • Quick analysis • High resolution • Less cumbersome • More reproducibility
  • 20. Disadvantages • Disadvantages of HPLC are; • Tedious to detect co-elution. • High cost. • Complex to operate.