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    Pharmacognosy

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    Fizzah Razzaq
    This document provides an overview of high performance liquid chromatography (HPLC). It describes the basic components and principles of HPLC including the stationary and mobile phases, modes of separation, solvents, columns, and major detectors. The document also discusses applications of HPLC and concludes that it is a highly sophisticated technique that has revolutionized the pharmaceutical industry through its ability to efficiently separate mixture components.

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    Pharmacognosy

    Uploaded by

    Fizzah Razzaq
    131 views23 pages
    This document provides an overview of high performance liquid chromatography (HPLC). It describes the basic components and principles of HPLC including the stationary and mobile phases, modes of separation, solvents, columns, and major detectors. The document also discusses applications of HPLC and concludes that it is a highly sophisticated technique that has revolutionized the pharmaceutical industry through its ability to efficiently separate mixture components.

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    pharmacognosy

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    This document provides an overview of high performance liquid chromatography (HPLC). It describes the basic components and principles of HPLC including the stationary and mobile phases, modes of separation, solvents, columns, and major detectors. The document also discusses applications of HPLC and concludes that it is a highly sophisticated technique that has revolutionized the pharmaceutical industry through its ability to efficiently separate mixture components.

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    131 views23 pages

    Pharmacognosy

    Uploaded by

    Fizzah Razzaq
    This document provides an overview of high performance liquid chromatography (HPLC). It describes the basic components and principles of HPLC including the stationary and mobile phases, modes of separation, solvents, columns, and major detectors. The document also discusses applications of HPLC and concludes that it is a highly sophisticated technique that has revolutionized the pharmaceutical industry through its ability to efficiently separate mixture components.

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    You are on page 1of 23

    HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

    By Khizar Abbas

    HPLC
    TECHNIQUE USED TO SEPARATE COMPONENT OF MIXTURE BY USING A VARIETY OF CHEMICAL INTERACTIONS B/W THE SUBSTANCE BEING ANALYZED & THE CHROMATOGRAPHIC COLUMN

    Types of HPLC
    1. 2. 3. 4. 5. Normal phase chromatography Reversed phase chromatography Size exclusion chromatography Ion exchange chromatography Bioaffinity chromatography

    Mode of separation
    It depends upon nature of stationary phase. Five modes Adsorption Partition Ion exchange Size exclusion affinity

    1. 2. 3. 4. 5.

    PRINCIPLE
    SAMPLE TO BE ANALYZED IS INTRODUCED IN SMALL VOLUME IN THE MOBILE PHASE RETARDED BY SPECIFIC CHEMICAL OR PHYSICAL INTERACTION WITH STARIONARY PHASE

    THE AMOUNT OF RETARDATION DEPENDS ON 1. THE NATURE OF ANALYTE 2. NATURE OF STATIONARY PHASE 3. COMPOSITION OF MOBILE PHASE

    SOLVENTS USED
    Miscible combination of water Various organic liquids Methanol Acetonitrile Water may contain salt or buffer like Trifluroacetic Acid which assist in the separation of analyte component

    STATIONARY PHASE
    The stationary phase is packed in column In NP-HPLC It may be Silica,Alumina or porous graphite In RPC-HPLC it is modified Silica,Alumina or Porous Graphite For ion-exchange it may be Resin or polymer with Acid or Base group In SEC porous silica or polymers are used In Chiral Chromatography cellulose or amylase derivatives are used

    PARTS OF HPLC

    MAJOR PARTS OF HPLC


    1. 2. 3. 4. 5. 6. SOLVENT RESVIOR PUMP SAMPLE INJECTORS SAMPLING VALVES & LOOPS COLUMN DETECTORS

    SOLVENT RESVIOR
    HPLC uses a liquid to push a sample This liquid is called MOBILE PHASE or simply solvent

    PUMP
    PUMPS are used to pump the solvent through the HPLC The pressure used is sometime so high 1900 psi

    Different types of Pumps


    Reciprocating piston pump Syringe type pump Constant pressure pump

    SAMPLE INJECTORS
    Syringe is used to inject the sample into HPLC Normal syringe is used to inject sample by hand Auto injector can automatically inject a whole series of sample

    COLUMN
    Most important part of HPLC Where work gets down It is a long open tube that is coated with a stationary phase Mobile phase pushes the sample into the column Small molecules in sample pass very quickly Large molecules get caught in stationary phase

    TYPES OF COLUMN
    1. 2. 3. 4. GUARD COLUMN DERIVATIZING COLUMN CAPILLARY COLUMN MICROBORE OR SMALL BORE COLUMN 5. FAST COLUMN 6. PREPARATORY COLUMN

    DETECTORS
    Detectors are the component of an HPLC that emits a response due to eluting sample & subsequently signals a peak on the chromatogram

    TYPES OF DETECTORS
    1. 2. 3. 4. Refractive index detector Ultra-Violet detector Fluorescent detector Radiochemical detector

    5.
    6. 7. 8. 9.

    Electrochemical detector
    Mass Spectroscopy detector Nuclear Magnetic Resonance detector Light Scattering detector Near Infrared detector

    RETENTION TIME
    Time taken for a particular compound to travel through the column to the detector. Retention time depend on Pressure used Nature of stationary phase Composition of mobile phase Temperature of the column

    Applications;
    HPLC is used in analysis, separation and purification of a large verity of components, like Proteins, Peptides and Amino Acids Nucleic Acids Saccharides and Organic Acids Drugs, Metabolites and Chiral Compounds Vitamins, Hormones and Lipids Pesticide Residue, Synthetic Polymers Anions and Cations

    Conclusion
    It is highly sophisticated technique which has revolutionized pharmaceutical industry. It is time saving process Further research is going in this field would lead to more reliable and efficacy of results.

    References
    Fast and Ultrafast HPLC on sub-2 m Porous Particles Where Do We Go From Here? - LC-GC Europe Xiang, Y.; Liu Y. and Lee M.L. (2006). "". Journal of Chromatography A 1104 (1-2): 198-202. Horvth, Cs.; Preiss B.A. and Lipsky S.R. (1967). "". Analytical Chemistry 39: 14221428.

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