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    HPLC

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    kushal
    HPLC is a type of column chromatography used to separate and analyze mixtures of chemical compounds. It works by pumping a pressurized liquid solvent containing the sample mixture through a column containing a solid adsorbent material. Compounds within the mixture interact differently with the solid material and elute from the column at different rates, allowing for separation. HPLC is useful for analyzing both volatile and non-volatile compounds, and can be used for qualitative and quantitative analysis in various fields such as pharmaceuticals, biology, and environmental science.

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    HPLC

    Uploaded by

    kushal
    82 views12 pages
    HPLC is a type of column chromatography used to separate and analyze mixtures of chemical compounds. It works by pumping a pressurized liquid solvent containing the sample mixture through a column containing a solid adsorbent material. Compounds within the mixture interact differently with the solid material and elute from the column at different rates, allowing for separation. HPLC is useful for analyzing both volatile and non-volatile compounds, and can be used for qualitative and quantitative analysis in various fields such as pharmaceuticals, biology, and environmental science.

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    HPLC PPT

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    HPLC is a type of column chromatography used to separate and analyze mixtures of chemical compounds. It works by pumping a pressurized liquid solvent containing the sample mixture through a column containing a solid adsorbent material. Compounds within the mixture interact differently with the solid material and elute from the column at different rates, allowing for separation. HPLC is useful for analyzing both volatile and non-volatile compounds, and can be used for qualitative and quantitative analysis in various fields such as pharmaceuticals, biology, and environmental science.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    82 views12 pages

    HPLC

    Uploaded by

    kushal
    HPLC is a type of column chromatography used to separate and analyze mixtures of chemical compounds. It works by pumping a pressurized liquid solvent containing the sample mixture through a column containing a solid adsorbent material. Compounds within the mixture interact differently with the solid material and elute from the column at different rates, allowing for separation. HPLC is useful for analyzing both volatile and non-volatile compounds, and can be used for qualitative and quantitative analysis in various fields such as pharmaceuticals, biology, and environmental science.

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    of 12

    HIGH PERFORMANCE LIQUID

    CHROMATOGRAPHY [HPLC]
    HPLC
     HPLC is a High Performance liquid Chromatography.
     High Pressure Liquid Chromatography.
     High Priced Liquid Chromatography.
     It is column chromatography.
     It is Liquid Chromatography.
     It is modified from of gas chromatography, it is applicable for both Volatile as well as
    Non volatile compound.
     It can mainly divided by two types 1. Normal phase HPLC 2. Reversed Phase HPLC.
     It is having a high resolution and separation capacity.
     It is used as qualitative as well as quantitative analysis.
     High performance liquid chromatography (HPLC) is a chromatographic technique used
    to separate a mixture of compounds in analytical chemistry and biochemistry with the
    purpose of identifying, quantifying or purifying the individual components of the
    mixture.
    7
    PRINCIPLE
     High Performance Liquid Chromatography [HPLC] is principle is based on
    adsorption as well as partition chromatography is depending on the nature
    of stationary phase, if stationary phase is solid principle is based on adsorption
    chromatography and if stationary phase is liquid principle is based on partition
    chromatography.
     It is important for determination of volatile and non volatile compounds.

     It is important for determination qualitative and quantitative analysis.


     It is important for determination of Retention Time (the time is required , after
    sample injection maximum angle peak reaches to detector)

    Common mobile phases used include any miscible combination of water with various
    organic solvents (the most common are acetonitrile and methanol)

    A separation in which the mobile phase composition remains constant throughout the
    procedure is termed isocratic (meaning constant composition).

    A separation in which the mobile phase composition is changed during the separation
    process is described as a gradient elution
    ADVANTAGES
     It is simple, rapid , reproducible.
     High sensitivity.
     High performance.
     Rapid process and hence time saving.
     It is having a high resolution and separation capacity.
     Accuracy and Precision.
     Stationary phase was chemically innert.
     Wide varities of stationary phase.
     Mobile phase was chemically innert.
     Less requirement of mobile phase in developing chamber.
     Early recovery of separated component.
     Easy visualization of separated components.
     It is having Good reproducibility and repeatability.
     It is analytical technique is important for validation of product, quality control
    studies of product.
     It is important for qualitative and quantitative analysis.
     It is used for both analytical and preparative purpose.
    TYPES OF HPLC SEPARATIONS
     Normal Phase: Separation of polar analytes by partitioning onto a polar, bonded
    stationary phase.

     Reversed Phase: Separation of non-polar analytes by partitioning onto a non-polar,


    bonded stationary phase.

    WHY USE HPLC


     Simultaneous analysis
     High resolution

     High sensitivity

     Good repeatability

     Moderate analysis condition

     Easy to fractionate and purify

     Not destructive
    INSTRUMENTATION OF HPLC

     Solvent storage bottle


     Gradient controller and mixing unit

     De-gassing of solvents

     Pump

     Pressure gauge

     Pre-column

     Sample introduction system

     Column

     Detector

     Recorder
    SELECTION OF DETECTORS
    Detectors Type of compounds can be detected
    UV-Vis & Compounds with chromophores, such as aromatic rings or
    Photo diode array multiple alternating double bonds.
    detector
    Radio freqency Fluorescent compounds, usually with fused rings or highly
    detector RF conjugated planar system.

    Conductivity Charged compounds, such as inorganic ions and organic


    detector CDD acid.
    Electron capture
    detector ECD For easily oxidized compounds like quinones or amines.

    Refractive index (detects the change in turbidity)


    Mass spectrometry (HPLC-MS)
    COMPARISION BETWEEN GC AND HPLC

    SR.NO. GC HPLC

    1 Gas Chromatography High Performance Liquid


    Chromatography
    2 Less resolution High resolution
    3 Limited Flexibility Extreme Flexibility
    4 Determination of Volatile Determination of Volatile and Non
    compounds Volatile Compounds
    APPLICATION
     Drug Discovery
     Clinical Analysis
     Proteomics
     Forensic Chemistry
     Drug Metabolism study
     Environmental chemistry
     Diagnostic studies
     Cosmetic analysis
     Structural Determination
     Pharmaceutical Applications
     Clinical Applications
     Biochemical Genetics
     qualitative and quantitative analysis
     Therapeutic Drug Monitoring

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