High Performance Liquid Chromatography HPLC- UV-VIS Detector Animation

00:05:45
https://www.youtube.com/watch?v=eCj0cRtJvJg

Sintesi

TLDRHigh-Performance Liquid Chromatography (HPLC) is a crucial analytical method used to effectively separate, identify, and quantify components within mixtures. The system consists of a mobile phase, which is pushed through the system by a pump, and a stationary phase contained in a column. Sample introduction can be automatic or manual, and the system incorporates a detector for analyzing the separated components post-column. The differentiation of sample components relies on their varying affinities toward the mobile and stationary phases. HPLC can be performed in isocratic or gradient modes, affecting how solvents interact during the separation process. Two primary forms of HPLC include reverse phase, with a non-polar stationary phase, and normal phase, where the stationary phase is polar. Various detectors can be integrated, such as UV detectors, to quantify samples based on specific wavelength absorption. Finally, the output data is presented through a chromatogram, detailing the retention times and quantifying the analytes present by measuring peak areas.

Punti di forza

  • 🔬 HPLC is key for separating and quantifying mixtures.
  • 🚰 The system uses a mobile phase pushed by a pump.
  • 🔄 Sample can be injected manually or automatically.
  • 🧪 Detection occurs after separation in the column.
  • ⚙️ Two main types: reverse phase and normal phase.
  • 📈 UV detectors measure absorbance in light spectra.
  • 📊 Chromatograms display peaks corresponding to components.

Linea temporale

  • 00:00:00 - 00:05:45

    High-performance liquid chromatography (HPLC) is a technique in analytical chemistry used for the separation, identification, and quantification of components in a mixture. The process involves a mobile phase, pumped through the system, and a stationary phase, usually a column located in an oven with controlled temperature. Samples can be injected automatically via an auto sampler or manually using a syringe. A detector measures the separated analytes. An integral part of the system includes a vacuum pump, which removes dissolved gases from the solvents and ensures that the solvent flow to the mixing chamber occurs under high pressure. HPLC typically utilizes isocratic or gradient elution for analysis. In isocratic mode, the mobile phase composition stays constant, while in gradient mode, it changes during separation. Sample introduction can occur via an injection valve, ensuring a smooth transition of the sample into the column. The separation process relies on the differential partitioning of components between the mobile and stationary phases, where components with lower affinity for the stationary phase elute faster.

Mappa mentale

Video Domande e Risposte

  • What is HPLC?

    HPLC stands for High-Performance Liquid Chromatography, a technique used to separate, identify, and quantify components in a mixture.

  • What are the main components of an HPLC system?

    The main components include a mobile phase, stationary phase (column), pump, injector, and detector.

  • What are the two main types of HPLC?

    The two main types are reverse phase HPLC and normal phase HPLC.

  • What is the function of the detector in HPLC?

    The detector measures the analytes after they have been separated in the column.

  • What is the difference between isocratic and gradient elution?

    In isocratic elution, the mobile phase composition remains constant, while in gradient elution, the mobile phase composition changes during separation.

  • How are samples introduced into the HPLC system?

    Samples can be introduced automatically using an auto sampler or manually with a syringe.

  • What types of detectors can be used in HPLC?

    Common detectors include UV detectors that measure absorbance in the ultraviolet or visible regions.

  • What indicates the number of components in a mixture in an HPLC chromatogram?

    The number of peaks in the chromatogram indicates the number of components present in the mixture.

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Sottotitoli
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Scorrimento automatico:
  • 00:00:00
    high-performance liquid chromatography
  • 00:00:01
    is a technique and analytical chemistry
  • 00:00:04
    used to separate identify and quantify
  • 00:00:06
    each component in a mixture as well as
  • 00:00:09
    other forms of chromatography in HPLC
  • 00:00:12
    there is a mobile phase which forced by
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    a pump to pass through the system and
  • 00:00:16
    there is a stationary phase called a
  • 00:00:18
    column which located in an oven where
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    the temperature can be controlled
  • 00:00:24
    the sample can be automatically injected
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    into an HPLC system by the use of HPLC
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    auto sampler
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    or can be manually introduced into the
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    injector using a syringe
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    there is also a detector attached to the
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    HPLC system which measures the analytes
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    after its separation in the column
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    the pump forces the mobile phase through
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    the column and then the detector under
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    high pressures
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    in the HPLC system a vacuum pump and a
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    dagger sir are connected to the pump and
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    used to remove dissolved gases from the
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    solvents the pump drives each solvent to
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    the mixing chamber where mixing takes
  • 00:01:09
    place under higher pressures HPLC
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    analysis commonly uses isocratic or
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    gradient elution an isocratic mode the
  • 00:01:18
    mobile phase composition remains
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    constant throughout the procedure
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    whereas in gradient mode the mobile
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    phase composition is changed during the
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    separation process
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    sample introduction can be accomplished
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    in various ways the simplest method is
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    to use an injection valve in the load
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    position the high-pressure el Yuting
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    solvent flows to the column directly the
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    loop is loaded at the atmospheric
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    pressure from a syringe via the needle
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    port excess sample exits the loop via
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    vent port after loading the sample the
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    valve is switched to the inject position
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    the flow delivered by the pump flows
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    through the loop forcing the sample
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    ahead of it flowing to the column
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    then the valve returns to the load
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    position and the mobile phase moves the
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    sample through the column
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    the separation is based on differential
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    partitioning of the sample components
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    between the mobile and stationary phases
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    the component which has more affinity to
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    the mobile phase consequently less
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    affinity to the stationary phase travels
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    faster and eluded out first and the
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    component which has more affinity to the
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    stationary phase consequently more
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    interaction travels slower and diluted
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    later this separation can be carried out
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    according to which type of HPLC used
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    there are two main types of HPLC reverse
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    phase and normal phase reversed phase
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    has a non-polar stationary phase and
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    moderately polar mobile phase one common
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    stationary phase is a silica which has
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    been modified by attaching a straight
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    chain alkyl group to its surface such as
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    the ITAT as'll group c18 or the octal
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    group c8
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    in this case the more hydrophobic the
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    analytes are the more retained they will
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    be on the stationary phase the more
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    polar they are the more they will prefer
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    the mobile phase
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    then normal phase chromatography the
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    stationary phase is polar and the mobile
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    phase is non-polar this method separates
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    analytes based on their affinity for a
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    polar stationary surface such as silica
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    in this case the more polar the analytes
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    are the more retained they will be on
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    the stationary phase the more
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    hydrophobic they are the more they will
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    prefer the mobile phase
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    as compounds ellyiot from the column
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    they interact with the detector
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    different types of detectors can be used
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    such as the UV this detector which
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    showing an absorption spectrum in the
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    ultraviolet or visible region for UV
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    detection a deuterium discharge lamp as
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    a light source is used and four
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    components detection and visible region
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    a tungsten lamp is used then UV this
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    detector we can also find entrance slit
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    lens prism or diffraction grating exit
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    slit flow cell and detector for
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    absorption measurements
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    light from the lamp is shown onto the
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    prism and dispersed according to
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    wavelength when the measurement is
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    performed with a specific wavelength the
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    angle of the prism is adjusted so that
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    the light of this wave can shine on the
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    flow cell as the compound zealot from
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    the column they enter the flow cell
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    where the bonding and non-bonding
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    electrons of these compounds can absorb
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    energy in the form of ultraviolet or
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    visible light
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    the manner in which the final data is
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    displayed is based on the computer in
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    software
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    the number of Peaks present can indicate
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    how many components are in the mixture
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    usually the x axis of the HPLC
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    chromatogram shows the amount of time
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    taken for the analytes to pass through
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    the column and reach the detector
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    typically the y-axis or the area of the
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    peak is a reflection of the amount of a
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    specific analyte that's present
Tag
  • HPLC
  • chromatography
  • analytical chemistry
  • separation
  • detection
  • mobile phase
  • stationary phase
  • isocratic
  • gradient elution
  • UV detector