CHEM 2010: Liquid Chromatography Lecture 5
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Questions and Answers

How does decreasing particle size in a packed column affect high performance liquid chromatography (HPLC)?

  • Decreases selectivity during separation
  • Increases sensitivity of detection systems
  • Reduces resistance to mobile phase flow
  • Improves efficiency of the packed column (correct)

    • What is the main characteristic of isocratic elution in chromatography?

  • Maintains constant mobile phase composition during the run (correct)
  • Utilizes multiple solvents for separation
  • Varies the mobile phase composition during the run
  • Involves non-destructive detection systems

    • What is a key requirement for HPLC pumps to be effective?

  • They should be made of a porous material
  • They must be able to generate pressures below 3,000 psi
  • They must generate pressures of up to 6,000 psi or more (correct)
  • They must use only volatile solvents

    • Which statement about HPLC detection systems is accurate?

    <p>They tend to be non-destructive for samples</p> Signup and view all the answers

    What is a significant advantage of high performance liquid chromatography in analytical applications?

    <p>It provides high sensitivity for a wide range of compounds</p> Signup and view all the answers

    What does the retention factor (Rf) indicate in Thin Layer Chromatography?

    <p>The distance moved by the compound relative to the solvent</p> Signup and view all the answers

    Which of the following is NOT an advantage of Thin Layer Chromatography?

    <p>Requires expensive equipment</p> Signup and view all the answers

    What materials can serve as the stationary phase in Thin Layer Chromatography?

    <p>Silica gel and cellulose</p> Signup and view all the answers

    Which step in column chromatography involves the separation of the adsorbed substance?

    <p>Continuous flow of mobile phase</p> Signup and view all the answers

    What is the primary purpose of developing a TLC plate?

    <p>To identify unknown compounds</p> Signup and view all the answers

    In column chromatography, which factor plays a crucial role in the qualitative analysis of solute?

    <p>The configuration of the stationary phase</p> Signup and view all the answers

    Which industry primarily utilizes Thin Layer Chromatography for its applications?

    <p>Pharmaceutical industry</p> Signup and view all the answers

    Which of the following statements about mobile phase in Thin Layer Chromatography is FALSE?

    <p>It must always be non-polar.</p> Signup and view all the answers

    What is the primary distinction between Column Chromatography and Planar Chromatography?

    <p>Column Chromatography employs a cylindrical tube for stationary phase, while Planar uses a flat surface.</p> Signup and view all the answers

    In High Performance Liquid Chromatography (HPLC), what characterizes the mobile phase?

    <p>It is a liquid under high pressure.</p> Signup and view all the answers

    Which term in the van Deemter Equation corresponds to the resistance to mass transfer?

    <p>Cu</p> Signup and view all the answers

    What factor primarily influences the retention time (tR) in chromatography?

    <p>The capacity factor k′.</p> Signup and view all the answers

    What is the main difference between Liquid-Liquid Chromatography and Liquid-Solid Chromatography?

    <p>Liquid-Liquid utilizes a liquid stationary phase; Liquid-Solid uses a solid phase.</p> Signup and view all the answers

    In terms of efficiency, when does the resolution (Rs) significantly improve?

    <p>At small values of the capacity factor k′.</p> Signup and view all the answers

    Which component is NOT typically part of liquid chromatography instrumentation?

    <p>Heating coils</p> Signup and view all the answers

    What common application is found in industries utilizing chromatography?

    <p>Determining alcohol levels in blood samples.</p> Signup and view all the answers

    Study Notes

    CHEM 2010: Chemical Analysis A - Chromatography

    •  Course: CHEM 2010: Chemical Analysis A
    •  Lecture: Liquid Chromatography A
    •  Lecturer: Dr. Joyann Marks
    •  Email: [email protected]
    •  Lecture number 5 of 8

    Van Deemter Equation

    •  H = A + B/u + Cu
    •  A: Eddy diffusion term
    •  B/u: Longitudinal diffusion term
    •  Cu: Mass transfer term (resistance to)

    Variation of Retention (Elution) Time and Resolution with Capacity (Retention) Factor

    •  Resolution (R) improves greatly at small values of k'
    •  But above k' > 5 (or 10), no improvement in R, but longer elution time (tR)
    •  Optimal k' is usually between 1 and 5 (or 10)

    Lesson Objectives

    •  Lecture 5 focuses on liquid chromatography: planar, column, and high-performance liquid chromatography (HPLC)
    •  Topics include instrumentation, operating principles, columns, detectors, sample preparation, and applications
    •  Students will understand the operating principles of liquid chromatography
    •  Students will explore the components of liquid chromatography instrumentation.

    Types of Chromatography

    •  Chromatography encompasses various techniques - Supercritical-fluid chromatography (SFC) - Gas chromatography (GC) – - Gas-solid chromatography (GSC) - Gas-liquid chromatography (GLC) - Liquid chromatography (LC) – - Liquid-solid chromatography (LSC) - Liquid-liquid chromatography (LLC) - Ion exchange chromatography - Gel permeation chromatography (GPC) / gel filtration chromatography - Thin-layer chromatography (TLC) - Paper chromatography

    Liquid Chromatography

    •  Mobile phase: Liquid
    •  Separation is on a column or a plane
    •  High-performance liquid chromatography (HPLC) uses small packing particles and high pressure.

    Liquid Chromatography Classifications

    •  Liquid-liquid chromatography (partitioning)
    •  Liquid-solid chromatography (adsorption)
    •  Column chromatography vs planar chromatography
    •  Thin-layer chromatography (TLC) vs Paper chromatography (PC)
    •  HPLC

    Column Chromatography

    •  Stationary phase is held in a cylindrical tube
    •  Mobile phase is forced through by gravity or pressure

    Planar Chromatography

    •  Stationary phase is supported on a flat plate or in the pores of paper
    •  Mobile phase moves through the stationary phase by capillary action or gravity

    Thin Layer Chromatography (TLC)

    •  A separation technique with a stationary phase present as a plane.

    •  Includes paper chromatography and thin-layer chromatography (TLC)

    •  Types include paper chromatography, thin-layer chromatography (TLC) (solid particles on a flat support)

    •  Different compounds travel at different rates depending on how strongly they interact with the stationary phase compared to the mobile phase

    •  Retention factor (Rf) is used to compare components

    •  Widely used in pharmaceutical and food industries

    •  Advantages for TLC:

    • Simple, rapid, and cheap.

    • Faster runs

    • Better separations.

    • Choice between different adsorbents.

    • Better resolution

    • Allows for quantification

    •  Stationary phase: Adsorbent powder (SiO2 or Al2O3) on a solid plate (glass, aluminum, or plastic)

    •  Used to identify unknown compounds and determine purity of mixtures

    •  Sample solution is spotted on plate, evaporated

    •  TLC plate: Aluminum or glass coated with the stationary phase

    •  Coated material thickness: 0.1-0.3 mm

    •  Fluorescent indicator for UV light exposure

    •  Stationary phase: Silica gel, alumina, or cellulose on a flat, inert substrate

    •  Mobile phase: Volatile organic solvents

    TLC Plate Development

    •  The process of carrying a sample through a stationary phase using a mobile phase.
    •  The TLC plate is tilted at an angle in a solvent-containing chamber.
    •  The solvent ascends the plate, carrying the sample components with it.

    Components in Thin Layer Chromatography

    •  Mobile phase: Solvent (A, B, and C) moving across the plate.
    •  Adsorbed solvent (A, B, C): Liquid solvent on the plate.
    •  Plate Surface: Plate where the adsorbed solvents are.
    •  Adsorbent: Solid phase coated on the plate surface.

    Thin-Layer Chromatography Calculation

    •  Rf = dR/dm –dR = Distance moved by compound –dm = Distance moved by solvent

    Column Chromatography

    •  Adsorption/retention of substance on the stationary phase.
    •  Separation of the adsorbed substance using a mobile phase.
    •  Recovery of individual components via a continuous flow of mobile phase
    •  Quantitative and qualitative analysis of solutes recovered.

    HPLC (High-Performance Liquid Chromatography)

    •  Efficiency of a packed column increases as particle size decreases.
    • Smaller particle size leads to increased resistance to mobile phase flow.
    •  New technology features 3mm diameter particles, and pressures up to 6000 psi

    HPLC Advantages

    • High sensitivity
    • Applicable to non-volatile and thermally unstable compounds
    • Applicable to compounds of wide interest and importance.
    • Detection systems tend to be non-destructive.
    • Options for separation: Isocratic elution (constant mobile phase composition) and Gradient elution (varying mobile phase composition).

    HPLC Instrument Components

    •  Solvent/Solvent reservoir
    •  Pump (Gradient)
    •  Injection valve
    •  Column
    •  Detectors
    •  Recorder/data processing

    Solvent/Solvent Reservoir

    •  Adequate capacity for the reservoir
    •  Degassing of solvents is crucial
    •  High purity and particulate-free solvent are essential.

    HPLC Pump Requirements

    •  Inert or corrosion-resistant material construction
    •  Ability to generate pressure up to 6,000 psi (or higher in some cases)
    •  Pulse-free output (low noise)
    •  Delivery of flow rates from 0.1 to 10 mL/min, with increments
    •  High flow reproducibility (0.5% or better)
    •  Gradient capability for varying mobile phase composition

    Injection Valve (Sample Introduction)

    - Six-port, rotary action  - Two positions (Load and Inject)  - Sample volume determined by loop volume.

    Injection Valve- Considerations

     - Stainless steel construction to withstand high pressures

    • Very reproducible sample delivery
    • Sample volume within loop can be altered for varied analyses.
    • Relatively easy to use compared to syringe injection for GC (Gas Chromatography).

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    Lecture 5: Liquid Chromatography A PDF

    Description

    This quiz covers key concepts from Lecture 5 of CHEM 2010: Chemical Analysis A, focusing on liquid chromatography including its types, principles, and the Van Deemter Equation. Students will enhance their understanding of chromatography's practical applications and factors affecting retention time.

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