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 (C)</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 (B)</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 (B)</p> Signup and view all the answers

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

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

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

<p>Silica gel and cellulose (A)</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 (C)</p> Signup and view all the answers

What is the primary purpose of developing a TLC plate?

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

Which industry primarily utilizes Thin Layer Chromatography for its applications?

<p>Pharmaceutical industry (C)</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. (C)</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. (A)</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. (A)</p> Signup and view all the answers

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

<p>Cu (C)</p> Signup and view all the answers

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

<p>The capacity factor k′. (C)</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. (B)</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′. (D)</p> Signup and view all the answers

Which component is NOT typically part of liquid chromatography instrumentation?

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

What common application is found in industries utilizing chromatography?

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

Flashcards

Liquid Chromatography (LC)

A separation technique using a liquid mobile phase and a stationary phase (column or plane).

Mobile Phase (LC)

The liquid that flows through the stationary phase in liquid chromatography.

High Performance Liquid Chromatography (HPLC)

A type of liquid chromatography that uses high pressure to force the mobile phase through the column.

Column Chromatography

A type of liquid chromatography where the stationary phase is held in a cylindrical tube.

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Planar Chromatography

A type of liquid chromatography where the stationary phase is on a flat surface (like a plate or paper).

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Retention Factor (k')

A measure of how strongly a compound interacts with the stationary phase.

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Resolution (Rs)

A measure of how well two separated components can be distinguished.

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Van Deemter Equation

An equation that relates the plate height (H) of a chromatographic column to the linear velocity (u) of the mobile phase.

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HPLC Efficiency

The efficiency of a packed column in HPLC increases as the particle size of the stationary phase decreases. This is because smaller particles provide more surface area for interaction with the analyte, leading to better separation.

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HPLC Pressure

HPLC requires high pressure to force the mobile phase through the column, especially when using small particle sizes which have higher resistance to flow. Systems with pressure up to 6,000 psi are common, while 'Ultra High Performance Liquid Chromatography' systems can reach 14,000 psi.

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Why degas solvents?

Solvents used in HPLC must be degassed to remove dissolved gases, which can cause bubbles in the system, affecting flow, pressure, and detection. Degassed solvents also improve the performance and lifespan of the HPLC pump.

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Isocratic vs Gradient Elution

Isocratic elution uses a constant mobile phase composition throughout the run, while gradient elution changes the mobile phase composition during the run. Gradient elution offers improved selectivity by varying the eluting strength of the mobile phase, allowing for better separation of complex mixtures.

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HPLC Pump Requirements

HPLC pumps must be made from inert or corrosion-resistant materials to avoid contamination of the mobile phase. They need to be able to generate high pressure, up to 6,000 psi for standard systems and even higher for Ultra High Performance Liquid Chromatography.

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Thin Layer Chromatography

A planar chromatography technique where a thin layer of adsorbent material (like silica gel) is spread on a plate (glass, aluminium, or plastic).

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Stationary Phase (TLC)

The adsorbent material (like silica gel or alumina) that's coated on the TLC plate. It's a solid.

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What are the advantages of Thin Layer Chromatography?

TLC is a simple, rapid, and inexpensive technique that provides good separation, multiple choices of adsorbents, and greater resolution.

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Applications of Thin Layer Chromatography

TLC can identify unknown compounds, determine the purity of a mixture, and examine fractions collected from other separation techniques.

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TLC Plate Development

The process in TLC where the mobile phase carries the sample components through the stationary phase.

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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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