Pharmaceutical Analysis Lecture 2: Chromatographic Theory
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Questions and Answers

What does the capacity factor (k) measure in chromatography?

  • The amount of time an analyte spends in the solvent
  • The ratio of retention time to void time for the analyte (correct)
  • The total volume of the solvent used
  • The efficiency of the chromatography column

    • If the void volume (Vo) of a column is 1 mL and the retention time (tr) for a compound is 6 minutes, what is the retention volume (Vr) if k is 5?

  • 9 mL
  • 5 mL
  • 6 mL
  • 7 mL (correct)

    • In a chromatography context, what does the term selectivity (α) refer to?

  • The overall efficiency of the chromatography column
  • The spacing between two peaks in a chromatogram (correct)
  • The total retention time of all compounds in the mixture
  • The amount of solvent used in the experiment

    • How is the column efficiency expressed in chromatography?

    <p>In theoretical plates per meter</p> Signup and view all the answers

    What is the formula for calculating the number of theoretical plates (n) in a chromatography setup?

    <p>n = 5.54 (tr/W1/2)2</p> Signup and view all the answers

    What does the 'plate' theory of chromatography primarily describe?

    <p>The efficiency of separation in chromatography.</p> Signup and view all the answers

    Which factor is crucial for achieving high resolution in chromatography?

    <p>Column temperature.</p> Signup and view all the answers

    Which of the following components of a chromatogram indicates the presence of a particular compound?

    <p>The retention time.</p> Signup and view all the answers

    What information can be derived from the Van Deemter equation in the context of HPLC?

    <p>The optimal flow rate for maximum efficiency.</p> Signup and view all the answers

    In the context of a HPLC operation, what role does the mobile phase 'A' play when combined with 'B'?

    <p>Provides the main solvent for dissolution of solutes.</p> Signup and view all the answers

    What is a common application of gas chromatography (GC)?

    <p>Separation of opiates and related substances.</p> Signup and view all the answers

    What parameter is critical to ensure consistency in chromatographic results?

    <p>Calibration of the detector.</p> Signup and view all the answers

    Which type of chromatography is best suited for analyzing non-volatile compounds?

    <p>High Performance Liquid Chromatography (HPLC).</p> Signup and view all the answers

    What is the relationship between efficiency and the length of the column in chromatography?

    <p>Efficiency is inversely proportional to column length.</p> Signup and view all the answers

    Which term represents the distance required for a single partition step to occur in a chromatography column?

    <p>Height Equivalent of a Theoretical Plate (HETP)</p> Signup and view all the answers

    What does band broadening in chromatography primarily result from?

    <p>Variability in time spent by individual molecules in the column.</p> Signup and view all the answers

    In the Van Deemter equation, what does the term 'B' refer to?

    <p>Rate of diffusion of molecule in the mobile phase</p> Signup and view all the answers

    How does a higher linear velocity (u) of the mobile phase affect band broadening?

    <p>It reduces the time analyte spends on the column.</p> Signup and view all the answers

    Which factor does NOT contribute to resistance to mass transfer in stationary phase?

    <p>Nature of the mobile phase solvent</p> Signup and view all the answers

    What effect does a thinner and uniform stationary phase have on band broadening?

    <p>It reduces the resistance to mass transfer.</p> Signup and view all the answers

    Which of the following terms describes the effect of different paths taken by solute molecules in the column?

    <p>Eddy diffusion term (A)</p> Signup and view all the answers

    What happens to the efficiency of chromatographic separation when longitudinal diffusion is decreased?

    <p>Efficiency increases.</p> Signup and view all the answers

    Which of the following factors is a part of the Van Deemter equation that contributes to band broadening?

    <p>Eddy diffusion term</p> Signup and view all the answers

    Which term significantly contributes to band broadening at low flow rates?

    <p>B term</p> Signup and view all the answers

    What is baseline resolution achieved when Rs is greater than or equal to?

    <p>1.5</p> Signup and view all the answers

    In the Van Deemter equation, which term corresponds to the eddy diffusion?

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

    Which type of gas is most commonly used for good efficiency without reducing flow rate in GC?

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

    What characteristic of the stationary phase contributes to increased column efficiency?

    <p>Small, uniformly-shaped particles</p> Signup and view all the answers

    What does the selectivity factor (α) describe in chromatography?

    <p>Separation of band centers</p> Signup and view all the answers

    To increase retention factor (k) in gas chromatography (GC), what is a recommended action?

    <p>Change the temperature</p> Signup and view all the answers

    Which factor is more influential in enhancing resolution than efficiency in chromatographic processes?

    <p>Increasing the capacity factor</p> Signup and view all the answers

    What effect does using capillaries with smaller internal diameters have in GC?

    <p>Reduces transverse diffusion effect</p> Signup and view all the answers

    Which mobile phase has gained popularity in GC for high efficiency at high flow rates?

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

    Study Notes

    Pharmaceutical Analysis Lecture 2: Chromatographic Theory

    • Recommended Texts:

      • Pharmaceutical Analysis by David G. Watson (various editions)
      • Quantitative Chemical Analysis by Daniel C. Harris (8th edition, 14 copies available in the Drill Hall Library)

    • Learning Outcomes:

      • Students should understand the information presented in a chromatogram.
      • Students should understand the plate theory of chromatography.
      • Students should understand the rate theory of chromatography.
      • Students should understand the Van Deemter equation's implications in High-Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC).
      • Students should understand the significance of resolution in chromatography.

    HPLC Chromatogram (Barbiturates)

    • Separation: Barbiturates were separated using high-performance liquid chromatography.
    • Column: Discovery C18, 15 cm × 4.6 mm ID, 5 μm particles.
    • Column Temperature: Ambient.
    • Mobile Phase: A mixture of methanol and water (45:55).
    • Flow Rate: 1 mL/min.
    • Injection Volume: 10 μL.
    • Detector: UV at 214 nm.
    • Separation: Opiates and related substances were separated using gas chromatography.
    • Column: 15 m × 0.25 mm × 0.25 μm Rxi-5ms
    • Additional Details: Included specific compounds like Hydrocodone, Diazepam, THC, Fentanyl, Codeine, Morphine, Oxycodone, Flunitrazepam, and Benzoylecgonine.

    Chromatograms: Production and Information

    • Production: Chromatograms are generated by injecting a sample into a stream of mobile solvent. The components are separated based on their interactions with the stationary phase.
    • Information Gained: Chromatograms provide information concerning the retention volume/time, necessary for identification and to quantify different components

    Chromatograms: Capacity Factor (k)

    • Definition: The capacity factor (k) measures the fraction of time an analyte spends in the stationary phase relative to the mobile phase.
    • Formula: k = (Vᵣ - V₀) / V₀ = (tᵣ - t₀) / t₀
      • Vᵣ = analyte retention volume
      • V₀ = void volume
      • tᵣ = analyte retention time
      • t₀ = retention time of unretained compound

    Chromatography: Selectivity (α)

    • Definition: Selectivity (α) is a measure of the separation of two peaks.
    • Formula: α = k(B) / k(A)
      • k(B) = capacity factor of component B,
      • k(A) = capacity factor of component A

    Chromatography: Column Efficiency (n)

    • Definition: Measures the number of theoretical plates (n) in a column, correlating with the peak width. Efficiency is usually expressed in units of theoretical plates per meter [n x 100 / L].
    • Formula: n = 5.54 (t'/W½)²
      • t' = adjusted retention time
      • W½= peak width

    Rate Theory of Chromatography

    • Description: A more detailed explanation of chromatographic processes, accounting for equilibration times between the mobile and stationary phases.
    • Factors affecting peak shapes: Equilibrium rate and paths in the column.

    Van Deemter Equation

    • Purpose: Explains how various factors contribute to band broadening.
    • Components:
      • A = Eddy diffusion term (variation in path lengths).
      • B = Longitudinal diffusion term (diffusion of analyte).
      • C = Resistance to mass transfer in stationary phase.

    Peak Characteristics & Band Broadening

    • Peak Asymmetry (A): Measure of peak shape, ideally close to 1.
    • Band Broadening: Occurs due to variations in movement times of individual molecules within the same compound.

    Factors Affecting Column Efficiency

    • Stationary Phase: Thin, uniform coatings of small, regularly shaped particles improve efficiency.
    • Mobile Phase: High diffusion coefficient results in better efficiency.
    • Temperature: Higher temperatures generally improve efficiency in GC.

    Resolution (RS):

    • Expression 1 Formula: RS = 2(tRB - tRA)/(WA + WB)
    • Baseline Resolution: When RS ≥ 1.5.
    • Expression 2 Formula: RS = 1.18(tRB - tRA)/(WA0.5 + WB0.5).
    • Effect on R: Efficiency increase improves resolution only by a 1.41 factor. Capacity factor changes have a stronger impact.

    Resolution Equation:

    • Description: A fundamental equation for relating resolution to important column parameters (number of plates, selectivity factor, and retention factors)
    • Practical Implications: Increasing efficiency slightly improves the resolution, but altering retention factors will result in greater changes to resolution.

    Practical Considerations:

    • Increasing 'k' in LC: Modifying the mobile phase composition.
    • Increasing 'k' in GC: Changing the temperature.

    Resolution Example

    • Mobile Phase Changes in LC: Example details using adjusted water/acetonitrile ratios

    Peak Asymmetry (AS)

    • Importance: Critical for proper peak analysis; relates to peak shape and symmetry.
    • Characteristics: Values of 1.0 to 1.05 are considered excellent, while values higher than 2.0 indicate unacceptable shape

    Take Home Points

    • Chromatogram Information: Components' identification and quantification from retention/time data.
    • Chromatographic Theories: Understanding the fundamentals of plate and rate theories.
    • Van Deemter Equation: Recognizing its role in band broadening, and how factors like stationary phases, mobile phases and temperature affect column efficiency.
    • Resolution Significance: Understanding the importance of resolution, its formulas (expressions 1 and 2) and implications and examples for optimizing separations.

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    Related Documents

    MSOP1015 Pharmaceutical Analysis Lecture 2: Chromatographic theory 2024-2025 - PDF

    Description

    This quiz focuses on the fundamental concepts of chromatographic theory as outlined in the second lecture of Pharmaceutical Analysis. It covers key elements like plate theory, rate theory, and the Van Deemter equation as they pertain to HPLC and GC. Understanding chromatograms and resolution will also be emphasized.

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