CHROMATOGRAPHY

Lecture 3: Optimising LLE SPE Separation Using pH

Summary

  • Recap of Liquid-Liquid Extraction (LLE)
  • Effect of pH on acidic and basic analyte solutions
  • Optimising Liquid-Liquid Extraction
  • Optimising Solid Phase Extraction (SPE)

Recap: Liquid-Liquid Extraction (LLE)

  • Definition: LLE involves two immiscible liquids, typically an aqueous layer and a non-aqueous layer (organic).
  • The analyte and impurities partition into the separate layers during extraction.
    • Extraction Methods: Components to be extracted are dissolved in a polar (aqueous) and non-polar (organic) solvent.

Process of LLE

  1. Add a sample containing the analyte to the solvent.
  2. Mix the two layers.
  3. Allow separation and carefully transfer the organic layer.
    • Outcomes:
      • Analyte + impurities in one layer
      • Pure analyte collected from the other layer.

Assumptions in LLE

  • Analyte Conditions: Analyte is assumed to be completely non-polar (non-ionised).
  • Impurities: All impurities are polar (ionised).
  • Reverse Extraction:
    • Possible to have impurities in the organic layer and analyte in the aqueous layer.

Partition Coefficient

  • Definition: A measure of the ratio of concentrations of a substance in two immiscible phases at equilibrium. Extraction recovery depends on the partition coefficient.

Effect of pH on Analyte Solutions

  • The pH can significantly impact the extraction process.
  • pH changes alter the polarity of the analyte:
    • Ionised (Polar) Form: Increased solubility in polar solvents.
    • Non-ionised (Non-polar) Form: Increased solubility in non-polar solvents.
  • Changes affinity of analytes for the solvent types based on their ionisation state.
  • Changes the partition coefficient and thus the separation efficiency.
  • Important factors:
    • Whether the analyte is a base or an acid.
    • The analyte's pKa value.

Acidic and Basic Analytes

Acidic Analytes
  • General Reaction: HA + H₂O ⇌ A¯ + H₃O⁺
    • Non-ionised Form: Not charged, hydrophobic, lipophilic.
    • Ionised Form: Charged, hydrophilic, lipophobic.
  • More non-ionised at lower (acidic) pH.
Basic Analytes
  • General Reaction: B + H₂O ⇌ BH⁺ + OH⁻
    • Non-ionised Form: Not charged, hydrophobic, lipophilic.
    • Ionised Form: Charged, hydrophilic, lipophobic.
  • More non-ionised at higher (basic) pH.

Le Chatelier’s Principle

  • Principle: A system at equilibrium will respond to disturbances by opposing the change.
  • Example for Acid Analytes:
    • Adding Acid: Increases H₃O⁺, shifts equilibrium left.
    • Adding Base: Decreases H₃O⁺, shifts equilibrium right.

Rules for Analytes and pH

  • Acid Analytes:
    • Become more non-ionised in acidic pH.
    • Become more ionised in basic pH.
  • Basic Analytes:
    • Become more non-ionised in basic pH.
    • Become more ionised in acidic pH.
  • Visual Representation: Understand pH environments and their effects on ionisation states.

Recap of Charged vs Non-charged Analytes

  • Charged Analytes:
    • Ionised, prefer aqueous solvents.
  • Non-charged Analytes:
    • Non-ionised, prefer non-polar solvents.

Required Information for Extraction Optimization

  1. Determine if the analyte is an acid or a base.
  2. Identify the pKa of the analyte.
  3. Measure the pH environment.

Weak Acids and Dissociation Constants

  • For weak acids, the dissociation constant (Ka) is low, e.g. aspirin has Ka = 3.16 × 10⁻⁴.
  • Strong acids fully dissociate, leading to infinite Ka.
  • Use of pKa: Preferentially used as it provides better insight into the concentration equilibrium.

pH and Half-Equivalence Point

  • Definition: pH = pKa at the half-equivalence point means:
    • 50% of HA is unreacted (non-ionised), 50% is dissociated (ionised).
  • Changes in pH will adjust these proportions.

Henderson-Hasselbalch Equation

  • Used to calculate the pH relative to pKa and dissociated concentrations during titration.

Predicting Ionisation and Environmental Impact

  • Using the nature of the analyte, pKa, and pH, one can:
    • Predict if the analyte is primarily ionised or non-ionised.
    • Estimate necessary pH for liquid-liquid extraction.

Optimising Liquid-Liquid Extraction (LLE) with pH

  • Rule of Thumb:
    • Alternative pH ranges for ionisation:
    • pKa - 3: 0.1% non-ionised acidic molecules, 99.9% non-ionised basic molecules.
    • pKa - 2: 1% non-ionised acidic, 99% basic.
    • pKa: 50% non-ionised.
    • pKa + 3: 99.9% non-ionised acidic, 0.1% non-ionised basic.

Solid Phase Extraction (SPE)

Overview of SPE
  • Components:
    • Cartridge, vacuum pump, syringes, luer fittings.
  • Mechanism: Utilizes solid-phase for extraction, typically reverse-phase (C-18) for non-polar interactions.
Steps in SPE Process
  1. Conditioning with a polar solvent.
  2. Waste collection post conditioning.
  3. Adding the sample matrix.
  4. Washing to remove impurities.
  5. Collecting eluted analyte.
Types of SPE
  • Reverse Phase SPE:
    • Polar liquid phase, non-polar solid phase.
    • Utilizes hydrophobic interactions—applicable in various analyte contexts, such as drugs and amino acids.
  • Normal Phase SPE:
    • Non-polar liquid phase, polar solid phase.
    • Utilizes polar-polar interactions.
Ion Exchange SPE
  • Mechanism: Utilizes electrostatic attractions for separation of anions (negative) and cations (positive).
    • Anionic exchange needs non-ionised analytes with proper pH adjustments above pKa for elution; cationic exchange requires similar principles but in reverse.