Chromatography 7

Introduction to Separation and Chromatography

  • Instructor: Marwa Fouad, PhD

  • Contact: marwa.fouad@ngu.edu.eg

  • Module: PHAR 201

Learning Outcomes (LOs)

  • a3: Demonstrate understanding of various quantitative analytical techniques.

  • a4: Integrate organic and analytical chemistry knowledge for pharmaceutical analysis.

  • b2: Apply quality control requirements in pharmaceutical material analysis.

  • b3: Recognize principles of various instruments and select techniques for synthesis and analysis of pharmaceuticals.

Separation Science

  1. Key Concepts

    • Polarity, Miscibility, and Solubility

    • Partitioning and Extraction

    • Liquid/Liquid Extraction

  2. Chromatography

    • Classes of Chromatography:

      • Thin Layer Chromatography (TLC)

      • Column Chromatography

    • Instrumental Techniques:

      • High-Performance Liquid Chromatography (HPLC)

      • Gas Chromatography (GC)

Fundamental Principles of Chromatography

Visualization Experiment

  • Illustration: Placing a drop of ink on tissue paper demonstrates dye migration through solvent channels.

Forces in Chromatography

  • Movement Mechanism: Solutes move along channels (stationary phase) filled with solvent molecules (mobile phase).

  • Key Forces:

    • F1: Solvent flow (mobile phase solubility influence)

    • F2: Retardation forces (affinity to stationary phase)

      • Interactions include:

        • Hydrogen bonds

        • Dipole forces

        • Hydrophobic interactions

Stationary and Mobile Phases

Solid Stationary Phase

  • Suggestion: Performing many small extractions is more effective than one large extraction.

  • Mechanism: Utilize solid stationary phase while running liquid mobile phase through it.

Classes of Chromatography

  1. Normal Phase Chromatography

    • Principle: Adsorption with silica as stationary phase.

    • Types of Interactions include:

      • Ionic

      • Hydrogen bonding

      • Dipole-dipole interactions

  2. Reversed Phase Chromatography

    • Principle: Partitioning using a non-polar stationary phase (bonded lipophilic groups).

    • Mechanism: Separation based on solubility differences in the mobile and stationary phases.

  3. Exclusion Chromatography

    • Principle: Size exclusion with uniform, porous stationary phases.

    • Application: Separates by molecular size and shape; ideal for macromolecules like proteins.

  4. Ion Exchange Chromatography

    • Principle: Separation based on ionic forces at charged stationary phases.

    • Mechanism: Separates cations or anions based on their exchange affinities.

Specific Chromatography Examples

Thin Layer Chromatography (TLC)

  • Understanding Rf Value:

    • Formula: Rf = Distance travelled by sample/Distance travelled by solvent.

    • Rf values provide insight into the sample's properties and interactions with the solvent.

    • Example: Separation of water-soluble vitamins using TLC.

Separation Techniques by Chromatography

  • Case Study: Separation of opioids using normal phase chromatography

    • Highlights differences in molecular interaction and retention.

Advanced Techniques and Reading Recommendations

Column Chromatography

  • Types include Adsorption/Partition, Exclusion, and Ion exchange chromatography; note that the last two require 3D stationary phases.

Suggested Reading

  • Books:

    • Florence AT & Attwood D. "Physicochemical Principles of Pharmacy" (Chapter 5)

    • Martin, A. "Physical Pharmacy" (Chapter 10)

    • Skoog, West, Holler & Crouch. "Fundamentals of Analytical Chemistry" (Chapters 30-32)