Chromatography

Chromatography Lectures by Dr. Mustafa Ozel

Learning Outcomes

  • Introduction to Chromatography

    • Familiarization with chromatography methods.

  • Understanding Phases

    • Differentiation between mobile and stationary phases.

  • Chromatographic Terminology

    • Learn key terms such as:

      • Chromatographic methods

      • Retention time

      • Column efficiency

      • Plate theory

      • Selectivity

      • Resolution

  • Types of Chromatography

    • Thin Layer Chromatography (TLC)

    • High Performance Liquid Chromatography (HPLC)

    • Gas Chromatography (GC)

    • Types of chromatography separation:

      • Adsorption

      • Partition

      • Ion exchange

      • Size exclusion

      • Spectroscopic methods

      • Chromatographic methods

Basics of Chromatography

  • Definition

    • Chromatography is a method of separation. The Greek words suggest: "chroma" = color and "graphein" = writing.

  • Mechanism

    • Analytes distribute between two phases: the stationary phase and the mobile phase.

  • Types of Chromatography

    • Thin layer chromatography (TLC)

    • Column chromatography

Terminology

  • Analytes: The compounds of interest in a mixture.

  • Separation Process: Based on differences in migration rates of compounds in a mobile phase through a stationary phase.

  • Stationary Phase: Can be solid, liquid, or gel, that interacts with analytes; if packed in a tube, it's called a column.

  • Mobile Phase: A solvent or gas moving through the supporting medium.

  • Supporting Medium: The solid surface where stationary phase is bound or coated.

Purpose of Chromatography

  • Determining the identity, purity, and amount of impurities in samples.

    • Protect public health concerning pharmaceuticals and clinical products.

    • Critical in Quality Control (QC) and Quality Assurance (QA).

    • Important in drug discovery processes, including ADME (Absorption, Distribution, Metabolism, Elimination).

  • Quality Control Factors:

    • Ensuring API is within specifications (usually 100-110% of stated amount).

    • Identifying and quantifying other active ingredients in medicines.

Historical Context

  • Invention of Chromatography:

    • Invented in 1906 by Russian botanist Michael Tswett who used a glass column with calcium carbonate to separate plant pigments.

  • Original Experiment:

    • Used powdered limestone to observe colored bands indicating different pigments: chlorophylls, xanthophylls, and carotenoids.

  • Nobel Prize in Chemistry 1952:

    • Awarded to Archer John Porter Martin and Richard Laurence Millington Synge for the invention of partition chromatography.

Common Types of Chromatography

  • Based on Tswett’s Technique (Liquid Chromatography):

    • Paper chromatography

    • Thin Layer Chromatography (TLC)

    • Liquid Chromatography (LC, HPLC, UPLC)

    • Gas Chromatography (GC, GC-MS)

    • Supercritical Fluid Chromatography (SFC)

Chromatographic Process

  • How It Works:

    • Mobile phase transports the sample through a stationary phase in a column or solid surface.

    • Affects how fast or slow sample components interact and separate.

  • Separation Mechanism:

    • Based on differences in affinity for mobile and stationary phases; those with higher affinity to the stationary phase move slower.

  • Quantifying Separation:

    • Retention Time (RT) and Baseline Detection: To ensure good separation, the detector must record peaks that return to baseline.

Chromatographic Dynamics

  • Physical Equilibrium:

    • Expressed as the distribution coefficient or partition ratio:
      K=racC<em>SC</em>MK = rac{C<em>S}{C</em>M}

    • Where CS = molar concentration in stationary phase, and CM = molar concentration in mobile phase.

  • Component Separation:

    • Each component has a different distribution coefficient, affecting how long it is retained in the stationary phase versus carried by the mobile phase.

  • Theoretical Plates (N):

    • Measurement of column efficiency based on retention time and peak widths.

Peak Resolution and Efficiency

  • Resolution (R):

    • Rs measures peak separation, baseline separation occurs when Rs >= 1.5.

    • Capacity factor (Retention factor):

      • k′=ract<em>R−t</em>mtmk' = rac{t<em>R - t</em>m}{t_m}

      • High k value indicates a sample is highly retained in the stationary phase.

    • Selectivity:

      • ext{Selectivity} (α) > 1

      • α=rack<em>2k</em>1α = rac{k<em>2}{k</em>1}

      • High α indicates good separation power.

Causes of Peak Broadening

  • Eddy Diffusion:

    • Peak broadening caused by multiple flow paths in a packed column.

  • Mobile Phase Mass Transfer:

    • Occurs when equilibrium isn't established before the mobile phase moves on, which leads to broadened peaks.

Column Efficiency Metrics

  • Theoretical Plates Number (N):

    • N=16rac(tR)2(w)2N = 16 rac{(t_R)^2}{(w)^2}

  • Plate Height (H):

    • H=racLNH = rac{L}{N}

  • Selectivity:

    • α > 1

  • Column Resolution (R):

    • R=rac2(t<em>R)</em>B−(t<em>R)</em>Aw<em>A+w</em>BR = rac{2(t<em>R)</em>B - (t<em>R)</em>A}{w<em>A + w</em>B}

HPLC Example and Calculations

  • Example Calculation:

    • For a column with a length of 50 mm and 785 theoretical plates, column efficiency, extNext{N}, is:

    • N=racLHN = rac{L}{H} ; Solving gives
      H=rac50mm785=0.06mmH = rac{50mm}{785} = 0.06 mm

TLC Overview

  • Process of Thin Layer Chromatography (TLC):

    • Stationary phase is a thin layer on a glass or alumina plate.

    • Samples are deposited as spots, then developed in a mobile phase tank.

    • Advantages of TLC:

      • Inexpensive, simple, effective for strongly retained analytes.

    • Disadvantages:

      • Higher detection limits compared to HPLC, GC, or CE.

  • Visualization Methods:

    • Physical: UV light.

    • Chemical: iodine, sulfuric acid, pH indicators.

Reporting Results in TLC

  • Retention Factor (Rf):

    • Calculation:
      Rf=racextdistancetravelledbysoluteextdistancetosolventfrontR_f = rac{ ext{distance travelled by solute}}{ ext{distance to solvent front}}

    • Rf values range from 0 to 1, indicating the interaction strength with the stationary phase.

High-Performance Thin Layer Chromatography (HPTLC)

  • Definition:

    • Automated version of TLC.

    • Reduces inconsistencies in manual processes.

    • Example: Paracetamol and lornoxicam in tablets (500mg PCM, 8mg LORN).

Recommended Readings

  • Introduction to Pharmaceutical Chemical Analysis;

    • Authors: S. Hanson, S. Pedersen-Bjergaard, K. Ramussen - Chapters 14; Wiley 2012 (available online)

  • Analytical Chemistry;

    • Author: Christian D. Gary - Chapter 19 (available in the library - recommended purchase).