CHEM220-Section07

Chromatography Overview

  • Chromatography: A physical separation method involving two phases.

    • Phases:

      • Stationary phase (solid or viscous liquid).

      • Mobile phase (liquid or gas).

    • Components separated based on structure/composition differences.

Basic Principles

  • Procedure Example:

    • A solution containing compounds A and B is applied to a column.

    • Solute A is more strongly adsorbed, taking longer to elute than solute B.

  • Important Terms:

    • Eluent: Fluid entering the column.

    • Eluate: Fluid exiting the column.

    • Elution: Process of passing fluid through the chromatography column.

Chromatographic Techniques

Classification by Equilibrium Process

  1. Adsorption Chromatography:

    • Solid stationary and liquid/gas mobile phase.

  2. Partition Chromatography:

    • Thin liquid stationary phase on solid support.

  3. Ion-Exchange Chromatography:

    • Ionic groups on stationary phase attract solute ions.

  4. Molecular Exclusion Chromatography:

    • Separates by size; larger molecules pass quickly.

  5. Affinity Chromatography:

    • Specific interactions between solute and stationary phase.

The Chromatogram

  • Definition: Detector's response vs. time (retention time).

  • Important Parameters:

    • Retention Time (tR): Time for solute to reach the detector.

    • Mobile Phase for an Unretained Compound (tM): Time for the mobile phase to travel through the column.

    • Actual Retention Time (t’R): Calculated as tR - tM.

Column Efficiency

  • Theoretical Plates:

    • Discrete section for solute equilibration.

    • A higher number indicates a more efficient column.

    • Calculation: N = 16 (tR/w)^2.

Resolution in Chromatography

  • Defines the space between adjacent peaks. Higher resolution indicates better separation.

  • Calculation: Resolution = (tR2 - tR1) / (w1 + w2) / 2.

Band Broadening

  • Causes for band broadening in columns:

    • Diffusion: Broadens due to longitudinal diffusion.

    • Multiple Flow Paths: Irregular paths increase spreading.

    • Mass Transfer Between Phases: Slower equilibration can cause lag.

Liquid Chromatography

  • Traditional vs. High‐Performance Liquid Chromatography (HPLC):

    • HPLC offers better resolution and speed through high pressure and smaller particles.

    • HPLC components include:

      • Solvent delivery system.

      • Sample injection valve.

      • Column.

      • Detector.

Detection Methods

Common Detectors in HPLC

  1. Ultraviolet (UV) Detector:

    • Measures UV light absorption.

  2. Refractive Index Detector:

    • Detects changes in light deflection resulting from different solutes.

  3. Fluorescence Detector:

    • Sensitive to fluorescent compounds.

Gas Chromatography Basics

  • Function: Separates volatile components using a gaseous mobile phase.

  • Carrier Gases: Must be pure and chemically inert.

  • Injection Types:

    • Split Injection: Only a portion of the sample reaches the column.

    • Splitless Injection: Maximizes sample on column.

    • On-Column Injection: Directly injects into the column, minimizing sample loss.

Types of Gas Chromatography Columns

  1. Packed Columns: Fill with small particles, used less frequently today.

  2. Capillary Columns: Offer better separation due to longer lengths and lack of multiple flow paths.

Conclusion

  • Chromatography is integral for both qualitative and quantitative analysis of compounds across various applications, allowing for effective separation and identification based on different physical principles.