MEVE 018: Unit 03 - Chromatography Techniques

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UNIT 3: CHROMATOGRAPHY TECHNIQUES

3.0 Introduction

Chromatography has evolved into a fundamental method in analytical chemistry. Modern chromatographic techniques, such as Gas Liquid Chromatography (GLC), High-Performance Liquid Chromatography (HPLC), and Supercritical Fluid Chromatography (SFC), offer precise, sensitive, and high-throughput capabilities for separating and analyzing complex mixtures. These methods play a pivotal role in environmental monitoring, pharmaceutical quality control, food safety, and biomedical research.

3.1 Objectives

Upon completion of this unit, learners will be able to:

  • Understand the principles behind modern chromatographic techniques.
  • Explain the working and instrumentation of GLC, HPLC, and SFC.
  • Differentiate between types of HPLC.
  • Identify applications of chromatography in environmental and industrial contexts.

3.2 Gas Liquid Chromatography (GLC)

Principle:

GLC separates volatile organic compounds based on their distribution between a gaseous mobile phase (carrier gas) and a liquid stationary phase coated on the surface of an inert solid within a column.

Instrumentation:

  • Carrier gas (e.g., Helium or Nitrogen)
  • Injector for sample introduction
  • Column (either packed or capillary)
  • Oven to maintain optimal temperature
  • Detector (commonly Flame Ionization Detector or Thermal Conductivity Detector)
  • Data System for analysis

Applications:

  • Environmental analysis (e.g., VOCs in air)
  • Food and flavor testing
  • Forensic analysis
  • Petrochemical testing

3.3 High-Performance Liquid Chromatography (HPLC)

HPLC is a versatile technique ideal for separating compounds that are thermally unstable or non-volatile, unlike those analyzed by GLC.

3.3.1 Principle of HPLC:

HPLC separates components based on their differential interactions with a liquid mobile phase and a solid stationary phase under high pressure, allowing for efficient and fast analysis.

3.3.2 Instrumentation:

  • Solvent Reservoirs: Contain mobile phase (usually a mixture of solvents like water, methanol, acetonitrile).
  • Pump: Forces the mobile phase through the column under high pressure (up to 6000 psi).
  • Injector: Introduces the liquid sample into the mobile phase stream.
  • Column: Stainless steel tube packed with stationary phase (e.g., silica particles).
  • Detector: Commonly UV-Vis, Refractive Index (RI), or Fluorescence.
  • Data System: Computer software for acquiring and analyzing chromatograms.

3.3.3 Types of HPLC:

  • Normal Phase HPLC: Polar stationary phase (e.g., silica) and non-polar mobile phase.
  • Reverse Phase HPLC (RP-HPLC): Non-polar stationary phase (e.g., C18 column) and polar mobile phase — most widely used.
  • Ion Exchange HPLC: Based on ionic interactions; used for inorganic ions and biomolecules.
  • Size Exclusion HPLC: Based on molecular size; used for polymers and proteins.

3.3.4 Application of HPLC:

  • Environmental Monitoring: Detection of pollutants, pesticides, herbicides.
  • Pharmaceuticals: Drug purity and stability testing.
  • Food Industry: Detection of preservatives, vitamins, and additives.
  • Clinical Diagnostics: Measurement of hormones, metabolites.

3.4 Supercritical Fluid Chromatography (SFC)

Principle:

SFC uses a supercritical fluid (typically CO₂) as the mobile phase. A supercritical fluid is a substance above its critical temperature and pressure, exhibiting properties of both liquids and gases.

Advantages:

  • Faster separations due to low viscosity.
  • Ideal for thermally labile compounds.
  • Environmentally friendly (less organic solvent used).

Applications:

  • Separation of chiral compounds.
  • Analysis of pharmaceuticals, polymers, lipids.
  • Green chemistry and sustainability-focused research.

3.5 Application of Chromatographic Techniques in Environmental Monitoring

Chromatographic techniques are indispensable in environmental science for the accurate and sensitive detection of pollutants.

Key Applications:

  • Water Quality Testing: Detection of trace organic compounds (e.g., pesticides, pharmaceuticals).
  • Air Pollution Studies: Monitoring volatile and semi-volatile compounds using GC.
  • Soil Analysis: Identification of hydrocarbon contamination using GC-MS or HPLC.
  • Wastewater Treatment: Monitoring degradation products and persistent organic pollutants.

Example Scenarios:

  • HPLC used for identifying pesticides in agricultural runoff.
  • GLC for quantifying BTEX (benzene, toluene, ethylbenzene, xylene) in air samples.
  • SFC for separation of persistent organic pollutants (POPs) in soil samples.

3.6 Let Us Sum Up

Modern chromatographic techniques, including GLC, HPLC, and SFC, provide powerful tools for the analysis of complex mixtures in various fields. Their principles rely on the interactions of analytes with both mobile and stationary phases, which vary depending on chemical properties. Each technique has specific strengths: GLC for volatile organics, HPLC for thermally sensitive compounds, and SFC for rapid, environmentally friendly separations. Their application in environmental monitoring ensures regulatory compliance, pollution tracking, and ecological protection.

Keywords

  • GLC (Gas Liquid Chromatography)-A chromatographic technique for separating volatile compounds using a gas mobile phase.
  • HPLC (High-Performance Liquid Chromatography)-A technique using high pressure to separate, identify, and quantify non-volatile compounds.
  • SFC (Supercritical Fluid Chromatography)-Chromatography using supercritical fluids, combining gas and liquid properties for separation.
  • Mobile Phase-The fluid that carries the analytes through the stationary phase.
  • Stationary Phase-The solid or liquid phase that remains fixed in place inside the column.
  • Detector-Device that identifies and quantifies the separated components.
  • Environmental Monitoring-The use of analytical techniques to measure pollutants in air, water, and soil.
  • Reverse Phase HPLC-A type of HPLC where the stationary phase is non-polar and the mobile phase is polar.

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