MEV 013: Unit 14 - Chromatography Techniques

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UNIT 14: CHROMATOGRAPHY TECHNIQUES

14.0 Introduction

Chromatography is a vital analytical tool used for the separation, identification, and quantification of components in a mixture. It plays a crucial role in environmental monitoring, pharmaceutical analysis, food safety, and biomedical research. This unit introduces key chromatographic methods, focusing on Gas Liquid Chromatography (GLC), High-Performance Liquid Chromatography (HPLC), and Supercritical Fluid Chromatography (SFC).

14.1 Objectives

After completing this unit, you will be able to:

·         Understand the basic principles of different chromatographic techniques.

·         Describe the instrumentation and operation of GLC, HPLC, and SFC.

·         Explain the applications of chromatography in environmental analysis and other fields.

·         Differentiate between various types of HPLC.

14.2 Gas Liquid Chromatography (GLC)

GLC is a widely used technique for the separation of volatile compounds.

14.2.1 Principle

Gas Liquid Chromatography works on the principle of partitioning between a stationary liquid phase and a mobile gas phase. The analyte is vaporized and carried by an inert gas through a column coated with a stationary liquid. Separation occurs based on differences in volatility and interaction with the stationary phase.

14.2.2 Instrumentation

Major components of a GLC system include:

·         Carrier Gas Supply (e.g., helium, nitrogen)

·         Injector (for vaporizing the sample)

·         Chromatographic Column (packed or capillary)

·         Oven (temperature-controlled chamber for the column)

·         Detector (e.g., Flame Ionization Detector - FID, Thermal Conductivity Detector - TCD)

·         Data System (for recording and analyzing chromatograms)

14.2.3 Application of GLC

GLC is used in:

·         Air quality analysis

·         Monitoring of volatile organic compounds (VOCs)

·         Food flavor analysis

·         Detection of hydrocarbons in environmental samples

·         Forensic and pharmaceutical applications

14.3 High-Performance Liquid Chromatography (HPLC)

HPLC is a technique for separating non-volatile or thermally unstable compounds using a liquid mobile phase.

14.3.1 Principle

HPLC is based on the partitioning of analytes between a liquid mobile phase and a solid stationary phase under high pressure. Separation depends on the analyte's affinity for the stationary phase and solubility in the mobile phase.

14.3.2 Instrumentation

An HPLC system includes:

·         Solvent Reservoirs

·         Pump System (to generate high pressure)

·         Injector

·         Chromatographic Column (usually packed with silica or polymer particles)

·         Detector (e.g., UV-Vis, Refractive Index, Fluorescence)

·         Data Acquisition System

14.3.3 Types of HPLC

·         Normal Phase HPLC: Polar stationary phase, non-polar mobile phase

·         Reverse Phase HPLC (most common): Non-polar stationary phase, polar mobile phase

·         Size Exclusion Chromatography (SEC): Separates molecules by size

·         Ion Exchange Chromatography: Based on charge interactions

·         Affinity Chromatography: Based on biological specificity

14.3.4 Application of HPLC

·         Analysis of pesticides, herbicides, and heavy metals

·         Pharmaceutical product testing

·         Water and wastewater quality assessment

·         Detection of pollutants and trace organic compounds

·         Analysis of biological fluids

14.4 Supercritical Fluid Chromatography (SFC)

SFC utilizes supercritical fluids (commonly CO₂) as the mobile phase, combining advantages of both gas and liquid chromatography.

Principle:

Supercritical fluids have high diffusivity and low viscosity, allowing fast separation with efficient resolution. SFC is suitable for separating:

·         Chiral compounds

·         Thermally labile substances

·         Non-polar or moderately polar analytes

Applications:

·         Environmental testing of pesticides and pollutants

·         Pharmaceutical and cosmetic compound analysis

·         Separation of lipids and essential oils

14.5 Application of Chromatographic Techniques in Environmental Monitoring

Chromatography is indispensable in detecting and quantifying environmental contaminants such as:

·         Airborne pollutants (e.g., VOCs, PAHs)

·         Pesticides and herbicides in water and soil

·         Heavy metals and industrial effluents

·         Endocrine-disrupting chemicals (EDCs)

·         Monitoring of environmental samples for compliance with legal standards

It supports environmental impact assessment (EIA) and regulatory monitoring programs.

14.6 Let Us Sum Up

In this unit, you have learned:

·         The fundamentals and instrumentation of Gas Liquid Chromatography (GLC), High-Performance Liquid Chromatography (HPLC), and Supercritical Fluid Chromatography (SFC).

·         Various types of HPLC and their specialized uses.

·         The applications of chromatographic techniques in environmental, pharmaceutical, and industrial fields.

·         How chromatography plays a crucial role in environmental monitoring and pollution control.

14.7 Keywords

  • Chromatography – A technique used to separate, identify, and quantify components in a mixture based on differential partitioning.
  • Gas Liquid Chromatography (GLC) – A chromatographic method where the mobile phase is a gas and the stationary phase is a liquid on a solid support.
  • High-Performance Liquid Chromatography (HPLC) – A high-pressure technique to separate, identify, and quantify compounds in liquid mixtures.
  • Supercritical Fluid Chromatography (SFC) – Chromatography using supercritical fluids (like CO₂) as the mobile phase for separation.
  • Stationary Phase – The solid or liquid phase in chromatography that stays fixed inside the column.
  • Mobile Phase – The phase (gas or liquid) that moves through the stationary phase carrying analytes with it.
  • Retention Time – The time a compound takes to pass through the chromatography column to the detector.
  • Detector – A device in chromatography that identifies and quantifies separated components.
  • Elution – The process of extracting one material from another by washing with a solvent in chromatography.
  • Environmental Monitoring (via Chromatography) – Use of chromatographic methods to detect pollutants or contaminants in environmental samples (air, water, soil).

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