Sunday, 11 May 2025

High-Performance Liquid Chromatography (HPLC)

High-Performance Liquid Chromatography, commonly known as HPLC, is a powerful technique used in laboratories around the world to separate, identify, and quantify components in a mixture. Whether it’s used in pharmaceuticals, food industries, environmental analysis, or plant research, HPLC plays a key role in modern science.

In this article, we will explain:

  • What HPLC is
  • How it works (with a diagram)
  • Types of HPLC
  • Applications
  • Advantages and Limitations

What is HPLC?

HPLC stands for High-Performance Liquid Chromatography. It is an analytical technique used to separate and analyze compounds that are dissolved in a liquid.

Basic Principle of HPLC

HPLC works on the principle of separation based on the interactions of molecules with two phases:

  • Stationary Phase: A solid or liquid fixed inside a column.
  • Mobile Phase: A liquid solvent that flows through the column.

Different compounds in the sample move through the column at different speeds depending on their affinity (interaction) with the stationary and mobile phases. This leads to separation.

HPLC Diagram and Components

Here is a simple diagram of an HPLC system:



Main Components of HPLC:

  1. Solvent Reservoir – Contains the mobile phase (liquid solvent).
  2. Pump – Pushes the mobile phase through the system.
  3. Injector – Introduces the sample into the mobile phase.
  4. Column – Filled with stationary phase material where separation occurs.
  5. Detector – Detects the separated compounds.
  6. Data System (Computer) – Displays and analyzes the results as a graph called a chromatogram.

Types of HPLC

There are different types of HPLC based on the type of stationary and mobile phases used:

  1. Normal Phase HPLC
    • Stationary phase: polar (e.g., silica)
    • Mobile phase: non-polar (e.g., hexane)
    • Used for separating polar compounds.
  2. Reverse Phase HPLC (RP-HPLC)
    • Stationary phase: non-polar (e.g., C18 column)
    • Mobile phase: polar (e.g., water, methanol)
    • Most commonly used type in pharmaceuticals and plant research.
  3. Size-Exclusion HPLC
    • Separates molecules based on size.
    • Large molecules elute first, small ones later.
  4. Ion-Exchange HPLC
    • Separates based on charge (ions).
    • Useful for amino acids and proteins.

What is a Chromatogram?

A chromatogram is the output of an HPLC analysis. It looks like a graph with peaks. Each peak represents a different compound, and:

  • The retention time (Rt) tells us when the compound came out of the column.
  • The area under the peak tells us how much of that compound is present.

Applications of HPLC

HPLC is used in many fields:

  • Pharmaceutical Industry: To check drug purity and content.
  • Plant Biotechnology: To identify and quantify plant metabolites.
  • Food Industry: To detect preservatives, sugars, and nutrients.
  • Environmental Testing: To monitor pollutants in water or soil.
  • Clinical Research: To measure biomarkers in blood or urine.

Advantages of HPLC

  • High accuracy and precision
  • Fast analysis
  • Can handle complex mixtures
  • Works for heat-sensitive samples (unlike gas chromatography)

 Limitations of HPLC

  • Equipment is expensive
  • Requires trained personnel
  • Uses a lot of solvents (which may be toxic)
  • Regular maintenance need


 

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