High-Performance Liquid Chromatography (HPLC)

High-Performance Liquid Chromatography (HPLC)

Understanding HPLC

  • High-Performance Liquid Chromatography (HPLC) is an analytical chemistry technique used in the separation, identification and quantification of each component in a mixture.
  • It is a type of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column containing the stationary phase.
  • Retention time is the measure of time that a compound remains in the liquid phase, and it can help identify the compound.

Principles of HPLC

  • The principle behind HPLC is the interaction of the molecules of the sample with the stationary and mobile phases.
  • It relies on pumps to pass a pressurised liquid solvent containing the sample mixture through a column filled with a solid adsorbent material.
  • Each component in the mixture interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out of the column.

Key Components of HPLC

  • An injector system is employed to introduce the mixture into the flow stream in a reproducible and quantifiable manner.
  • The high-pressure pump is responsible for moving the mobile phase and sample components through the column where separation happens.
  • The stationary phase or column performs the separation of sample components.
  • The detector senses the presence of the components and sends signals to the data analysis system.

Types of HPLC

  • There are several types of HPLC techniques which are selected based on the nature of the mixture and the components:
    • Normal-phase HPLC (NP-HPLC),
    • Reversed-phase HPLC (RP-HPLC),
    • Size-exclusion HPLC (SE-HPLC),
    • Ion-exchange HPLC (IE-HPLC).

Practical Applications of HPLC

  • HPLC is a popular method of quantitative chemical analysis and has a wide range of applications:
    • In research laboratories, it is used for separation and identification of compounds.
    • In industries like food, cosmetic, and pharmaceutical, it is used for quality control or to detect the presence of certain key compounds.
    • In environmental analysis, it is used to identify and quantify pollutants and other chemical compounds.

Importance of Safety and Quality Control in HPLC

  • With any practical lab technique, safety is paramount: proper personal protective equipment (PPE) should be used and safe lab procedures followed when using HPLC systems.
  • Quality control processes, such as routine checks on equipment and repeat analyses, ensure the robustness, validity, and reliability of HPLC results.