PA TOPIC 5 PART 1

• Separation in chromatography

  Separation is based on the differences in the rates of migration of the components
• Gas Chromatography (GC)

  1. Mobile phase (inert carrier gas eg Hydrogen, Nitrogen, Helium) flows under pressure through a heated column

  2. Stationary phase: Non-volatile liquid that is added as a coating on the inside of a capillary column

  3. Sample is injected and vaporized at the injector port

  4. The vaporized sample is then carried through the column by the carrier gas

  5. Compounds in the sample are partitioned between the two phases and separated
• Gas Chromatography (GC)

  • Separation is based on the unique affinities of each compound for the stationary phase
• Gas Chromatography (GC) - Advantages

  • Short analysis time
  • Requires very small sample size
  • Can be used to separate very similar compounds
  • Can be used for purpose of positive identification of compounds
• Gas Chromatography (GC) - Limitations

  • Only thermally stable and volatile compounds can be analysed
  • Requires an additional step of sample preparation
• Gas Chromatography (GC) - Applications

  • Detection of process impurities
  • Limit test for solvent residues/volatile impurities
  • Detection & quantification of drugs and their metabolites in biological fluids
  • Quantification of drugs in formulations
• High Performance Liquid Chromatography (HPLC)

  1. Mobile phase (liquid) flows under pressure through a stainless steel column

  2. Stationary phase: Porous particles with a large surface area, contains different active groups for the components in a sample to interact with

  3. Sample is dissolved in a liquid that is miscible with the MP & injected at the injector port

  4. The sample is then carried through the column by the MP which flows through the column under high pressure

  5. Compounds in the sample are partitioned between the two phases and separated
• High Performance Liquid Chromatography (HPLC) - Advantages

  • Well developed method
  • Readily automated
  • Less risk of sample degradation due to high temperature during analysis
  • Provides accurate, precise and robust method for separation and quantitative determination of drugs
• High Performance Liquid Chromatography (HPLC) - Disadvantages

  • Need to extract the drugs prior to analysis
  • Generates large amount of organic solvents as waste
• High Performance Liquid Chromatography (HPLC) - Applications

  • When coupled with UV/Vis detection, it can be used for quantitative analysis of pharmaceutical products
  • To monitor the stability of drugs in formulations
  • To quantify the amount of drugs and metabolites in biological fluids
• Thin Layer Chromatography (TLC)

  1. Mobile phase: liquid

  2. Stationary phase: Spread as a thin layer on a plate made of glass or alumina foil

  3. Sample is prepared in a volatile solvent and placed as spots on the plate

  4. MP travels up the plate by capillary action

  5. The sample migrates up the plate depending on the relative affinity of the sample for the SP vs MP
• Thin Layer Chromatography (TLC) - Advantages

  Robust, simple and cheap
• Thin Layer Chromatography (TLC) - Disadvantages

  • Poor sensitivity
  • Not suitable for volatile samples
• Thin Layer Chromatography (TLC) - Applications

  • Determination of impurities in raw materials and pharmaceutical products
  • Basic identity check on pharmaceutical raw materials