Pharmacy Courses

Basic Knowledge on Chromatographic Technique (HPLC & GC)

Chromatography is a method in which separation of components take place between two phases – a stationary phase and a mobile phase.

  • Stationary phase: the substance on which adsorption of the analyte takes place.
  • Mobile phase: the solvent which carry the analyte.
  • Analyte: the substance whose chemical constituents are being identified and measured.
  • Retention time is the time require for elute the analyte. Which takes more time to elute, its retention time is more.
  • Absorption: assimilation
  • Adsorption: accumulation on surface

Usually there are four types of chromatography technique - high-performance liquid chromatograph, gas chromatography, thin-layer chromatography, and paper chromatography. Among them high-performance liquid chromatograph and gas chromatography highly used in pharmaceutical industry. 

HPLC means ‘high performance liquid chromatography or high pressure liquid chromatography’. Where high pressure is used to pass the target molecule with mobile phase through compacted or densely packed stationary phase. HPLC types are usually falls in – 

  1. Normal phase: Mobile phase is Non-polar, Stationary phase is Polar.
  2. Reverse phase: Mobile phase is Polar, Stationary phase is Non-polar.

Commonly  there are 6 column lines of HPLC. Where 4 lines for mobile phase, 1 line for needle wash and 1 line for cell/pump.

  • Back pressure showed by stationary phase against high pressure.
  • Mobile phase component include usually – Salt and Solvent.
  • Column is cleaned through 5% organic solvent or hot water to solve the column blockage.
  • A guard column is a protective column or cartridge installed between the injector and the analytical column.

For important terms for information -

  • Column branching increase the separation time and show clear graph. 
  • Elution strength increase the retention time.
  • Resolution indicates how well peak is separate.
  • Run time is equal to inject time.
  • Dead time is equal the time until sample reach the detector.
  • Capacity factor or retention factor means the position of a sample peak in the chromatogram. 
  • Selectivity factor also called the separation/selectivity coefficient.
  • Theoretical plates characterize the quality or efficiency of a column. If width of peak is more, theoretical plate will decrease. (Theoretical plate should be - NLT 200)
  • The tailing factor is the coefficient of the peak symmetry. Which is measured by simply the entire peak width divided by twice the front half-width (where, std. value=1)

Column efficiency depends on

  • Particle size
  • Particle size distribution

Usually universal wave length is selected and λmax is not essential. Where following detector may used – 

  • UV detector
  • IR
  • Electrochemical detector

Gas Chromatography 

Gas chromatography (GC) is a common type of chromatography used in analytical chemistry for separating and analyzing compounds depending on the volatility of sample.

  • Mainly used to separate volatile compounds (Methanol, Heptane, and Acetone etc.).
  • Column is like coil/folding – Packed column (Glass or SS, 1 to 3 m), Capillary column (Fused silica, Quartz, 10-100 m)
  • Stationary phase: Silicon, grease
  • Mobile phase: Helium, Nitrogen
  • More volatile materials pass first
  • Commonly used detector in GC: FID (Flame ionization detector), Hydrogen & Oxygen

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