Glossary of HPLC Terms

  • HPLC – separation technique for components of organic mixtures involving retention of components on stationary phase packed inside column on the basis of physico – chemical interactions followed by sequential elution
  • Stationary phase – solid bed inside column whose particles are coated with the retention phase.
  • Mobile phase – liquid carrier medium used for transporting the sample through the HPLC system,
  • Normal phase separation – separation mode in which the retention material is polar and mobile phase is nonpolar. Retained sample components are eluted in ascending order of polarity.
  • Reverse phase separation – separation mode in which the stationary phase is nonpolar and mobile phase is polar. Elution order of components is in decreasing order of polarity.It is the most commonly used mode of HPLC separations.
  • Column efficiency – expressed in terms of HETP which expresses resolution power of the HPLC column.
  • Column – a steel tube packed with the stationary phase for separation of sample components.
  • Autosampler – a device for automated precise selection and introduction of programmed sample volume into the HPLC system
  • Injector – manual or automated device capable of precise sample volume injection of sample into the HPLC system
  • Filter – frit fitted with a screen membrane to remove solid suspensions from mobile phase of sample
  • Degassing – procedure for removal of dissolved air from mobile phase using vacuum filtration, helium purging or online degassing.
  • Pump – a module of the system for metering controlled flow rate mobile phase through the system
  • Detector – a device for providing response of the component after separation by the HPLC column
  • Column Oven – a housing for the HPLC column which can be maintained at a constant temperature. It permits constant flow rate of mobile phase.
  • Ion-exchange chromatography – branch of HPLC involving separation of charged sample components by attraction to the oppositely charged stationary phase
  • Gel permeation chromatography – separation technique in which separation is based on molecular size of component molecules. Smaller molecules get trapped in pores of stationary phase and are eluted after the large molecules.
  • Chiral chromatography – chromatographic separation technique for optically active sample components
  • Analytical scale separations – separations in the mass range mg-ng involving use of columns packed with 3-5μm size particles
  • Semi-preparative HPLC – HPLC separations involving mass range from mg-gm involving collection of separated fractions.
  • Preparative HPLC – mass range gm-kg involving collection of separated fractions
  • UHPLC – Ultra-high-Pressure or Performance HPLC. Particle size is less than 2 µm. Such systems and columns are capable of handling back pressures in the range of 15,000 up to 18,000 psi
  • Isocratic Elution – mobile phase composition does not change during the chromatographic run.
  • Gradient Elution – mobile phase composition varies during the run and can be programmed before starting the chromatographic run
  • Retention time – time taken between injection and the maximum of the peak response of a component
  • Mobile phase reservoir – a container that is filled with the mobile phase
  • Fraction collector – a carousel packed with empty vials in which different components get collected after separation on the HPLC column
  • Syringe – hand held glass device capable of injecting selected volume of sample into the chromatograph
  • Bulk property detector – detector based on detection of total property of mobile phase and sample component as a single system
  • Specific property detector – detector based on specific property of eluting molecules and is independent of changes in the mobile phase composition
  • HETP – Height equivalent to theoretical plate. It is a measure of column efficiency and is expressed as a numerical value without units

HETP = L/N

Where L is column length and N the number of theoretical plates.The larger the number of theoretical plates the lower is HETP and better is the column efficiency.

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