A Guide to Modern HPLC

05 Nov, 2010 | Guides & Resources

HPLC stands for high performance liquid chromatography. It is a chromatographic technique that can separate a mixture of compounds. This technique is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture, particularly in the separation of amino acids and proteins due to their different behaviour in solvents related to the amount of electronic charge of each one.
Like liquid chromatography, HPLC uses a liquid mobile phase to transport the sample mixture. However, HPLC is a step up from liquid chromatography in several ways.

  • Size matters: HPLC generally uses very small packing articles compared to liquid chromatography. A particle size analyzer can easily determine the size of these particles. Because the particles are smaller, there is greater surface area for interactions between the stationary phase and the molecules flowing past it, allowing for better separation of the components.
  • High Pressure: The solvent does not drip through the column under gravity in HPLC. Instead, it is forced through under high pressures of up to 400 atmospheres, quickening the entire process.
  • Stationary Phases: HPLC also utilises different types of stationary phases. The most common stationary phase is the hydrophobic saturated carbon chain but others such as a pump that moves the mobile phase and analyte through the column and a detector that provides a characteristic retention time from the analyte are also used.

How HPLC Works

  • The molecule of interest is held in the liquid state.
  • The sample is injected into the HPLC instrument.
  • The sample preparation passes through a column. Molecules are partitioned based on size and reasons of polarity interactions. Basically the column allows smaller molecules to pass through quickly and holds onto bigger molecules longer.
  • After each molecule is partitioned, it passes through the column and heads toward the detector. The sample is carried past the detector by the mobile phase.
  • The detector emits light in the range of 190-700nm. When the molecule of interest passes the detector it responds electronically with the light. The intensity of the response relates directly to the concentration of that molecule in the sample preparation.
  • The software plots the intensity of the molecule, on the y-axis. The software also records the time that the compound passed the detector. This is the “elution time,” or the characteristic time for that molecule, and represents the x-axis.

Four main types of HPLC

    • Partition

This was the first kind of chromatography that chemists developed. The partition method separates analytes based on polar differences.

    • Adsorption

Also known as normal-phase chromatography, this method separates analytes based on adsorption to a stationary surface chemistry and by polarity.

    • Ion-exchange

This is commonly used in protein analysis, water purification and any other technique that can be separated by charge

    • Size Exclusion or Gel

Size exclusion chromatography separates particles based on size. To determine the size of the particles, a commonly used technique is laser diffraction as it is able to measure the size of a wide range of particles from very fine to very coarse Size exclusion chromatography is generally a low-resolution technique that is usually reserved for the final “polishing” step of purification. This method is useful for determining the tertiary and quaternary structures of purified proteins.

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