Understanding of HPLC (High Performance Liquid Chromatography)

What is HPLC?
  • HPLC is an abbreviation for High Performance Liquid Chromatography. "Chromatography" is a technique for separation, "chromatogram" is the result of chromatography, and "chromatograph" is the instrument used to conduct chromatography.
  • Among the various technologies developed for chromatography, devices dedicated for molecular separation called columns and high-performance pumps for delivering solvent at a stable flow rate are some of the key components of chromatographs. As related technologies became more sophisticated, the system commonly referred to as High Performance Liquid Chromatography, simply became referred to as "LC". Nowadays, Ultra High Performance Liquid Chromatography (UHPLC), capable of high-speed analysis, has also become more wide-spread.
  • Only compounds dissolved in solvents can be analyzed with HPLC. HPLC separates compounds dissolved in a liquid sample and allows qualitative and quantitative analysis of what components and how much of each component are contained in the sample.

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What is the principle of HPLC?
  • The separation principle of HPLC is based on the distribution of the analyte (sample) between a mobile phase (eluent) and a stationary phase (packing material of the column). 
  • Depending on the chemical structure of the analyte, the molecules are retarded while passing the stationary phase.

How Does HPLC Work?
  • In column chromatography a solvent drips through a column filled with an adsorbent under gravity. HPLC is a highly improved form of column chromatography. A pump forces a solvent through a column under high pressures of up to 400 atmospheres. The column packing material or adsorbent or stationary phase is typically a granular material made of solid particles such as silica or polymers.
  • The pressure makes the technique much faster compared to column chromatography. This allows using much smaller particles for the column packing material. The smaller particles have a much greater surface area for interactions between the stationary phase and the molecules flowing past it. This results in a much better separation of the components of the mixture.
  • The pressurized liquid is typically a mixture of solvents such as water, acetonitrile and/or methanol and is referred to as the mobile phase.
  • The components of a mixture are separated from each other due to their different degrees of interaction with the absorbent particles. This causes different elution rates for the different components and leads to the separation of the components as they flow out the column. Compared to column chromatography, HPLC is highly automated and extremely sensitive.

Types of HPLC
The two most common variants are normal-phase and reversed-phase HPLC.

Normal-Phase HPLC
  • The column is filled with tiny silica particles, and a non-polar solvent, for example, hexane. A typical column has an internal diameter of 4.6 mm or smaller and a length of 150 to 250 mm. 
  • Non-polar compounds in the mixture will pass more quickly through the column, as polar compounds will stick longer to the polar silica than non-polar compounds will.

Reversed-Phase HPLC
  • The column size is the same. The column is filled with silica particles which are modified to make them non-polar. This is done by attaching long hydrocarbon chains (8–18 C atoms) to its surface. A polar solvent is used, for example, a mixture of water and an alcohol such as methanol. 
  • Polar compounds in the mixture will pass more quickly through the column because a strong attraction occurs between the polar solvent and the polar molecules in the mixture.
  • Non-polar molecules are slowed down on their way through the column. They form varying degrees of attraction with the hydrocarbon groups principally through van der Waals dispersion forces and hydrophobic interactions. They are also less soluble in the aqueous mobile phase components facilitating their interactions with the hydrocarbon groups.
  • Reversed phase HPLC is the most commonly used form of HPLC

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