High performance liquid chromatography

Download a printable version of this document here

HPLC in the analytical facility at the School of Chemistry, University of Birmingham

High performance liquid chromatography (HPLC) is often used in synthetic chemistry to separate polar organic molecules. HPLC is a very efficient method of chromatography, and is often used for reverse-phase chromatography.

In HPLC, solvents are used as the mobile phase, under pressure, to elute the mixture through the solid phase in the column.  The column is made of a stainless steel tube packed with extremely fine spherical particles, typically 5-10 μm in diameter. Due to the tiny size of these particles, any solvent that soaks into the particles is close to the surface of the particle, and can equilibrate readily with the mobile phase. This makes HPLC much more efficient than standard adsorption column chromatography, in which the larger particles (60 μm) have deep pools of stagnant solvent. The column can be seen in the photograph to the left, as the door is open to the column chamber.

The tiny particles are highly packed in the column, and therefore high pressures are necessary to force the eluent through the column.

HPLC can be analytical, or preparative, the latter just requires higher pressure and a larger column.

HPLC systems are commercially available with a variety of different setups, but they all work in the manner shown in the schematic below:hplc

HPLC can be run in ‘normal phase’ or ‘reverse phase’, depending on the polarity of the compounds that you are trying to separate. The ‘normal phase’ setup is analogous to adsorption column chromatography, in which polar compounds move slower and polar eluents have greater eluting power. An example solvent system used in normal phase is hexane/isopropanol.

In the ‘reverse phase’ setup silica is functionalised with a long chain hydrocarbon (C18), and non-polar compounds move slower. Therefore non-polar solvents have greater eluting power. An example solvent system used is water/acetonitrile. Usually with HPLC a gradient solvent system is used; the proportion of one of the solvents is increased gradually during the run, and it is key to optimise these conditions for the best separation of a mixture.

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.