High Performance Liquid Chromatography

High performance liquid chromatography is a chromatographic technique used to separate a mixture of compounds. HPLC is also used in analytical chemistry and biochemistry to quantify as well as identify and purify individual components of a mixture. This makes use of various stationary phase types which in turn moves analyte and mobile phases through columns and a detector provides characteristic retention time. Additional information relating to the analyte may also be provided by the detector.

Depending on the strength of the interactions with the stationary phase will depend on the analyte retention time as the ratio of the flow rate and solvents used of the mobile phase. Liquid chromatography uses smaller size columns and smaller media in the columns as well as increased mobile phase pressures. As opposed to gravity a pump produces high pressure needed to move the analyte and mobile phase through the densely packed column. Smaller particles sizes increase the density which allows for separation in columns with a shorter length in comparison to normal column chromatography.

A small amount of the sample that needs to be analyzed is introduced into the stream of the mobile phase. When the column is in a stationary phase the solution moving through the column is slowed down by specified physical and chemical interactions. The nature of the sample will depend on the velocity of the solution as well as on the compositions of the stationary phase.

Retention time is the time it takes the sample to come out at the end of the column and this retention time identifies the characteristics of the sample under certain conditions. When making use of smaller sized columns, this will increase the linear velocity.

In turn this causes the components to diffuse in less time inside the column which in turn improves the resolution of the chromatogram. Salts or buffers may be contained in the water which aids the separation of compounds or sample pieces and in turn acts as an pairing ion agent such as trifluoroacetic acid.

An additional refinement of high performance liquid chromatography during the analysis is to vary the mobile phase composition and gradient. For reversed chromatography the normal gradient may begin at five percent progress linearly and methanol up to fifty percent methanol over a period of twenty five minutes. Depending on how hydrophobic the sample is will define the gradient. This separation process is almost the same as the process that occurs during a liquid to liquid extraction the only difference is that this process is not step wise but continuous.

The nature of the sample and the column will depend on the choice of solvents, gradients and additives. The sample is tested as well as a number of trial runs are done in order to define the high performance liquid chromatography method which will produce the best peak separation. The very first HPLC was developed by chemists. The NP HPLC was made redundant in the late 70's due to the lack of reproducibility retention time and replaced with HPLC.