High Performance Liquid Chromatography Lab (HPLC)

High-performance liquid chromatography or commonly known as HPLC, is an analytical technique used to separate, identify or quantify each component in a mixture.

The mixture is separated using the basic principle of column chromatography and then identified and quantified by spectroscopy. HPLC is thus basically a highly improved form of column liquid chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures.

Normal Phase Chromatography
Normal phase is a specific type of partitioning chromatography where the stationary phase is polar, and the mobile phase is non-polar. While the compounds selected might not be particularly polar or non-polar, their relative polarities are the most important. As long as the stationary phase is more polar than the mobile phase, it is considered normal phase chromatography. The stationary phase is usually a column packed with silica particles that have R groups attached. Common R groups for normal phase are -C2H4CN or -C3H6NH2. When the mixture passes through the column, the polar components will be more attracted to the stationary phase and the non-polar components will be attracted to the mobile phase. This will lead the non-polar components to flow through (or elute) the column first. The more polar a compound is, the longer it will take to elute. By increasing the polarity of the mobile phase, the bound polar component will partition more into the mobile phase and elute from the column.

Reverse Phase Chromatography Reversed phase chromatography is another type of partition chromatography but is opposite from normal phase. The mobile phase is more polar than the stationary phase. In this case, the most polar components will elute first. Common R groups found on the silica particles for reverse phase are -n-octyl (C8) or -n-octyldecyl (C18).