How High Performance Liquid Chromatography Is Being Used In The Lab

High performance liquid chromatography, or HPLC, is becoming one of the most widely used tools in chemical analysis. This powerful instrumentation uses many same means as thin layer chromatography and column chromatography, in which it is based.

To understand HPLC, we must first understand chromatography basics. Chromatography is the separation of chemicals or materials that are mixed together. For example, if you have a mixture of red dye and blue dye, you would have a purple-colored mixture. To separate each of the colors, one must understand that the red dye has different physical properties than the blue, so when a solvent is used to mix the dyes, they can be separated using thin layer chromatography.

Thin layer chromatography is when the solvent flows up a thin plate due to capillary action of the solvent. The solvent carries each dye with it, eventually separating them due to their physical properties. What is left would be a spot on the plate that is red, and possibly above it, a spot of blue. You have separated the dyes into their basic components.

In columnar chromatography, the principle is the same, except you use a glass tube, or column, to separate the chemicals. There is still a solvent involved, but instead of the chemicals flowing up, they flow downward by the use of natural gravity or fluidic pumps. Each chemical is separated within the solvent substrate, and can be purified in this manner.

In high performance liquid chromatography, this whole process is speeded up, due to the use of high pressures for the solvent to run through the column. The pressures used are about 400 times the earth's atmosphere, so speed is naturally the result.

The state of phase of the materials being separated or tested is important in HPLC. A liquid phase is the most common and easiest to separate, so we will use it as an example. Under pressure, particulates that are to be separated can be smaller, and the interactions of any special coatings on the inside surface of the column and the latter to be separated is made much more sensitive.

In Normal Phase HPLC, silica particles are used with a positive polarity, and the solvent is a non-polar hexane-type. The materials that need separation tend to stick to the silicates rather than to the solvent, so they are easily demarked and will flow as a purified solution out of the column.

In Reverse Phase HPLC, the solvent will be the carrier of the separated molecules instead of the silica particles. This is most commonly used when extracting special chemicals from a mixture. An example would be to extract the common chemicals from plants that are beneficial to humans, like, say, aspirin.

This technique is used all over the world for the extraction of many beneficial chemicals for pharmaceuticals, cosmetics, and chemical production. It is used by nearly every chemical research laboratory on earth, and is useful in the biochemical and biomedical fields. Without HPLC, processes to extract materials or separate chemicals would be nearly non-existent.