| High performance liquid chromatography, or HPLC, is | | | | pressures for the solvent to run through the column. |
| becoming one of the most widely used tools in | | | | The pressures used are about 400 times the earth's |
| chemical analysis. This powerful instrumentation uses | | | | atmosphere, so speed is naturally the result. |
| many same means as thin layer chromatography and | | | | The state of phase of the materials being separated |
| column chromatography, in which it is based. | | | | or tested is important in HPLC. A liquid phase is the |
| To understand HPLC, we must first understand | | | | most common and easiest to separate, so we will use |
| chromatography basics. Chromatography is the | | | | it as an example. Under pressure, particulates that are |
| separation of chemicals or materials that are mixed | | | | to be separated can be smaller, and the interactions of |
| together. For example, if you have a mixture of red | | | | any special coatings on the inside surface of the |
| dye and blue dye, you would have a purple-colored | | | | column and the latter to be separated is made much |
| mixture. To separate each of the colors, one must | | | | more sensitive. |
| understand that the red dye has different physical | | | | In Normal Phase HPLC, silica particles are used with a |
| properties than the blue, so when a solvent is used to | | | | positive polarity, and the solvent is a non-polar |
| mix the dyes, they can be separated using thin layer | | | | hexane-type. The materials that need separation tend |
| chromatography. | | | | to stick to the silicates rather than to the solvent, so |
| Thin layer chromatography is when the solvent flows | | | | they are easily demarked and will flow as a purified |
| up a thin plate due to capillary action of the solvent. | | | | solution out of the column. |
| The solvent carries each dye with it, eventually | | | | In Reverse Phase HPLC, the solvent will be the carrier |
| separating them due to their physical properties. What | | | | of the separated molecules instead of the silica |
| is left would be a spot on the plate that is red, and | | | | particles. This is most commonly used when extracting |
| possibly above it, a spot of blue. You have separated | | | | special chemicals from a mixture. An example would |
| the dyes into their basic components. | | | | be to extract the common chemicals from plants that |
| In columnar chromatography, the principle is the same, | | | | are beneficial to humans, like, say, aspirin. |
| except you use a glass tube, or column, to separate | | | | This technique is used all over the world for the |
| the chemicals. There is still a solvent involved, but | | | | extraction of many beneficial chemicals for |
| instead of the chemicals flowing up, they flow | | | | pharmaceuticals, cosmetics, and chemical production. It |
| downward by the use of natural gravity or fluidic | | | | is used by nearly every chemical research laboratory |
| pumps. Each chemical is separated within the solvent | | | | on earth, and is useful in the biochemical and |
| substrate, and can be purified in this manner. | | | | biomedical fields. Without HPLC, processes to extract |
| In high performance liquid chromatography, this whole | | | | materials or separate chemicals would be nearly |
| process is speeded up, due to the use of high | | | | non-existent. |