High performance liquid chromatography is a powerful tool in analysis. This page looks at how it is carried out and reveals how it employs the very same principles as in thin layer chromatography and column chromatography. High Performance liquid chromatography is essentially an extremely improved form of column chromatography. Rather than a solvent being permitted to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much quicker. It Also permits you to use a very much smaller particle size for the column packing material which provides a much greater surface area for connections between the stationary phase and the molecules flowing past it. This permits a much greater separation of the components of the mixture.
The Other key advancement over column chromatography concerns the detection methods that could be used. These methods are highly automated and extremely sensitive. Confusingly, there are two variants being used in HPLC based on the relative polarity of the solvent as well as the stationary phase. This Is basically just the same as you will already have read about in thin layer what is chromatography. Even though it is described as normal, it is not the most widely used kind of HPLC. The Column is full of tiny silica particles, and the solvent is non-polar – hexane, for example. A normal column has an inner diameter of 4.6 mm and may be less than that, and a length of 150 to 250 mm. Polar Compounds in the mixture being passed through the column will adhere longer to the polar silica than non-polar compounds will. The non-polar ones will pass quickly through the column.
In This case, the column size is identical, but the silica is altered to make it non-polar by attaching long hydrocarbon chains to its surface – typically with either 8 or 18 carbon atoms in them. A polar solvent is used – for instance, a combination of water and an alcohol such as methanol. In This case, there will be a strong attraction between the polar solvent and polar molecules in the mixture being passed through the column. There will not be as much fascination between the hydrocarbon chains attached to the silica the stationary phase and the polar molecules in the solution. Polar molecules in the mix will therefore spend most of their time going with the solvent.