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Friday, March 5, 2010

Normal and Reversed Phase Simplified


Normal phase HPLC
Although it is described as "normal", it isn't the most commonly used form of HPLC.
The column is filled with tiny silica particles, and the solvent is non-polar - hexane, for example. A typical column has an internal 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 stick longer to the polar silica than non-polar compounds will. The non-polar ones will therefore pass more quickly through the column.


Reversed phase HPLC
In this case, the column size is the same, but the silica is modified 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 example, a mixture 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 won't be as much attraction between the hydrocarbon chains attached to the silica (the stationary phase) and the polar molecules in the solution. Polar molecules in the mixture will therefore spend most of their time moving with the solvent.
Non-polar compounds in the mixture will tend to form attractions with the hydrocarbon groups because of van der Waals dispersion forces. They will also be less soluble in the solvent because of the need to break hydrogen bonds as they squeeze in between the water or methanol molecules, for example. They therefore spend less time in solution in the solvent and this will slow them down on their way through the column.
That means that now it is the polar molecules that will travel through the column more quickly.
Reversed phase HPLC is the most commonly used form of HPLC.

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