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what is HPLC
Risun Bio-Tech Inc  Sep 21, 2016

What is HPLC?

 

    High performance liquid chromatography is basically a highly improved form of column chromatography. Instead of a solvent being

allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it 

much faster.


    It also allows you to use a very much smaller particle size for the column packing material which gives a much greater surface area

 for interactions between the stationary phase and the molecules flowing past it. This allows a much better separation of the 

components of the mixture.


    The other major improvement over column chromatography concerns the detection methods which can be used. These methods 

are highly automated and extremely sensitive.



The column and the solvent


    Confusingly, there are two variants in use in HPLC depending on the relative polarity of the solvent and the stationary phase.


Normal phase HPLC


    This is essentially just the same as you will already have read about in thin layer chromatography or column chromatography. 

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.