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Changing the Organic Modifier

If the desired resolution cannot be achieved with the first organic modifier chosen it is possible to change to an alternative solvent.  It is important to note that both retention and selectivity will change if the organic:aqueous ratio is altered (Figure 1).

From the separation of neutral chromatographic test compounds shown, it is clear that methanol is the ‘weakest’ solvent chromatographically and that THF is the ‘strongest’. It should also be noted that there are marked differences in the selectivity of each chromatogram.

The selectivity of the separation changes due to the differences in acidity, basicity and polarity of each of the common organic modifiers. Each of the solvents interacts in subtly different ways with the analyte molecules as each analyte has a different chemistry (dipole, acidic, basic, neutral etc.).

A solvent selectivity triangle is shown (Figure 2) on which the properties of common organic solvents are plotted.  Comparison of methanol, acetonitrile, and THF shows that THF has the most basic character (proton acceptor) and that acetonitrile has the largest molecular dipole.  Proton donor characteristics represent the acidity of the solvent.

 

Relative solvent properties

Figure 1: Relative solvent strength of some typical reversed phase HPLC solvents.

 

Figure 2: Solvent selectivity triangle.

 
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