Substituting Acetonitrile With Another Solvent

Replacing acetonitrile with other HPLC solvents can be considered for both HPLC and UHPLC applications. For any change in mobile phases, it is important to consider how the change will affect not only retention factors but also selectivity, peak shapes, and resolution. In general, elutropic and polarity index values are a good first reference for consideration. The relationship between polarity indexes (P’) and retention factors in reversed phased liquid chromatography can be expressed as



where k’ represents the retention factor of an analyte in two different mobile phases. The polarity index for the mobile phase can be easily calculated knowing the polarity indexes of each one of the components and the composition of the mobile phase



If we consider these parameters as a starting point, methods that employ acetonitrile (P’=5.8) could be evaluated with methanol (P’=5.1) as possible replacement solvent.

For example, using these equations, a mobile phase comprising 50% water : 50% acetonitrile would give the same elutropic strength as a mobile phase comprising 44% water : 56% methanol.

Below is a plot which correlates the composition of several water-miscible solvents which could be used to replace the acetonitrile in a mobile phase.



It is important to consider that any solvent change affects the maximum pressure achieved by a method (due to changes in mobile phase viscosity). In addition, the actual results obtained can not be modelled or predicted based solely on solvent polarity index and strength parameters; rather, these parameters should be used as a starting point in the evaluation of a modified method.

Hints and tips for method development

Use a good quality column - Firstly, choose a new generation column such as Hypersil GOLD which gives good peak shape. The narrow, symmetrical peaks will help facilitate method development by offering improved resolution over older silica based media.

Optimise the mobile phase - Don’t forget that the mobile phase plays a key role in analyte retention and selectivity so optimize the mobile phase pH and buffer to obtain the necessary separation.

Evaluate an alternative stationary phase - Try a different column chemistry to see which gives the best selectivity with the new solvent. Hypersil GOLD columns are available with 6 different bonded phases.

Choose the appropriate UV wavelength - When working with UV detection, don’t forget to consider the chromophore properties of your analytes. The UV cutoff for methanol is 205nm and for acetonitrile is 190nm, so this may be an important consideration when changing mobile phase as part of method development.