Mobile phase has been rightly termed as the lifeline of the HPLC system. It plays the important role of transport of the sample through the separation column and subsequently to the detector for identification of the separated components.
Mobile phase is seldom a single solvent. It consists of combination of water with organic solvents, aqueous buffers with polar solvents or mixtures of organic solvents in desired proportions. The two operational modes commonly used are:
Isocratic mode – in the Isocratic mode the composition of the mobile phase remains constant throughout the analytical run ,i.e, the proportion of solvents in the mixture is pre-decided and remains unchanged during analysis
Gradient Elution – the composition is varied by the software through the analytical run in a predetermined mode and at the end of the run the proportion of solvents is different from the initial proportion.
The objective of using different solvent mixtures is to achieve the desired polarity of mobile phase for complete miscibility of the sample and control interaction of sample components with the stationary phase to achieve the desired degree of resolution of separated component peaks in the shortest possible time.
In order to meet the required objectives the mobile phase should have the following essential features:
- The sample should be fully soluble in the mobile phase. Any insolubility will result in flow restrictions. Always check sample solubility in mobile phase before injection into the system.
- Mobile phase components should be non-hazardous and non-toxic. They should not pose any health hazard to the operator.
- Mobile phase shouId be inert towards sample constituents and the stationary phase. Any reactions can lead to formation of insoluble suspensions which can result in column blockages.
- The mobile phase should not give its own response on passing through the detector. In other words the detector signals should reflect only the response of the sample constituents. This, however, is not applicable in bulk property detectors such as refractive index detector which respond to overall changes in refractive index of the mobile phase containing the eluting compound.
- The mobile phase should be affordable and the proportion of solvents used should make the analysis economically viable.
It is important to mix the solvents of specified purity and from same source to get consistency of results and also to adopt the same off-line mixing technique to avoid errors due to heat of mixing errors. In case buffers are to be used always adjust the pH of the aqueous phase prior to making up the final volume with the organic phase.
HPLC technique holds great promise for applications in diverse fields. Control of operational conditions including consistency of mobile phase will ensure repeatability of results.
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