Selection of solvents for use as a mobile phase in HPLC analysis is a key component of method development. It is not possible to hava a universal solvent which will meet all applications and more than often a combination of solvents is decided based on the analysis requirements. Selection of suitable solvents is based on their physical properties and compatibilities with the sample and column stationary phase.
The article discusses some of the essential considerations that play an important role in deciding suitability of solvents as mobile phase.
Cost is an important consideration as HPLC requires superior purity grade solvents and it is common to see dozens of HPLC systems operating round the clock in large laboratories. This means consumption of high grade solvents in bulk quantities and therefore cost considerations play a vital role.
The sample should be completely soluble in the mobile phase. Slightest insolubility will result in phase separations or suspensions which will contribute to operational problems.
Generally the detectors used in HPLC are based on absorbance of light by sample contituents. The intrinsic absorbance of the mobile phase components in the selected wavelength range should not interfere with the absorbance of the sample. The mobile phase solvent should ideally have no absorbance at the wavelength of interest.
The mobile phase solvents should have low volatility especially for use with light scattering detectors. Highly volatile solvents can lead to compositional changes in mobile phase composition over use and storage. This can lead to poor reproducibility of chromatograms.
The selected solvents should have low viscosity so that flow through the column does not lead to development of high back pressures.
The selected solvents should be inert to sample components, column packing and column material. Any reactivity with any of these components can lead to formation of precipitates, gases or other reaction products which can upset the system performance. The solvents should not form separate phase on coming in contact with the sample. In other words there should be complete miscibility of solvents.
Water is also an important component of reverse phase mobile phase composition either in combination with other solvents or as buffer media. Water purification systems are commercially available. The water generated by such systems should be used at the earliest possible so that its quality does not degrade over the storage period.
HPLC technique is highly sensitive to changes in the composition of mobile phase reaching the detector so it is essential to filter the solvents through 0.45μm filter to remove any solid suspensions. Degassing is also an essential requirement to remove any trace of dissolved air which can lead to flow restriction or spurious peaks.