Concepts of mobile phase selection in development of an HPLC method of analysis

Abstract

Introduction. Chromatography is the separation of analytes from a mixture using a stationary phase, into which the analyte is retained and a mobile phase that transports them through the column. The drug substances have different chemical affinity for the phases, those with a greater affinity for the mobile phase will elute earlier than those with a less affinity. The solvents used for the mobile phase, and the ratio in which they are used are very important as it can be tuned to change the relative analyte affinity and hence the retention time and selectivity of the separation. Aim of study. To highlight the concepts of selection of mobile phase in development of an HPLC method of analysis. Methods and materials. The electronic databases that were used: Medline, While, Scopus and Springer. The information was searched by using the keywords: “HPLC solvents”, “mobile phase solvents”, “acetonitrile” and “methanol”. Also, the search was conducted by using printed pharmaceutical and chemical journals. 78 bibliographic sources were eligible for our study. Results. It is considered that the solvent selection in development of a new HPLC method is perhaps the most commonly overlooked parameter, although the importance is major. Typically acetonitrile or methanol would be selected as hydrophobic solvents and would be mixed with water in different proportions for obtaining the highest resolution of separation. The separation process is influenced by many factors and using the only acetonitrile or methanol may not be enough. The solvents are selected by taking into account characteristics such as viscosity, refractive index, noncorrosiveness, toxicity, miscibility, transparency, reasonable price, commercial availability etc. To choose the most efficient solvents for the mobile phase, it should respect some rules: (1) the mixture of solvents of the mobile phase must be miscible. For example it have to avoid the mixture of acetonitrile with cyclopentane, cyclohexane, heptane, hexane, octane and pentane or the mixture of methanol with cyclopentane, cyclohexane, heptane, hexane. Water can be mixed with tetrahydrofuran, i-propyl-alcohol, methanol, ethanol, dioxane, dimethylformamide, acetic acid, acetonitrile, acetone; (2) mobile phase depends on both the choice of organic solvent and its concentration – it has been observed that on lowering the concentration of organic solvents the retention time increases; (3) buffered mobile phase should be used when the analyte contains acidic or basic moieties and a buffer should never be used outside 1.0 pH units around its pKa; (4) the mobile phases must be filtered through a 0.45 or 0.2 μm membrane filter and degassed. Conclusion: The solvent selection in development of a new HPLC method is a difficult process, but a very important stage. There is a choice among hundreds of solvents for different applications of HPLC and to select the most efficient solvents for the mobile phase, it should respect the main rules.