Practical applications of the solvophobic theory to the analytical separation of biomolecules by reversed phase HPLC

Reversed phase high performance liquid chromatography (RP-HPLC) is widely applied to analyze a very broad range of molecules including charged and polar compounds. The separation mechanism, which is based on the interactions of the analytes with the hydrophobic chromatographic support in a polar mobile phase, has been deeply described by Csaba Horváth on the basis of the solvophobic force theory [1]. Accordingly, the distribution of a given analyte between the two phases depends on its polarity, the binding properties of the medium and the composition of the mobile phase, consisting of a hydro-organic mixture, which might contain a suitable buffer to control the protonic equilibrium. Decreasing the mobile phase polarity by adding more organic solvent reduces the hydrophobic interaction between the stationary phase and the analyte, resulting in weaker retention. The more hydrophobic the analyte the more time it will spend on the stationary phase and the higher the concentration of organic solvent that is required to promote elution. This communication discusses the dependence of retention behaviour of a variety of biomolecules in RP-HPLC on the experimental parameters, such as flow rate, column length and ID, dwell volume, temperature, isocratic and gradient elution mode, variation of organic solvent concentration in gradient elution mode (gradient shape and duration). The influence of the considered parameters on the chromatographic behaviour of the selected compounds is discussed in the framework of the hydrophobic theory, both by changing one of the above parameters while keeping constant all the others. Also discussed is the use of DryLab modelling software, which allows the development of methods concordant with a Quality by Design (QbD) criteria, increasing flexibility in routine operations. The state-of-the-art will be illustrated with a few applications in the field of food and phytochemical analysis. [1] Cs. Horvàth, W. Melander, I. Molnar, J. Chromatograph. 125, (1976) 129 - 156