Separation of f-block elements is a daunting task due to their similar chemical behaviour. Among these, the separation of uranium from a thorium matrix is one of the persisting challenges. This finds application both in the processing of thorium ores e.g. monazite, as well as in the separation of U-233 from irradiated Th-232. U/Th separation has been conventionally achieved by solvent extraction using tri-n-butyl phosphate (TBP). This work is an extensive study on the use of tri-sec-butyl phosphate (TsBP), which shows significantly higher selectivity for U(VI) over Th(IV), leading to strikingly superior separation factors. This selectivity can be attributed to the combination of structural and electronic effects near the binding site. Systematic and comprehensive studies at both liquid-liquid as well as solid -liquid interfaces have been carried out under a wide range of experimental conditions. The effect of extractant concentration on selectivity towards U(VI) has been examined at the liquid interface. Further, the extractants TBP and TsBP were impregnated on to a XAD-7 polymer support and the separation of U(VI) from Th(IV) matrices of various U/Th ratios has been achieved using extraction chromatography. Strategically optimized conditions of loading and elution stages for varying U/Th feed ratios have been demonstrated using TsBP coated columns for efficient separation of U(VI) from a Th(IV) matrix. Additionally, electronic structure calculations using density functional theory (DFT) have been carried out to shed light on the energetics and specific complexation of U(VI) over Th(IV) with the extractant TsBP and compared with TBP. Theoretically calculated, solvent corrected complexation energies for the formation of U(VI) and Th(IV) complexes with TBP and TsBP are in good agreement with the experimental observations.