Molecules move along temperature gradients through a process called thermophoresis or the Soret effect. This phenomenon has been studied extensively, but its theoretical foundation in liquids remains debated. Using an all-optical microfluidic fluorescence method, researchers have experimentally demonstrated the Soret effect for DNA and polystyrene beads across a wide range of sizes, salt concentrations, and temperatures. The results support a unifying theory based on solvation entropy, where the Soret coefficient is given by the negative solvation entropy divided by kT. This theory predicts the thermodiffusion of polystyrene beads and DNA without any free parameters. The assumption of local thermodynamic equilibrium is valid for moderate temperature gradients below a fluctuation criterion. For both DNA and polystyrene beads, thermophoretic motion changes sign at lower temperatures, attributed to an increasing positive entropy of hydration and a generally dominating negative entropy of ionic shielding. Understanding thermodiffusion allows detailed probing of solvation properties of colloids and biomolecules. For example, the effective charge of DNA and beads was successfully determined over a size range not accessible with electrophoresis. The Soret coefficient scales linearly with particle surface and Debye length. The study shows that the Soret coefficient for DNA scales with the square root of its length, and for polystyrene beads, it scales with particle surface. The effective charge of DNA and beads was determined using the Soret effect, showing linear scaling with DNA length and particle surface. The findings provide a theoretical foundation for understanding thermodiffusion and its applications in colloid science and biotechnology.Molecules move along temperature gradients through a process called thermophoresis or the Soret effect. This phenomenon has been studied extensively, but its theoretical foundation in liquids remains debated. Using an all-optical microfluidic fluorescence method, researchers have experimentally demonstrated the Soret effect for DNA and polystyrene beads across a wide range of sizes, salt concentrations, and temperatures. The results support a unifying theory based on solvation entropy, where the Soret coefficient is given by the negative solvation entropy divided by kT. This theory predicts the thermodiffusion of polystyrene beads and DNA without any free parameters. The assumption of local thermodynamic equilibrium is valid for moderate temperature gradients below a fluctuation criterion. For both DNA and polystyrene beads, thermophoretic motion changes sign at lower temperatures, attributed to an increasing positive entropy of hydration and a generally dominating negative entropy of ionic shielding. Understanding thermodiffusion allows detailed probing of solvation properties of colloids and biomolecules. For example, the effective charge of DNA and beads was successfully determined over a size range not accessible with electrophoresis. The Soret coefficient scales linearly with particle surface and Debye length. The study shows that the Soret coefficient for DNA scales with the square root of its length, and for polystyrene beads, it scales with particle surface. The effective charge of DNA and beads was determined using the Soret effect, showing linear scaling with DNA length and particle surface. The findings provide a theoretical foundation for understanding thermodiffusion and its applications in colloid science and biotechnology.