Microscopic particle separation plays vital role in various scientific and industrial domains. In this Letter, we propose a universal non-equilibrium thermodynamic approach, employing the concept of Shortcuts to Isothermality, to realize controllable separation of overdamped Brownian particles. By utilizing a designed ratchet potential with temporal period , we find in the slow-driving regime that the average particle velocity , indicating that particles with different diffusion coefficients can be guided to move in distinct directions with a preset . Furthermore, we reveal that there exists an extra energetic cost with a lower bound , alongside a quasi-static work consumption. Here, is the thermodynamic length of the driving loop in the parametric space. We numerically validate our theoretical findings and illustrate the optimal separation protocol (associated with ) with a sawtooth potential. This study establishes a bridge between thermodynamic process engineering and particle separation, paving the way for further explorations of thermodynamic constrains and optimal control in ratchet-based particle separation.

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