Edgar A. Arriaga

Organelle size varies with normal and abnormal cell function. Thus, size‐based particle separation techniques are key to assessing the properties of organelle subpopulations differing in size. Recently, insulator‐based dielectrophoresis (iDEP) has gained significant interest as a technique to manipulate sub‐micrometer‐sized particles enabling the assessment of organelle subpopulations. Based on iDEP, we recently reported a ratchet device that successfully demonstrated size‐based particle fractionation in combination with continuous flow sample injection. Here, we used a numerical model to optimize the performance with flow rates a factor of three higher than previously and increased the channel volume to improve throughput. We evaluated the amplitude and duration of applied low‐frequency DC‐biased AC potentials improving separation efficiency. A separation efficiency of nearly 0.99 was achieved with the optimization of key parameters—improved from 0.80 in previous studies (Ortiz et al. Electrophoresis, 2022;43;1283–1296)—demonstrating that fine‐tuning the periodical driving forces initiating the ratchet migration under continuous flow conditions can significantly improve the fractionation of organelles of different sizes.