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Commercial C18 columns packed with superficially porous particles of different size and shell thickness (Ascentis Express, Kinetex, and Poroshell 120) or sub-2-μm totally porous particles (Acquity BEH) were systematically compared using a small molecule mixture and a complex natural product mixture as text probes.Significant efficiency loss was observed on 2.1-mm ID columns even with a low dispersion UHPLC system.The Kinetex 4.6-mm ID column packed with 2.6-μm particles exhibited the best overall efficiency for small molecule separations and the Poroshell 120 column showed better performance for mid-size natural product analytes.The Kinetex 2.1-mm ID column packed with 1.7-μm particles did not deliver the expected performance and possible reasons beside extra column effect have been proved to be frictional heating effect and poor column packing quality.Different column retentivity and selectivity have been observed on the four C18 columns of different brand for the natural product separation.Column batch-to-batch variability which has been previously observed on the Ascentis Express column was also observed on the Kinetex and Poroshell 120 column.