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Why do large molecules leave a size-exclusion chromatography column before small molecules?
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In size-exclusion chromatography, the stationary phase consists of a porous matrix. The separation of molecules is determined by their ability to penetrate these pores.12
Large molecules emerge from the column first because they are too bulky to enter the pores of the support material. Consequently, they are restricted to the volume of the mobile phase outside the pores and pass through the column more quickly.12
In contrast, smaller molecules take longer to elute because they can diffuse into and out of the porous structure. By traversing this additional volume within the matrix, their path through the column is effectively lengthened, causing them to appear later than the larger components.12