As indicated by the name, size-exclusion chromatography (SEC) separates macromolecules based on their size. To accomplish this, the sample is passed through a porous column and molecules are isolated in fractions from the eluate. The size of the pores can vary depending on the size of the desired product. For isolation of EVs from proteins and lipoproteins, pore sizes can vary from ~40-80 nm. As the sample passes through the column, molecules smaller than the pore size will get trapped in the pores, slowing them down, while larger molecules will pass through. This results in the larger molecules, such as EVs, eluting from the column in earlier fractions, while smaller molecules, such as proteins and protein aggregates, are eluted in later fractions. To optimally use SEC for dilute matrices, such as urine or cell culture media, first these samples must be concentrated by an ultracentrifugation or ultrafiltration step.