As a result of the synthesis protocol polyoxyethylene sorbitan monooleate (polysorbate 80, PS80) is a highly complex mixture of compounds. PS80 was therefore separated into its main constituents, e.g. polyoxyethylene isosorbide esters and polyoxyethylene esters, as well as mono- di- and polyesters using preparative high-performance liquid chromatography. In this comprehensive study the individual components and their ethoxylation level were verified by matrix assisted laser desorption/ionization time-offlight and their thermotropic behavior was analyzed using differential scanning calorimetry and X-ray diffraction. A distinct correlation was found between the average length of the ethylene oxide (EO) chains in the headgroup and the individual compounds? ability to crystallize. Importantly, a critical number of EO units required for crystallization of the headgroup was determined (6 EO units per chain or 24 per molecule). The investigation also revealed that the hydrocarbon tails only crystallize for polyoxyethylene sorbitan esters if saturated. PS80 is synthesized by reacting with approximately 20 mol of EO per mole of sorbitol, however, the number of EO units in the sorbitan ester in commercial PS80 products is higher than the expected 20 (5 EO units per chain). The complex behavior of all tested compounds revealed that if the amount of several of the linear by-products is reduced, the number of EO units in the chains will stay below the critical number and the product will not be able to crystallize by the EO chains. (c) 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).