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Different hydrophobic and hydrophilic functionalities are produced in the reaction mixture of Tween®surfactants which affect the physiochemical properties of the final product. Only hydrophilic fractions and monoesters were separated in the binary mobile phase. MALDI TOF-MS of Tween®40 revealed the identification of peaks separated in binary mobile phase (methanol-water). Liquid adsorption chromatography was performed on a gradient system on a SynergiTMfusion reverse phase column. Mobile phase composition used for this separation was a ternary phase gradient starting from 41 % methanol, 47 % acetone and going to 96 % acetone for the analysis time of 60 minutes. In the ternary mobile phase, gradient all the species such as polyethylene glycol, sorbitan and isosorbide ethoxylates, mono-, di- and triesters of sorbitan and isosorbide ethoxylates were well resolved in an of 60 minutes analysis time. The above separation mechanism can be employed for the analysis of polymeric multifunctional surfactant compositions.