Abstract The different oligomers of ethoxylated nonylphenols present in commercial surfactants can be separated by HPLC. While low ethylene oxide number (EON) species may be separated by isocratic HPLC on silica, intermediate EONs require gradient elution HPLC. The separation of higher oligomers can be carried out with an intermediate polarity column of the NH2 type and a solvent gradient. The suggested experimental conditions allow a satisfactory single run separation of a complex mixture with EONs ranging from 1 to 25 by using a linear gradient from 100% less polar solvent (n-heptane—chloroform—methanol 90/5/5) to its mixture with up to 20% of a more polar solvent made of chloroform—methanol (50/50). This method is used to analyze the EON distribution resulting from the mixing of different commercial surfactants. When two surfactants with very different EON distributions are mixed to produce a formulation scan, the occurrence of three-phase behavior at the optimum formulation of the surfactant—oil—water system is found to correlate with the absence of a gap in the overall EON distribution. Both the fractionation of low EON species into the oil phase and the fractionation of high EON oligomers into the water phase result in a depletion of the microemulsion phase.