Glycerol-based technical grade surfactants of industrial interest generally contain a mixture of components with diverse composition. Consequently, their use cannot be generalized since the properties or the technical grade product will likely depend on batches and source. Herein, we address this issue by deconvoluting the role of individual components and pondering their influence on the phase behavior of mixtures. Technical grade diglycerol-monoisostearate (C41) was separated into different fractions based on polarity differences. Three fractions are obtained, which contain mainly unreacted diglycerol, the monoester and higher esterification degree amphiphiles. The formation of a single-phase reverse hexagonal liquid crystal (H2) in the water/C41 system is hindered by the presence of unreacted diglycerol, which dehydrates the hydrophilic channels of the H2 phase. On the other hand, the neat monoester forms a thermotropic lamellar liquid crystalline phase that melts at c.a. 40 °C, as confirmed by X-ray scattering (SAXS) and differential calorimetry (DSC). The formation of the lamellar phase in the neat monoester is ascribed to branching in the alkyl chain. The monoester also forms lamellar liquid crystals mixed with water and remains stable up to high water contents. Moreover, the addition of the higher esterification degree amphiphiles to the monoester/water mixtures promotes a transition from the lamellar phase to a reverse hexagonal one, confirmed by polarized optical microscopy and SAXS. Such transitions can be somehow predicted by calculations of the molecular packing parameters of mixtures. Similar to the lamellar phase, the reverse hexagonal liquid crystal remains stable in excess water. Finally, the coexistence between lamellar or hexagonal phases in excess water is harnessed to produce unilamellar vesicles and hexosomes, as confirmed by dynamic light scattering, SAXS, and cryo-electron microscopy.