Among the steps of the transformation chain to produce second generation bioethanol, this work addresses experimental aspects of the final step of bioethanol purification with real fermentation broths whose reducing sugars come from wheat straw. The initial bioethanol diluted broth was preconcentrated up to a concentration close to the azeotropic point with a distillation column of 15 stages in continuous operation, following a process previously designed on Aspen Plus®. In comparison with the process carried out with a synthetic mixture, bioethanol preconcentration required a longer settling time, but reflux ratio and reboiler duty were slightly lower. This difference motivated the construction of an equilibrium curve based on bioethanol, through a batch distillation system, in which a smaller boiling point is seen in comparison with the one of a pure ethanol-water mixture. With respect to the ethanol dehydration using glycerol as entrainer, experimentation in the same batch distillation system was carried out with both preconcentrated bioethanol and synthetic ethanol, and different glycerol-ethanol ratios. The experimental outcomes show that although with bioethanol the distillation temperature is slightly lower than the one with synthetic ethanol, the experimentation time is much longer. In other hand, it is demonstrated that a glycerol-ethanol of 0.8:1 is enough to get a fuel grade ethanol, which is a ratio different resulting from an Aspen Plus® based study. These outcomes show that current studies based on ethanol-water mixture are just an approximation to real bioethanol systems and real fermented broths would be needed to obtain more accurate data.