Permeation experiments of aqueous solutions of diprotic amino acids (L-glutamine and glycine) were carried out with numerous commercial polymeric nanofiltration (NF) and tight ultrafiltration (UF) membranes. The aim of this study was to determine the permeate flux and the amino acid rejection as a function of increasing feed concentration and ionization state of the amino acids. So far, apart from a few limited studies, highly diluted Solutions have been considered in the literature, although separation and purification of concentrated systems possess particular industrial interests. The concentration of amino acids in the whole range of their solubility was studied with a stepwise pH scan ranging from 0 to -1 total net charge. Differences in separation behavior in between high feed concentration area and diluted systems are discussed. Considerable higher rejection and flux drop over the concentration was observed in higher pH range, where amino acids are present in dissociated form. Membranes with different type of active layer material show similar concentration dependent tendency in the permeation behavior. This phenomenon can be explained by the dissociation dependency of the osmotic pressure. An altered form of the van't Hoff law is reported to calculate the pH dependency of the osmotic pressure and verified by vapor pressure osmometry and reverse osmosis (RO) experiments.