Reactions of orthophthalaldehyde (OPA) with nucleophiles are used in analytical and biologically significant reactions for many years [1, 2]. Besides the analytical method for determination of amino acids, the solution of OPA is broadly applied in hospitals as a disinfecting agent for surgery instruments made from plastics. In the literature, there are several different protocols concerning the composition of the active solutions with different timing of the use. This situation is a consequence of the fact, that the mechanisms are not understood [3] and therefore the processes could not be optimized. OPA as a dialdehyde is a molecule with two communicating redox centers. The ortho-position of the two carbonyls enable intramolecular cyclization of intermediates, hence its reactivity with nucleophiles offers a number of structural and stoichiometric possibilities. In addition to this, hydration of the original structure plays a crucial role, since H2O is, in fact, the most abundant nucleophile in the system. Nevertheless, for the above mentioned use, the reaction of OPA with amines and particularly with amino acids is the most important. Due to the reducibility of carbonyl groups and their nitrogen analogues, the electrochemical approach is favorable for this mechanistic investigation. Besides the hydration(dehydration studies, the first series of experiments were focused on the reaction of OPA with aliphatic amino acids: eight simple amino acids with only one α-amino group (glycine, alanine, leucine, isoleucine, valine, norvaline, aminobutyric and aminoisobutyric acid), one ester preventing zwitterionic equilibrium (glycine ethyl ester) and lysine with two amino groups. Polarography and spectrometry were the main used techniques. Measurements were done in two phosphate buffers (pH 7,90 and 11,20) with pH above and under the pKa of amino groups in used amino acids. It was found out that the hydration of orthophthalaldehyde plays a very important role. If stock solution of OPA is prepared in acetonitrile, then in aqueous phosphate buffer the hydration of OPA competes with the reaction with amino acids more than it was expected in published articles [4]. If stock solution of OPA is prepared in water, the reaction with amino acids is faster than with non-aqueous stock solution. The main result is that according to our measurements the reactive species is not OPA as dialdehydic form but its (mono)hydrated form. At present the investigation continues with various aliphatic amino compounds, the relationship with hydration of OPA and the reaction products are presented and discussed. Acknowledgments The research was supported by GA CR (project No. 13-21704S) and by institutional support (RVO: 61388955).