Spectrophotometric determination of phenylalanine in aqueous solutions with different acidity

A method for the determination of phenylalanine using the spectrophotometric method without preliminary correction of the solutions pH and the use of auxiliary chemical reagents has been developed. A comparative analysis of the spectrophotometric determination of phenylalanine by varying acidity of the medium was carried out. The errors and the possibility of their minimization were analyzed. It was shown that the analytical signal and the results of amino acid determination in the solutions with different pH differ significantly due to the presence of phenylalanine in various ionic forms that vary in the magnitude of the molar absorption coefficient. It was found that the maximum value of the molar absorption coefficient decreases in the series ε(Phe−)> ε(Phe+)> ε(Phe±) by 20%. The maximum error in determining the phenylalanine concentration without taking into account the dependence of the optical density of the solution on the acidity of the medium was found in strongly alkaline solutions. The correlation dependences of the analytical wavelength and molar absorption coefficient of phenylalanine on the pH of the medium were obtained experimentally. A long-wavelength (bathochromic) shift of the absorption band maximum was established upon the transition from positive to negative charge of the phenylalanine molecule with increasing pH of the solution. Two ranges of pH values have been identified, in which a change in the acidity of the solutions did not cause spectral changes. These were the regions where the amino acid was predominantly found in cationic Phe+ and bipolar Phe± forms (pH < 8) and in the region of anionic Phe− (pH > 10) form. The regression equations that allow choosing the analytical wavelength at any arbitrary pH of the solution were presented. The advantage of the developed approach is the possibility of increasing the detection sensitivity and ensuring that the error in the amino acid determination in the solutions of arbitrary acidity is less than 0.5% with the variation of no more than 0.3%. The detection limit of amino acid, calculated by 3σ – test, is (1.4 − 1.6) × 10-5 M. The range of the determined phenylalanine contents is (0.5 − 5.3) × 10-3 M. The efficiency of the proposed approach for determining the content of phenylalanine in a sample of the "L-Phenylalanine" (Protein Company, Russia) drug by the method of standard addition was shown. The possibility of minimizing the error in determining the amino acid when applying the technique to control the content of components in the process of demineralization of water-salt solutions of phenylalanine by electrodialysis with experimental membranes has been proven.