Theoretical Analysis Concerning the Characteristics of a Dye-binding Method for Determining Serum Protein Based on Protein Error of pH Indicator: Effect of Buffer Concentration of the Color Reagent on the Color Development

In the dye-binding method based on protein error of a pH indicator, the color development has been reported to be markedly affected by the buffer concentration of the color reagent. In this study, the author analyzed this phenomenon by a theoretical calculation based on the chemical equilibrium of protein error. The calculation was performed on the assumption that both the dissociated dye anion and the anion contained in the buffer solution react with protein, forming a dye-protein complex and an anion-protein complex, respectively. The calculated results were compared with those obtained by the experiments using bromophenol blue, bromocresol green and bromocresol purple that are employed widely for determining the human serum albumin concentration clinically. The calculated results of this method are summarized as follows: (1) the color development decreases with the increase in the concentration of the anion contained in the buffer solution; (2) the calibration curve is more linear in the higher concentration of the anion than in the lower one. These calculated results agreed well with the experimental ones. From these results, it was concluded that the change in the color development by the buffer concentration of the color reagent is due to the change in the concentration of the buffer anion.