Analytical Sciences

The Upper Limit pH in the Dye-binding Method for the Determination of Serum Protein via Measurements of the Absorbance Increase Produced by Protein Error

Yuji SUZUKI

Saitama Prefectural University Junior College, Department of Medical Technology, 820, Sannomiya, Koshigaya, Saitama 343-8540, Japan

In the dye-binding method for determining the albumin concentration, the absorbance increase due to the change of the color shade by protein error of a pH indicator can be measured by a spectrophotometer. This absorbance increase is observed only in a restricted pH region, but this pH region is not theoretically studied yet. Thus, the author investigated the upper limit pH (pH_UL) at which the absorbance increase occurs by the theoretical calculation, and compared these results with those obtained experimentally using four pH indicators. The pH_UL is not affected by the dye or protein concentrations, or by the formation constant of the dye-protein complex; but the value changes according to the acid-dissociation constant of the dye (K_D) and the ratio of the molar absorptivities of the proton-dissociated dye anion (ε_D) and the dye-protein complex (ε_PD). The pH_UL value can be calculated by the equation, found theoretically. The calculated pH_UL values of BPB, BCG, BCP and BTB were 5.1, 4.8, 6.2 and 5.5, respectively. These values correlated with the experimental results of 4.5 for BPB, 4.7 for BCG, 5.9 for BCP and 5.2 for BTB, but were not associated with the pK_D values of each dye. The pH_UL of these dyes did not change significantly for various dye and protein concentrations, as was expected from the thoretical calculation.

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