pH‐stat methodology in continuous monitoring of the kinetics of hydrolysis of phosphate esters catalysed by alkaline phosphatase from human placenta: Limitations, advantages and theoretical aspects

• Published in: Polymer International
• Main Authors: Roig, M. G., Serrano, M. A., Bello, J. F., Cachaza, J. M., Kennedy, J. F.
• Format: Article in Journal/Newspaper
• Language: English
• Published: Wiley 1991
• Subjects: Carbonic acid
• Online Access: http://dx.doi.org/10.1002/pi.4990240108; https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1002%2Fpi.4990240108; https://onlinelibrary.wiley.com/doi/pdf/10.1002/pi.4990240108

Abstract The appropriateness of pH‐stat methodology in the dynamic monitoring of the kinetics of hydrolysis of phosphate esters catalysed by alkaline phosphatase (from human placenta) has been studied. This methodology involves autotitration of the H + released or consumed during a given reaction time. The lack of specificity of the method, titrating the total H + , together with the known effect of the dissolution of environmental CO 2 on the reacting system, giving rise to carbonic acid, implies an overtitration of the H + released in the hydrolysis reaction. Because of this, parallel to each series of reaction kinetics, the dissolution kinetics of environmental CO 2 under working conditions are quantified with suitable blanks, thus enabling one to subtract them from the global kinetics of the release of H + . Although no background buffers are added to the reacting system in order to maintain a constant pH, the buffering capacity of the system itself obliges one to quantify it by calibrations in order to calculate the true release rate of H + in the reaction. These two principal limitations of the pH‐stat methodology, the dissolution of environmental CO 2 and the inherent buffering capacity of the system, can be partially mitigated by working within an inert atmosphere of N 2 and having a good knowledge of the acid/base characteristics of the reagents and reaction products, which together will permit the choice of a working pH range in which the second limitation will be negligible. Having studied theoretically the acid/base balances of the system in reaction, it is concluded that optimum working conditions for the pH‐stat methodology are between pH 8·5 and 11·0, since over this pH range the buffering capacity is null (thus eliminating the need for calibrations) and the stoichiometry of H + ( n H + ) is close to unity. These theoretical predictions have been confirmed experimentally with calibrations and measurements of n H + at different pH values.