Detailed Report on the Use of Enzymatic Cycling for High Specificity and Precision in the Colorimetric Analysis of Ammonia

摘要

A new method was developed for the determination of ammonium ion concentrations in water in the range of 0.0001-0.00001 molar, based upon enzymatic cycling. The sensitivity of the method could be extended well below the ten nanomoles/sample limit of determination, using suitable modifications of the procedure. This two-step method involved the use of an ammonia-specific glutamate dehydrogenase enzyme reaction with simultaneous oxidation of NADH; followed by an enzymatic cycling reaction to measure oxidized NADH by means of measuring the cycling reaction product, a tetrazolium formazan, in the visible at 600 nm. Using the procedure, linear plots of absorbance versus original ammonium ion concentration had correlation coefficients of 0.997-0.9998. The precision (coefficient of variation) of the method on replicate analyses of samples containing 0.0000441 molar ammonium ion was 2%. A calibration curve for determination of unknown ammonium concentrations could be constructed from standard solutions carried through the procedure. Analysis time per sample was three hours, and all solutions were stable for at least two weeks.