Exp7 - EXPERIMENT 7 ALTERNATIVE DATA-PROCESSING OPTIONS FOR...

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1 EXPERIMENT 7 ALTERNATIVE DATA-PROCESSING OPTIONS FOR KINETIC DATA (ENZYMATIC DETERMINATION OF GLUCOSE) A. PURPOSES The principal purpose of this experiment is to illustrate the relative merits of two approaches to processing kinetic data, namely initial-rate and pseudo- equilibrium options . On one hand, the initial-rate option is expected to be much faster than the pseudo-equilibrium option . On the other hand, the pseudo- equilibrium option is expected to be influenced less by changes in experimental conditions than the initial-rate option . B. OVERVIEW Many different options have been developed for processing kinetic data. The most commonly used options include initial-rate and fixed-time methods. Principal advantages of these options include simplicity and short measurement times. Principal disadvantages include low sensitivity, large dependencies on signal noise and large dependencies on experimental variables such as temperature, pH, and reagent concentrations. Two less common options include a pseudo-equilibrium option and a two- rate option. Principal advantages of these options include higher sensitivity and less dependence on signal noise and experimental variables. Principal disadvantages include more complex data-processing requirements and longer measurement times. This experiment is designed to illustrate implementation and performance characteristics of two of these data-processing options, namely the initial-rate option and the pseudo-equilibrium option . Experimental data for the enzymatic determination of glucose using glucose oxidase will be used to evaluate and compare these two data-processing options. As with the experiment done earlier in the semester, changes in absorbance, A, are used to monitor the reaction as a function of time. Changes in glucose oxidase activity will be used to evaluate the effect of variables on each option. C. RATIONALE The rationale for variable dependencies is described below for the initial-rate and pseudo-equilibrium options. 1. Initial-rate option For low substrate concentrations, velocities (rates) of enzyme-catalyzed reactions tend to be proportional to substrate concentration and enzyme activity. For the glucose reaction, the initial rate of change in absorbance is approximated by:
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2 0 0 ) ( G GO C C k dt dA γ = (1a) where k is a pseudo-first-order rate constant, γ is proportionality constant between glucose concentration and absorbance, C GO is the activity of glucose oxidase, and C G 0 is the initial glucose concentration. Equation 1a is rearranged to be explicit in glucose concentration. GO G C k dt dA C γ 0 ) ( 0 = (1b) It is apparent from Eq. 1b that undetected changes in glucose oxidase activity are expected to cause errors in determined values of glucose concentrations.
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