Lab5_kineticsglucose - Chemistry 223 KINETIC DETERMINATION...

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1 Chemistry 223 KINETIC DETERMINATION OF GLUCOSE References: 1. C, Chap. 18, pp. 625-627. 2. SWH Chap. 22. Introduction The rate of a chemical reaction is usually a function of the concentration of species involved in that reaction. As a result, measurement of reaction rate can be used to quantitate concentrations. Enzyme-catalyzed reactions are especially useful in analysis because enzymes typically have high selectivity. A typical enzyme-catalyzed reaction can be represented with the Michaelis-Menten model of enzyme reactivity: P E ES S E 2 1 1 k k k where E is the enzyme, S is the substrate, ES is an activated intermediate complex, P is the product, and the k 's are the rate constants for each step. One can derive that the rate of reaction of substrate (-dS/dt) or formation of product (dP/dt) is Rate ] S [ ] S [ ] E [ P S 0 2 M K k dt d dt d where K M = ( k 2 + k -1 )/ k 1 and [E] 0 is the initial enzyme concentration. If the substrate concentration is small ([S] << K M ) this simplifies to Rate ] S [ ' ] S [ ] E [ P S 0 2 k K k dt d dt d M where 0 2 ] E [ ' M K k k Thus, for small substrate concentrations, the rate is linearly proportional to the substrate concentration. This is a first order reaction. Of course, as the substrate concentration decreases during the course of the reaction, the rate will decrease, until at equilibrium the rate is zero. However, at the beginning of the reaction, the initial rate will be directly proportional to the initial concentration of substrate, the concentration we wish to determine. In this experiment you will perform a fixed-time kinetic determination. The reaction is allowed to proceed for a fixed amount of time and then the amount of product formed will be determined. The higher the substrate concentration, the faster the reaction rate, and the more product is formed in the fixed time period.
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2 The glucose concentration of a sample will be determined by a procedure typical of the way glucose is determined in blood serum or plasma. Glucose oxidase is an enzyme used to selectively catalyze the oxidation of glucose to yield gluconic acid and hydrogen peroxide: Glucose + O 2 + H 2 O  oxidase glucose gluconic acid + H 2 O 2 The H 2 O 2 liberated by this first (slow) reaction is then determined by reducing the H 2 O 2 with a colorless dye to form a colored product in this much faster reaction: H 2 O 2 + Dye Red peroxidase H 2 O + Dye ox colorless colored The dye is o-dianisidine which yields a colored product that has an absorbance maximum at 540 nm.
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Lab5_kineticsglucose - Chemistry 223 KINETIC DETERMINATION...

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