UK CHE 226 - Kinetic Methods of Analysis - Enzymatic Determination of Glucose
Course Che 226-
Pages 11

Unformatted text preview:

Department of Chemistry University of Kentucky EXPERIMENT 9 Kinetic Methods of Analysis: Enzymatic Determination of Glucose SAFETY WARNING In this experiment you will be using syringes (glass, breakable) that have sharp pointy needles on them. Work carefully. Never use force on a syringe. Some of them might fit pretty tightly, especially when dry, but they need to slide relatively smoothly with a minimum of force. Please don’t spear yourself with the working end of a syringe. I don’t want to have to walk you over to the Student Health Service, where, if you weren’t sick or injured when you walked in, you will be so after you leave. The phenol and 4-aminoantipyrene in the “Enzyme Stock Solution” that you will be using are mildly toxic, but both are present in such low concentrations that this is not a major hazard problem at all. Nevertheless, you might wish to wear latex gloves when working with the reagents. Just stay alert and work in a controlled and neat manner. Keep things clean at all times. UNKNOWN Submit a clean, labeled 50-mL volumetric flask to the instructor so that your unknown iron solution can be issued. Your name, section number, experiment number, and locker number should be written legibly on this flask. The flask does not need to be dry on the inside, but needs to have been rinsed with distilled or deionized water after it has been washed. The flask must be turned in at least 1 lab period before you plan to do the experiment so that the Teaching Assistants will have time to prepare the unknown. Each student will have his or her own unknown to analyze even if you are working in pairs. [This year you do not need to submit a 50-mL flask for your unknown. This is a new experiment, it’s in the testing phase, and 50-mL flasks are not part of standard-issue student equipment.] INTRODUCTION Literally billions of clinical chemistry tests are performed annually in the United States. The bulk of these analyses are performed by use of spectrophotometric methods. Of these, ordinary absorption spectroscopy in the visible region is the one most commonly used. The instrumentation for visible absorption spectroscopy is simple, stable, relatively inexpensive, and easy to automate, thus it can handle the large numbers and variety of tests to be done. The method has a large number of advantages and very wide applicability. Many substances absorb light, and if the analyte itself does not absorb, it can often be treated with reagents to generate a product with suitable absorbance characteristics. CHE 226 – Analytical Chemistry Laboratory 50 Glucose KineticsDepartment of Chemistry University of Kentucky The vast majority of chemical analyses are performed on systems that are at equilibrium, clinical or otherwise. The chemistry is “over.” This is true whether the analysis method is spectrometric, electrochemical, chromatographic, calorimetric, or even radiochemical in nature. If not technically at complete equilibrium, as is the case in redox electrochemistry and radiochemistry, the analyses performed are under quasi steady-state conditions. When chemistry is performed to prepare samples for analysis, even with use of enzymes, the samples are normally allowed to react or to “incubate” for a sufficient period of time that the system is at or very near equilibrium. However, kinetic methods of analysis, wherein one measures the rate of an ongoing chemical reaction to determine the quantity or concentration of the analyte of interest, are becoming increasingly popular. The first, and probably most important, advantage of kinetic methods in general is speed. You don’t have to wait for the reaction to go to completion to make your measurement. Kinetic methods based on reaction-rate measurements are usually done only during the first 3 to 10% of the reaction, before possible back-reactions start to become significant. In this situation, one measures the so-called initial reaction-rate with little error. Kinetic methods are also particularly useful in samples in which some interferent is present, even if in large and variable concentrations from sample to sample. For example, take absorption spectroscopy as the method and a colored sample such as whole blood or bottled/canned soft drinks and juices. If you can develop a kinetic method that reacts only with the analyte, or at least does not react with the interferent, modern instruments with microprocessors can often succeed in providing you with a decent or even very good analysis by measuring the rate of change in absorbance of a sample undergoing a reaction without having to do complicated and time-consuming chemistry to eliminate the interfering colored background or to resort to some separation method. The primary objective of this experiment is two-fold: To provide you experience with a kinetic method of analysis and to acquaint you with the use of enzymes as an analytical tool or “reagent”. THE ENZYMATIC REACTION Glucose is a major component of animal and plant carbohydrates. Quantitative determination of glucose is important in clinical chemistry, biochemistry, and food science. In this experiment you will measure glucose levels using enzymatic reactions and visible absorption spectrophotometry. While there are several variants in enzymatic determinations of glucose, this one will use glucose oxidase (GOX) and horseradish peroxidase (HRP) enzymes. Essentially the initial rate of the first enzymatic reaction will be the basis for quantitation. Glucose is oxidized to δ-D-gluconolactone and hydrogen peroxide in the presence of GOX: GOX β-D-glucose +O2 + H2O Æ δ-D-gluconolactone + H2O2 (1) CHE 226 – Analytical Chemistry Laboratory 51 Glucose KineticsDepartment of Chemistry University of Kentucky Unfortunately, neither of the products absorbs light in the visible region. Therefore, the production of hydrogen peroxide is coupled to a reaction catalyzed by HRP: HRP H2O2 + phenol + 4-aminoantipyrene Æ 4-N-(p-benzoquinoneimine)-antipyrine + 4 H2O (2) The reaction product absorbs light maximally at λmax = 505 nm with a molar absorptivity ε = 1.27 x 104 L/mol-cm (M-1cm-1). Enzyme Concentrations Enzyme “concentrations” are usually expressed in units of IU. IU is an acronym for International Unit, which is essentially an enzymatic “activity” – how well the enzyme does its catalysis.


View Full Document

UK CHE 226 - Kinetic Methods of Analysis - Enzymatic Determination of Glucose

Course: Che 226-
Pages: 11
Download Kinetic Methods of Analysis - Enzymatic Determination of Glucose
Our administrator received your request to download this document. We will send you the file to your email shortly.
Loading Unlocking...
Login

Join to view Kinetic Methods of Analysis - Enzymatic Determination of Glucose and access 3M+ class-specific study document.

or
We will never post anything without your permission.
Don't have an account?
Sign Up

Join to view Kinetic Methods of Analysis - Enzymatic Determination of Glucose 2 2 and access 3M+ class-specific study document.

or

By creating an account you agree to our Privacy Policy and Terms Of Use

Already a member?