Flow Injection Determination of Sugars by Combining Immobilized Enzyme Reactors and Chemiluminescence Detection
Swindlehurst, Cathy A.
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Permalink
https://hdl.handle.net/2142/70415
Description
Title
Flow Injection Determination of Sugars by Combining Immobilized Enzyme Reactors and Chemiluminescence Detection
Author(s)
Swindlehurst, Cathy A.
Issue Date
1988
Doctoral Committee Chair(s)
Nieman, Timothy A.
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Analytical
Abstract
Chemiluminescence has long been known as a sensitive means of detection, but analytical applications have been limited due to a lack of selectivity. There has been much interest in recent years in combining chemiluminescence detection with the inherent selectivity of enzyme reactions. The determination of glucose by enzymatic oxidation in the presence of glucose oxidase to produce gluconic acid and hydrogen peroxide and subsequent detection of the hydrogen peroxide produced by chemiluminescent reaction with luminol has been widely used.
A flow injection method for the determination of the sugars commonly found in food products (glucose, sucrose, maltose, lactose, and fructose) using enzyme reactions and chemiluminescence detection has been developed. Glucose is oxidized to produce hydrogen peroxide directly in the presence of glucose oxidase. Other sugars are first enzymatically converted to glucose. Enzyme reactions are accomplished on-line using immobilized enzymes in flow-through reactors. For glucose, sucrose, and maltose the linear working range is approximately 0.2 $\mu$M to 1.0 mM with a detection limit of 0.1 $\mu$M. For lactose and fructose the linear working range is about 0.5 $\mu$M to 1 mM with a detection limit of 0.5 $\mu$M. Assay time is two minutes.
The methods for the determination of individual sugars were then extended to the determination for sugars in mixtures. For the determination of sugars in mixtures containing glucose, the glucose initially present was eliminated on-line using immobilized glucose oxidase and catalase. Then the sugar of interest was converted to glucose by the appropriate enzyme(s) and quantitated using glucose oxidase and chemiluminescence detection. Impurities in some of the enzymes lead to an apparent cross-reactivity which necessitated the use of a mathematical correction. The mathematical correction was applied to the determination of sugars in prepared mixtures. Experimentally determined values were found to be in good agreement with actual concentrations.
The usefulness of this method for the determination of sugars was demonstrated by application to the determination of sugars in food products. Several foods with a wide variety of sample matrices were assayed. In all cases sample preparation was relatively simple. The results obtained were in good agreement with expected values.
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