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United States Department of Agriculture

Agricultural Research Service

Title: Multiple Detection (Light Scattering, Fluorescence, Retractive Index, Uv) in Size-Exclusion Chromatography of Soluble Glucan Polymers

Authors
item Yokoyama, Wallace
item Knuckles, Benny

Submitted to: Book Chapter
Publication Type: Book / Chapter
Publication Acceptance Date: April 26, 2004
Publication Date: January 1, 2005
Citation: Yokoyama, W.H., Knuckles, B.E. 2005. Multiple Detection (Light Scattering, Fluorescence, Refractive Index and UV) in Size-Exclusion Chromatography of Soluble Glucan Polymers. ACS Book Chapter 8:141-149.

Interpretive Summary: Soluble polymers containing, ß-glycosidic linkages, can be characterized and quantified in biological and food matrices using a combination of detectors after separation by size exclusion chromatography. A combination of light scattering and refractive index is used to determine the polymer size distribution. Post-column reaction of the glucan polymer with calcofluor to form a fluorescent complex provides a means of positive identification of glucan polymers from other co-eluting polymers. Characterization of soluble polymers can provide information about the effects of food processing and enzymes that can reduce the polymer size and physical or biological properties of these polymers.

Technical Abstract: ß-Glucan ((1->3),(1->4)-ß-D-glucan) and methylcellulose are soluble dietary fibers that have physiological properties beneficial to human health. They are often studied for their nutritional properties but they are rarely characterized. We describe the use of size exclusion chromatography and multiple detectors to characterize these polymers: an 18 angle light scattering detector and refractive index detector to determine molecular weight distributions of the polymers, and a postcolumn reaction to form a fluorescent complex detected by a fluorescence detector. The fluorescence from the polymer specific complex enables positive identification of the glucan and/or methylcellulose peak of interest when multiple components elute upon chromatography of biological samples. Some applications of this system to characterize the affects of processing and digestion on ß-glucans and their relationship to cholesterol lowering; and the effects of colonic fermentation on methylcellulose are described.

Last Modified: 9/22/2014