Title: Separation, Detection and Quantification of Galacturonic Acid Oligomers with a Degree of Polymerization Greater Than 50 Authors
|Grohmann, Karel - RETIRED, USDA|
Submitted to: Journal of Liquid Chromatography and Related Technologies
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 26, 2004
Publication Date: February 1, 2005
Citation: Cameron, R.G., Grohmann, K. 2005. Separation, detection and quantification of galacturonic acid oligomers with a degree of polymerization greater than 50. Journal of Liquid Chromatography and Related Technologies. 28:559-570. Interpretive Summary: Pectin, a major component of citrus fruit peel, is used in the food industry as a gelling and stabilizing agent. Potential industrial uses for pectin, modified pectin or pectin containing bio-based products includes additives for a variety of manufactured products, ion exchange resins, flavor or aroma encapsulators and as coatings. The functional properties of pectin depend on its chemical structure. Small changes in its structural properties can produce very different functional properties. One method of changing its structural properties is by enzymatic demethylation. To characterize these small structural changes it is often necessary to separate and measure very small fragments of the original material. Detecting and quantifying these small fragments has been especially difficult. The techniques described in this work improve our ability to both detect and to quantify these small fragments. These techniques will allow us to reconstruct the modified structural features that resulted in new or improved functional qualities and to compare the way different demethylating enzymes affect pectin fine structure.
Technical Abstract: A High Performance Anion Exchange Chromatography--Evaporative Light Scattering Detector (ELSD) method was developed to detect, separate and quantify galacturonic acid (GA) oligomers. Following digestion of polygalacturonic acid with a monocomponent endo-polygalacturonase, more than 70 GA oligomer peaks could be resolved using a convex/linear ammonium formate gradient. Linear calibration curves were produced for 0.015%--1.0% mono-, di- and tri-GA. The mass response for mono-GA differed from di- and tri-GA as evidenced from the slope of the calibration curve regression lines (1.611 ± 0.0201 for mono-GA vs. 1.3068 ± 0.0291 and 1.3004 ± 0.0262 for di- and tri-GA respectively). The degree of polymerization (DP) appeared to affect mass response as the trend line for log transformed peak areas of DP 3, 4, 6 and 8 oligomers had a slope of -0.0304 ± 0.0032 (r2 = 0.98). Buffer concentration also affected mass response. ANOVA of peak areas from isocratic elution of trimer and hexamer with 50 mM to 0.8 M ammonium formate indicated mass response was dependent on buffer concentration for each oligomer (P < 0.005), although Duncan's Multiple Range Test described concentration ranges within which mass response was not affected (P < 0.05).