New method for a two-step hydrolysis and chromatographic analysis of pectin neutral sugar chains.
A new method for the determination of the main neutral sugars in pectin has been developed. The sample preparation involves a mild chemical attack followed by an enzymatic hydrolysis. The completeness and nondestructive character of the method are demonstrated by comparison of the results obtained with different acids such as H2SO4, HCl, and trifluoroacetic acid (TFA) at different concentrations (2, 1, or 0.2 M) at two temperatures (80 or 100 degrees C). The chemical hydrolysis of pectin neutral sugar chains with strong acid (1 or 2 M) and high temperature (100 degrees C) shows that the liberation of the pectin sugars is not realized at the same rate for each sugar. Different optimum conditions are thus obtained. However, the chemical pectin hydrolysis with 0.2 M TFA at 80 degrees C is characterized by the liberation of pectin neutral sugar side chains without any degradation within 72 h of hydrolysis. Under these conditions, the liberation of some pectin sugars, essentially galactose, glucose, and rhamnose, was not complete. An enzymatic hydrolysis is necessary to obtain a complete release of all the sugars. The combination of the two treatments, a chemical hydrolysis realized with diluted acid (0.2 M) for 72 h at low temperature (80 degrees C) on one hand and an enzymatic hydrolysis on the other hand, allow a total liberation of pectin sugars. The quantitative analysis of the carbohydrates is realized with accuracy, high selectivity, and sensitivity with high-performance anion-exchange chromatography with pulsed-amperometric detection. The sugars can be analyzed without any derivatization with a limit of quantification of 0.1 mM.
Garna
Haikel
H
Unité de Chimie Biologique Industrielle, Faculté des Sciences Agronomiques de Gembloux, Passage des Déportés 2, B-5030 Gembloux, Belgium. garna.h@fsagx.ac.be
Mabon
Nicolas
N
Wathelet
Bernard
B
Paquot
Michel
M
D000143:Acids; D002241:Carbohydrates / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005690:Galactose / Q000032:analysis; D005947:Glucose / Q000032:analysis; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D010368:Pectins / Q000737:chemistry*; D012210:Rhamnose / Q000032:analysis; D012680:Sensitivity and Specificity
D000143:Acids; D002241:Carbohydrates; D010368:Pectins; D005947:Glucose; D012210:Rhamnose; D005690:Galactose