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This method uses quantification of sugar released by endoglucanases from a disrupted cellulosic substrate as an indirect measure of the degree of amorphogenesis of the substrate induced by the non-hydrolytic disruptive proteins.
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The established GC/MS protocol allows quantification of sugars, sugar alcohols, organic and amino acids, ascorbate and some lipophilic compounds [87], [88], [89].
The filtrate was saved for quantification of sugars and extracellular metabolites and stored at -80°C.
Derivatisation/trimethylsilylation and quantification of sugars by GC-MS were performed following the published procedure [ 35] with the following modifications.
Absolute quantification of sugars was achieved by equating normalised areas to a set of standard curves for sucrose, glucose, and fructose.
The phytochemical analysis was performed by thin layer chromatography (TLC) and by the spectrophotometric quantification of sugars, proteins and phenolic compounds.
These DP data will be helpful for exploring whether there is any correlation between the DP and the measured reducing sugar ends in the pretreated FP and CL residues (as described in earlier section, "Quantification of reducing sugar ends in remaining filter paper and cotton linter".
External standards of arabinose, galactose, glucose, xylose and mannose were used at six different levels to develop calibration curves for quantification of the sugar concentrations.
Quantification of each sugar was accomplished by comparing the peak areas of the samples with those of standard solutions.
The identification and quantification of the sugars were carried out by ion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).
The diluted digestion-mixture aliquots were then filtered (0.2-μm AcrodiscR) before quantification of released sugars by HPLC.
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