I extracted chitosan from fungi and attempted depolymerization by acid hydrolysis. I then used the DNS assay to confirm the release of reducing sugars, but the color did not change. Does this indicate incomplete hydrolysis, or is DNS not suitable for detecting glucosamine under these conditions?
My goal is to confirm chitosan purity by HPLC after breaking it down into monomers. What would be the most reliable method of depolymerization for this purpose? Are there better alternatives (e.g., nitrous acid, enzymatic, oxidative) if the aim is to get complete monomers for HPLC analysis?
Incomplete hydrolysis or unsuitability of the DNS assay for glucosamine likely caused the lack of color change. Nitrous acid or enzymatic depolymerization methods are more reliable than acid hydrolysis for producing chitosan monomers suitable for HPLC purity analysis.
Dear Suchithra,
I use HCl to depolymerize fungal biomass to measure glucosamine via GC-MS. Incubation for 2 hours in 2 M HCl at 100 °C (~ 100 µL per mg dry cell weight) should be enough. After filtering off the debris you should be able to detect glucosamine in the hydrolysate using a suitable HPLC setup (e.g. Aminex HPX-87H, 5 mM H2SO4, 0.6 mL/min, 65°C, RI detection).
References:
Article From 13 C-lignin to 13 C-mycelium: Agaricus bisporus uses po...
https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_1928.pdf
Good luck
Michael
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