Hey there all,
I need to prepare 0.5% chitosan substrate solution in 0.1 M Sodium acetate buffer (pH 5.6) for my enzymatic activity assay. The buffer is ready, the chitosan is ready (Sigma 448869, low molecular weight), yet it doesnt dissolve even a bit. I have tried to put it on a stirrer with 60 C temperature overnight, still nothing. Anyone with an experience on how to dissolve chitosan in something other than acetic acid, maybe?
Thanks a lot!
Hello Samet, chitosan is a weak base and very insoluble in water. However, it can dissolve, after pretreatment, in ethanol, methanol and chloroforn apart from the acetic acid you mentioned. To facilitate dissolution of chitosan, you could first depolymerize it by treating with hemicellulases and then dissolving in acetic anhydride to obtain N-acetylated chitosan. This link will give you the details. Cheers! https://www.sciencedirect.com/science/article/pii/S0144861705004571
Hello Samet:
The solubility of low molecular weight chitosan is ~80 mg / mL in 150 mM acetic acid at 25 ° C and only soluble in acetate buffer solutions of pH below 6.5, because above pH 6.5, the amino groups are less protonated compared to pH 3.0, causing the chitosan chains to be less flexible. In other words, it takes more time for chitosan chains to change their conformation. At pH 8.5 for example, most of the amine groups are not protonated and the solubility of chitosan decreases drastically, Also, at this pH, chitosan chains are too rigid to rearrange and form hydrogen bonds or exhibit noticeable hydrophobic attraction in a short time. In your case you use a pH of 5.6 and although it should dissolve at this pH does not, its solubility also depends on the degree of polymerization of chitosan, however you could try to dissolve the chitosan in a more acid solution of your buffer and then correct the pH, or first dissolve the chitosan in an aqueous solution of acetic acid at 2% and it can be delayed but in the end it is dissolved also leave it overnight under moderate agitation and then after dissolved the reagent adjust to the desired pH with the buffer and make up to your final volume.
good luck!
Check this link maybe useful
https://onlinelibrary.wiley.com/doi/pdf/10.1002/jbm.b.33204?cookieSet=1
regards