I also have another question.

why does the induced-purple color turn yellow when the solution is cooled to room temperature?

I have encountered this issue about glycine standard solution and low concentrated solution of gelatin-chitosan. the latter faded rapidly.

Dear Shadi Moshayedi, crosslinked (permanent bonds) polymers do not dissolve anymore once crosslinked, they may only swell at low or moderate crosslink density. My Regards

The change in color generally is an indication of complexation/aggregation process. More details are need to explain the change of color. My Regards

Abdelkader BOUAZIZ First, thanks for your quick reply. these days I have been trying to solve the problem, but I couldnt.

Then, I understood something. The dissolution of the crosslinked sample may not be the answer. may be the solid uv visible should be used, instead.

Have you come up with any new ideas?

Regards.

Dear Shadi Moshayedi, I have seen many publications doing the test at high temperature without complete dissolution rather high swollen scaffold. My Regards

Abdelkader BOUAZIZ

Yes you are right. but it did not work for me because there is no change in color during 20 mins, instead the sample itself has become colorful.

Well, I don't know exactly the origin of thé problem but you have to chack the degree of acetylation of the chitosan. My Regards

Dear Shadi Moshayedi I'm not an expert in this and I'm not sure if the link cited below is really helpful, but you can find numerous threads and publications around the ninhydrin test right here on RG. Here is one example:

https://www.researchgate.net/post/Why_is_my_Proline-ninhydrin_reaction_test_turning_purple