I am using the ninhydrin method as a way to quantify the amount of proline in a particular sample. I've started off testing out my method using pure L-proline as a test. However, the reaction quickly started turning dark purple, almost black. What could this mean? I'm using a pure proline sample so there shouldn't be any other amino acids in there. My best guess is contamination but I was very careful to avoid this. Even if there was some contamination the dark purple result was so far from what I was expecting that I have a feeling something else was wrong.
The reaction of amino acids with ninhydrin is colorimetric. It is used to detect ammonia or primary and secondary amines and a deep blue or purple color known as Ruhemann's purple is produced.
Andrei Blasko Yes but proline is supposed to be the exception because it’s side chain interferes with the amino group. It should in theory be the only amino acid that doesn’t turn purple, and yet my solution is turning dark purple
Kimberley,
All I can tell that I worked with proline and the I was able to determine it quantitatively using the colorimetric method.
Michael,
Very good insight on the mechanism.
Dear,
The ninhydrin, which is originally yellow, reacts with amino acid and turns ((deep purple)). It is this purple color that is detected in this method. Ninhydrin will react with a free alpha-amino group, NH2-C-COOH. This group is present in all amino acids including proline, proteins or peptides. Whereas, the decarboxylation reaction will proceed for a free amino acid, it will not happen for peptides and proteins. Theoretically only amino acids produce color with ninhydrin reagent. So nothing went wrong in your test
Greetings
I am triying to determine proline concentratin in leaves. I am preparing my ninhydrin stock solution by warming because I can not dissolve it using only glacial acetic acid and phosphoric acid 85% (60/40 v/v). I would like to know if this is correct or I must to dissolve only by agitation. Today I felt that my stock was light purple and I dont know if this preliminary color can disturber my proline test. Thanks very much
Could you do a control sample without proline and see what happens? I can only think of contaminating/interfering compounds for now. It should turn yellow for proline, not purple. Maybe your ninhydrin stock solution is contaminated/unfresh? Maybe your sample container is contaminated? Maybe you have found out by now, do let us know here, thanks. :)
Finally I observed that my ninhydrin stock turn purple only during the heating, after solution cool down is totally yellow, so I think that this turning color is a momentaneus process produced by warm, but it is necessary do it to dissolve ninhydrin in acids mentioned.
Nuria María Montes Osuna how long does it take for the colour to change back to yellow? That’s weird i thought it should stay purple since my protocol requires it to cool down first before adding alcohol and taking a wavelength read. Maybe the alcohol helps with preventing the colour change back to yellow?
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