Ninhydrin assay - secondary amines detection (Mar/20/2009 )

Dear all,

I have been trying to detect secondary amines with a ninhyndrin reagent kit (from sigma). However, I bumped into two main problems:
1) as a general fact, secondary amines detection leads to not-too-strong colors (red to yellow color instead of ruhemann's purple color for primary amines)
2) Ninhydrin reaction is optimum with pH5. But my samples are PBS or EtOH-based (meaning sample's pH is too basic => no difference of color when Ninhydrin reagent is added compared to control).

I already tried to change my sample pH by adding drops of glacial acetic acid to lower the pH at 4. I did the same for a control (pure PBS lowered @ pH4 with glacial acetic acid). The result was :
- sample with amine was red
- whereas the control was still yellow (which is not making any sense as I should have obtained the contrary).

Did anybody already encounter this problem? to measure secondary amines in basic buffer (pH7)?

Thank you all for any tip!

enguyen

The color issue for secondary amine can not be easily solved. However, from chemistry point of view I would say slightly basic pH is needed for the reaction.

Try this alternative method if you still have problem.

linkinghub.elsevier.com/retrieve/pii/0003269774901559

genehunter on Mar 20 2009, 03:10 PM said:

sure. This is an old one.

Chia-Mi L. Lin and Conrad Wagner
Analytical Biochemistry
Volume 60, Issue 1, July 1974, Pages 278-284

eh thank you so much again!

it is working waaaay better!

...*sigh*...

Dear colleagues

I need again some advice. A month ago, I was struggling detecting secondary amines. Now, Sodium nitroprusside (SNP) did work very well to detect aliphatic amines. However, I am trying now to detect Linear polyethyleneimine (which contains lots of secondary amines) and SNP assay is not working at all...

any clue why SNP does not react with my very long secondary amines chemical?

thanks again.

Check the pH. If these groups are in the salt form, I think they may not be as active.

***These secondary amines have substantial buffering capacity. ***


Also, when these groups are so close to each other the reaction may not go complete.


Isnt research fun!

*sigh*

pH again!?

thanks a lot genehunter.

In fact, LPEI did react with SNP but after 4 hours incubation (light purple color) which is not very good as the color is known to be unstable for long incubation time.

Ok, i'm going to check the pH and see if even after 4h hours, I can obtain a nice linear standard curve.

Cheers¨

I've checked the pH. It was indeed a little bit lower than required (8.5pH instead of 10pH). I tried to increase the pH to 10 but still no reaction within the first 30min. I've found this chemical reaction of the simon's test (upload file)...maybe someone can tell me why the hell LPEI reacts soooo slowly (normally, it takes, 10seconds for the blue color to appear! LPEI takes 4h...)