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Ninhydrin amino acid assay

Posted: Wed Nov 08, 2017 11:38 am
by bunnahabhain
I am trying to implement a colorimetric assay using ninhydrin for analysis of glycine. I know from student time that we did this in biochemistry basic training, and I remember that it was quite sensitive.
So I googled a bit and found several protocols that state a sensitivity of about 10 µM amino acid (= 750 µg/L glycine).
I tried it in the lab, but I just can't get it to work.
I have tested several protocols that I found in the web, some with sodium acetate buffer (pH 5.2 - 5.6), some with acetic acid added to the ninhydrin reagent, concentration of ninhydrin ranges from 0.1 to 2 % in ethanol or acetone, incubation times from 30 °C to boiling water bath, ...
To make a long story short: I am not able to see any colour development unless I use at least 200 mg/L glycine, which is 2.7 mM.
And yes, I tried two different batches of ninhydrin and two different batches of glycine.

Can anybody assist with a working protocol?
(or any other assay for glycine...)

Kind regards

Re: Ninhydrin amino acid assay

Posted: Thu Nov 09, 2017 8:39 pm
by James_Ball
Are you looking for visual color confirmation or through spectrometer?

If visual, could the lower limits possibly be taken from spectrometer?

Re: Ninhydrin amino acid assay

Posted: Fri Nov 10, 2017 7:36 am
by bunnahabhain
I am looking for photometric determination. However, as the color is well visible (570 nm) the photometer is not (much) more sensitive than the eye. Only quantification should be more precise. :)