Ninhydrin amino acid assay

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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
Are you looking for visual color confirmation or through spectrometer?

If visual, could the lower limits possibly be taken from spectrometer?
The past is there to guide us into the future, not to dwell in.
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. :)
3 posts Page 1 of 1

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