April 13, 2021, 04:15:57 AM
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Topic: Possibly changing the buffer conditions in the ninhydrin assay for amino acids  (Read 76 times)

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Offline Babcock_Hall

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“The analysis is performed by the addition of 0.2 mL of ninhydrin reagent (2 mg/mL of ninhydrin in 20 mM acetic acid—acetate buffer, pH 5) to1.0 mL of the amino acid solution.  The solution is heated 10 min at 100° C and cooled, and the absorbance is measured at 570 nm for all the amino acids except proline, which is measured at 440 nm.  A standard curve is prepared for the different amino acids.  The limit of sensitivity is 0.1 µmol.” From Robyt and White, p. 230 of Biochemical Techniques, 1987.

We would like a quantitative or semiquantitative assay for the fractions from a Dowex-50 column.  We are trying to prepare aspartate β-semialdehyde from a literature protocol (S Black and NG Wright 1955 J. Biol. Chem. 213 39-50.).  Fractions are eluted from the column using  4 M HCl.  We anticipate in general making two pools, one at high concentration of ASA and one at low concentration.  That is what the original authors did, and we also did so one time as well.  A reasonable guess is that the pooled fractions will be 50 mM in ASA.

Ninhydrin is a commonly used reagent with respect to amino acids in forensic chemistry and to detect amino acids in TLC experiments.  I don't have any experience with the quantitative Ninhydrin assay assay.  One problem I see in using this protocol is that there is very little buffer capacity, given that so much HCl is present.  For example, if we imagine using 1 µmol amino acid (ten times greater than the lower limit) that is 50 mM in concentration.  This is 8 x 10-5 moles of HCl, which is greater than tenfold more than the concentration of acetate ions.   If we neutralized the HCl for the aliquot of the fraction being tested, we would not have much margin of error, and I don't know how the presence of sodium or potassium ions from a hydroxide solution would affect this assay.

My initial thoughts would be to try the assay out on a commercially available amino acid (glycine, for example) before committing our sample.  Would anyone care to make a guess as to what would happen if we increased the acetate/acetic acid concentration?

Another possibility is to use a different assay, but it could not be anything too complex or expensive.  There would still be a potential problem because of the acid.

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