Ninhidrina

Microplate measurement of amino acids by ninhydrin
DISCLAIMER
Before using this or any other analytical method it is imperative that you check that it works with your
samples. The bare minimum isto test accuracy and precision.
· Test accuracy by creating a standard curve by serial dilution of a sample and/or via spike and
recovery tests. Both tests will show if the analysis is affected bythe sample matrix.
· Test precision by repeated analysis of the same sample. It s best to do separate precision tests for
the analytical method (replicate analyses of the same extract) and for theentire extraction and
analysis procedure (extract the same sample several times and carry each extract through the
analysis procedure). These tests will show you where poor precision is creeping intoyour
analysis.
Remember that your results are qualitative if you rely on a standard curve with a purified analyte.
Principle
The principle of this assay is formation of a purple compound whenninhydrin reacts with free alpha
amino acids.
The trick with this assay is that ninhydrin reacts with ammonium with around 50% of the sensitivity as
it reacts with amino acids. One approach is toremove ammonium (e.g. by adding MgO to volatilize
ammonium as ammonia, see notes below). Alternatively one may measure the concentration of
ammonium and use this to correct the apparent amino acidconcentrations. If you take the latter approach
you will need to carry ammonium standards through the ninhydrin procedure.
M ethod adapted from:
Jones DL, Owen AG, Farrar JF (2002) Simple method toenable the high resolution determination of total
free amino acids in soil solutions and soil extracts. Soil Biology and Biochem istry 34: 1893-1902
Moore S, Stein WH (1954) A modified ninhydrin reagentfor the photometric determination of amino
acids and related compounds. The Journal of Biological Chemistry 211: 907:913
Prepare stock solution s
Make 4 M Na -Acetate buffer:
· In a 100-mL...