AMINO ACIDS (TOTAL) IN XYLEM EXUDATE
NINHYDRIN ASSAY
Michael Nicodemus
4-26-06
REAGENTS:
DI water
Ninhydrin
2-methoxyethanol (spectrophotometric grade)
Ascorbic acid
Citric acid
Sodium hydroxide (NaOH)
60% ethanol
METHOD:
In a 10 ml test tube, add 100 µl of xylem exudate to 900 µl of DI water to provide 1 ml of
sample (these proportions can be adjusted if more exudate is available). To this add 1.2 ml of
ninhydrin reagent (0.575 g ninhydrin in 50 ml 2-methoxyethanol plus 0.02 g ascorbic acid in 2 ml
water made up to 60 ml with 2-methoxyethanol) and 0.5 ml 0.8 M citrate buffer (168 g citric acid
plus 64 g NaOH per l of water). The mixture will be heated on a hot plate at 100°C for 1 hour, with
a marble on top of each test tube to prevent sample evaporation. Then remove the test tubes from
the hot plate and allow to cool for at least 5 minutes. Add 3 ml of 60% ethanol to the test tubes.
After 30 minutes, transfer a 3 ml aliquot of the resulting solution into a cuvette and measure
absorbance on a spectrophotometer at 570 nm. Compare the recorded absorbance to a reference
curve derived from known concentrations of amino acid.