Primary Amine Content Assay

Ninhydrin assay

Ninhydrin Reagent Solution
Ninhydrin: 0.35 g
Add ethanol to: 100 ml (See Note 1.)

Procedures

Add 1 ml of the ninhydrin solution to 5 ml of sample. Cover the test tube with a piece of paraffin film to avoid the loss of solvent due to evaporation. A capped test tube can also be used instead.
With gentle stirring, react at 80-100ºC for 4-7 minutes. (How would one find out the amount of time needed to ensure a complete reaction?) If a large heated water bath is used for the entire class and if there is no good provision for holding the test tube in the hot water bath, the test tube may be held with a piece of wire and hang on the side of the water container. A clamp usually does not work too well.
After cooling to room temperature in a cold water bath, record the absorbance with a spectrophotometer. (How would you find out the best wavelength to use for this purpose? Hint: the final purple colored complex of ninhydrin absorbs the most amount of light at the wavelength of 570 nm.)