Practical 5

Photometric properties of the

ninhydrin amino acid complex
 Photometric techniques

• Based on color changes of the reaction mixture

• Used to determine:
• Reaction rates
• Concentrations of substances in solution

• Pharmaceutical application
• Quality control
• Determining concentrations of substances in pharmaceutical
products
In this test, ninhydrin (a chemical compound
with the formula C9H6O4; IUPAC name: 2,2-
dihydroxyindane-1,3-dione) is added to a test
solution of the analyte. The development of a
deep blue colour indicates the presence of
ammonia, primary/secondary amines, or
amino acids in the analyte.
 Color and wavelength

• Visible (white) light consist of a number of different wavelengths

• When visible light travels through a solution, some wavelengths are

absorbed

• Color of solution → determined by those wavelengths that are not

absorbed
 Absorption spectrophotometry

• Absorption spectrum
→gives the relation between the amount of light that was absorbed and
the wavelength of the incidental light
→used to identify compounds (each compound has its own absorption
spectrum)

• Absorbance
→ The amount of light that is absorbed at a specific wavelength
→ Used to determine the concentration of a solution
 Quantification of amino acids

• Ninhydrin forms a complex with -amino acids

• Original assays to test for amino acids
• Test can be used for amino acid conc at low as 10pmol
• -amino acid + 2 ninhydrin ---> CO2 + aldehyde + final complex
(purple-blue colour) + 3H2O
 Quantification of amino acids
• Colour reaction is the same for all the amino acids except for
proline.
• Differs due to the ring structure of the proline molecule.
• The ninhydrin-amino complex (purple colour) absorbs
maximally at 570 nm whereas the ninhydrin-proline complex
(yellow) absorbs maximally at 420 nm.