Quantitative Estimation of Amino Acids by Ninhydrin Method

Amino Acids 



Proteins are probably the most important class of biochemical molecules, although of course lipids and carbohydrates are also essential for life. Proteins are the basis for the major structural components of animal and human tissue.

Proteins are natural polymer molecules consisting of amino acid units. The number of amino acids in proteins may range from two to several thousand.

Amino Acids:

Although we are studying only about 20 amino acids, there are about six more found in the body. Many others are also known from a variety of sources. Amino acids are the building blocks used to make proteins and peptides. The different amino acids have interesting properties because they have a variety of structural parts which result in different polarities and solubilities.

Each amino acid has at least one amine and one acid functional group as the name implies. See graphic below. The different properties result from variations in the structures of different R groups. The R group is often referred to as the amino acid "side chain". Amino acids have special common names, however, a three letter abreviation for the name is used most of the time. 


Amino acid physical properties indicate a "salt-like" behavior. Amino acids are crystalline solids with relatively high melting points, and most are quite soluble in water and insoluble in non-polar solvents. In solution, the amino acid molecule appears to have a charge which changes with pH.

An intramolecular neutralization reaction leads to a salt-like ion called a zwitterion. The accepted practice is to show the amino acids in the zwitterion form.

(1) The carboxyl group can lose a hydrogen ion to become negatively charged.
(2) The amine group can accept a hydrogen ion to become positively charged.


Amino Acid Examples


The Ninhydrin Reaction

Common alpha-amino acids, except proline, undergo a unique reaction with the triketohydrindene hydrate known as ninhydrin. Among the products of this unusual reaction (shown below) is a purple colored imino derivative, which provides a useful color test for these amino acids, most of which are colorless. A common application of the ninhydrin test is the visualization of amino acids in paper chromatography, and for quantitative determination using spectrophotometry. As shown in the graphic below, samples of amino acids or mixtures thereof are applied along a line near the bottom of a rectangular sheet of paper (the baseline). The bottom edge of the paper is immersed in an aqueous buffer, and this liquid climbs slowly toward the top edge. As the solvent front passes the sample spots, the compounds in each sample are carried along at a rate which is characteristic of their functionality, size and interaction with the cellulose matrix of the paper. Some compounds move rapidly up the paper, while others may scarcely move at all. The ratio of the distance a compound moves from the baseline to the distance of the solvent front from the baseline is defined as the retardation (or retention) factor Rf. Different amino acids usually have different Rf's under suitable conditions. In the example below, the three sample compounds (1, 2 & 3) have respective Rf values of 0.54, 0.36 & 0.78.  The plate was sprayed with ninhydrin to visualize the 3 amino acids.


Paper Chromatography



Here is a link to a colorimetric analysis tutorial that shows how to graph absorbance data to find unknown concentrations.   Click Here.

Review Spectrophotometry (colorimetry) Experiment (Exp. 8) for a review of spectrophotometry principles.


A.  Read and review concepts in this handout.

B.  Access Quantitative Estimation of Amino Acids by Ninhydrin Method Simulation.   Click here.  Or type following URL address into your browser.

C.   Read THEORY section of Simulation.

D.   Read PROCEDURE section of Simulation.

E.   Run Simulation under the SIMULATOR tab.  Record observations and data in your Lab Notebook.

F.   Write a formal lab report.  Include a detailed discussion of amino acids, the ninhydrin reaction, and colorimetric analysis in the introduction.  Include the ninhydrin reaction.  Record ALL data in the experiment.   Prepare graphs in Excel and copy/paste to the Results section of your report.  Determine the concentration of the unknowns and include in the Results section.


Michael W.  McClure
Professor, Chemistry
Hopkinsville Community College
Phone:  270-707-3863


Copyright 2007  M.W. McClure, All Rights Reserved

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