An almost forgotten simplification in the detection of amino acids

Abstract: Ninhydrin spraying may be eliminated by the incorporation of ninhydrin into the chromatographic solvent.

A ninhydrin spray, with or without the addition of 2,4,6-trimethylpyridine [s-collidine]1, is commonly used to detect amino acids following chromatography on filter paper or on various other thin layer media.2 This exercise is found in both introductory organic chemistry and biochemistry labs.2 It is a good, cheap and colorful way to introduce students to both paper and thin layer chromatography. Students will also be interested to learn that this ninhydrin chemistry is used by forensic scientists as a sensitive probe for fingerprints — Google “ninhydrin fingerprints” for many procedures and images.

Rogers3 rediscovered an important simplification based on the fact that the reaction of amino acids with ninhydrin is rather slow at room temperature.4 If, therefore, chromatography is carried out fairly rapidly, the ninhydrin can be incorporated into the chromatographic solvent. This eliminates the extra step of spraying, the not inconsiderable cost of the sprayers, and the possible hazard of inhalation of the spray. This is a rediscovery because Nicholson first reported this idea in 1949.5 According to the Web of Knowledge, this paper was cited 8 times in the 1950s but hardly at all thereafter (nor by ref. 3). It is not mentioned in any of the standard books on chromatographic methods that I have looked at (such as reference 2) except for Hais.6 It is virtuous to honor our forebearers.


    1. A.J. Woiwod, Journal of Chromatography, 1960, 3, 278.
    2. H. Stübchen-Kirchner, Z. Hoppe Seylers Z Physiol Chem, 349,
      1049, 1968.
    1. I. Smith, Chromatographic and Electrophoretic Techniques,
      Vol. I, 3rd edition, Chapter 5, Interscience, NY, 1969.
    2. M.O. Hurst & D.K. Cobb, Journal of Chemical Education, 67, page 978, 1990.
    3. R. Switzer & L. Garrity, Experimental Biochemistry, 3rd edition, pages 111-121, W. H. Freeman, NY, 1999.
  1. P.W. Rogers, Journal of Chemical Education, 73, 189, 1996.
    1. C.E. Dent, Biochemical Journal, 43, 169, 1948.
    2. H. Meyer, Biochemical Journal, 67, 333, 1957.
    3. Z. Khan & A.A. Kahn, Journal of the Indian Chemical Society, 66, 454 1989.
  2. D.E. Nicholson, Nature, 1949, 163, 954, 1949.
  3. I.M. Hais, in Paper Chromatography, I.M. Hais & K. Macek, eds., Academic Press, NY, page 489, 1963.