The Histochemical Journal

, Volume 2, Issue 4, pp 329–342 | Cite as

A method for the determination of the molar extinction coefficient of structure-linked chromophores

  • J. P. R. Van Dalen
  • W. B. A. M. Ahsmann
  • P. Van Duijn


This paper describes a general method for the determination of the molar extinction ceefficient of a chromophore covalently bound to structure-linked groups, without isolating the compound formed. The method is illustrated by the determination of the molar extinction coefficient of the reaction product of 2,4-dinitro-1-fluorobenzene (DNFB) with films of aminoethyl-cellulose (AE-cellulose). The method is based on the relation between the decrease in extinction of a DNFB staining solution and the increase in extinction of the AE-cellulose after staining as measured in a film-spectrophotometer. In addition, the value of the molar extinction coefficient was used in establishing reaction conditions for a quantitative staining procedure for determining amino groups with DNFB and picric acid. Conditions of optimum DNFB staining were determined and the measured extinction was converted into concentration of amino groups using the molar extinction coefficient. The amino group concentration of the same batch of AE-cellulose was also determined, after finding the optimum reaction conditions, by staining with picric acid. The results, when compared, showed a linear relationship with a slope of unity for batches of AE-cellulose of varying amino group concentrations. This is consistent with the same stoichiometry in both cases and indicates that in both procedures one chromophore molecule has reacted with one amino group and that this reaction has proceeded to completion. The general applicability of the method is discussed.


Linear Relationship Extinction Coefficient Molar Extinction Coefficient Optimum Reaction Picric Acid 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Ahsmann, W. B. A. M. &van Duijn, P. (1968). Aminoacid cellulose films as models for histochemical protein reactions.Histochemie 12, 285–8.Google Scholar
  2. Dubin, D. T. (1960). The essay and characterization of amino by means of 2,4-dinitrofluoro benzene.J. biol. Chem. 235, 783–6.Google Scholar
  3. Duijn, P. Van, Den tonkellaar, E. M. &Hardonk, M. J. (1962). An improved apparatus for quantitative cytochemical model studies and its use in an experimental test of the two-wavelength method.J. Histochem. Cytochem. 10, 473–80.Google Scholar
  4. Fisher, E. R. &Lillie, R. D. (1954). The effect of methylation on basophilia.J. Histochem. Cytochem. 2, 81–7.Google Scholar
  5. Geiger, E. &Wissler, A. (1945). Beitrage zur Kenntnis von Oxy- und Hydrocellulose.Helv. chim. Acta 28, 1638–47.Google Scholar
  6. Hardonk, M. J. &van Duijn, P. (1964). Synthesis and properties of model systems, with their use in studying the Schiff Reaction in Histochemistry.J. Histochem. Cytochem. 12, 533–7.Google Scholar
  7. Maddy, A. H. (1961). 1-fluoro-2,4-dinitrobenzene as a cytochemical reagent. InGeneral Cytochemical Methods (ed. J. F. Danielli), Vol. 2, 259–85. New York: Academic Press.Google Scholar
  8. Mitchell, J. P. (1967). Combined protein and DNA measurements in plant cells using the dinitrofluorobenzene and Feulgen techniques.Jl R. microsc. Soc. 87, 1–7.Google Scholar
  9. Persijn, J. P. &Van Duijn, P. (1961). Studies of the Feulgen Reaction with the aid of DNA incorporated cellulose films.Histochemie 2, 283–97.Google Scholar
  10. Ploeg, M. Van Der &Van Duijn, P. (1968). Cytophotometric determination of alkaline phosphatase activity of individual neutrophilic leucocytes with a biochemically calibrated model system.J. Histochem. Cytochem. 16, 693–706.Google Scholar
  11. Samuelson, O. (1963). Determination of carboxyl groups. InMethods in Carbohydrate Chemistry (ed. R. L. Whistler), Vol. 3, pp. 31–8, New York: Academic Press.Google Scholar
  12. Sanger, F. (1945). The few amino groups of insuline.Biochem. J. 39, 507–15.Google Scholar
  13. Walker, P. M. B. (1958). Ultraviolet microspectrophotometry. InGeneral Cytochemical Methods (ed. J. F. Danielli), Vol. I, pp. 163–217. New York: Academic Press.Google Scholar
  14. Zerlotti, F. &Engel, M. B. (1962). The reactivity of proteins of some connective tissues and epithelial structures with 2,4-dinitrofluorobenzene.J. Histochem. Cytochem. 10, 527–46.Google Scholar

Copyright information

© Chapman and Hall Ltd 1970

Authors and Affiliations

  • J. P. R. Van Dalen
    • 1
  • W. B. A. M. Ahsmann
    • 1
  • P. Van Duijn
    • 1
  1. 1.Department of Pathology, Histochemical SectionUniversity of LeidenLeidenThe Netherlands

Personalised recommendations