, Volume 16, Issue 1, pp 68–84 | Cite as

Thin layer chrornatography and histochemistry of Sudan Black B

  • A. G. W. Lansink


Thin layer chromatography of commercial Sudan Black B on silica gel with chloroform-benzene (1∶1) as the developing solvent reveals two blue main fractions with Rf values of 0.49 and 0.19, SBB-I and SBB-II respectively. Furthermore at least eighteen secondary fractions or impurities have been found. SBB-I and SBB-II were isolated and purified by preparative thin layer chromatography. Commercial Sudan Black B consists of about 20 p.c. SBB-I, 60 p.c. SBB-II and 20 p.c. secondary fractions.

From spectrophotometrical and histochemical investigations it appeared that SBB-I stains lipids more pronounced than SBB-II; moreover SBB-I is more specific for neutral lipids than SBB-II, which fraction may also stain some proteins and acid mucopolysaccharides. Contrary to SBB-II the staining with SBB-I is fairly independent of pH. Finally, the colour of SBB-II changes under the influence of light and air, while SBB-I is much more stable.

A physico-chemical study of the nature of SBB-I and SBB-II, including spectrophotometry, chromatography, infrared spectroscopy and chemical analysis revealed, that SBB-II is a basic dye, while SBB-I in spite of the structural resemblance behaves as a neutral one, dissolving therefore better in neutral lipids.

As yet the chemical composition of SBB-I and SBB-II, and the relation to the scheme of synthesis of Sudan Black B has not been solved. The unspecificity of lipid staining by Sudan Black B is due to the basicity of SBB-II, and to the instability of this dye toward light and air. Moreover the some eighteen impurities may have some influence on the staining properties. The question of solubility or adsorption processes in the case of lipid staining by Sudan dyes is at least partially answered by the proposition of a dissolving fraction SBB-I and an adsorbed fraction SBB-II. The changing absorption spectra by the corresponding solvatochromic and metachromatic effects may give information about the nature of the lipids stained.


Lipid Thin Layer Chromatography Neutral Lipid Infrared Spectroscopy Main Fraction 
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. Bermes, E. W., and H. J. McDonald: Fractionation and characterization of the lipide stain Sudan Black B. Arch. Biochem. 70, 49–57 (1957).Google Scholar
  2. Christmas, J. J., and R. H. Trubey: Chromatography and biological stains. I. Paperchromatography as a means of determining purity and dye content of Sudans III and IV. Stain Technol. 27, 53–60 (1952).Google Scholar
  3. Fredricsson, B., T. C. Leurent, and B. Lüning: Decomposition of Sudan Black B causing an artefact in the staining of lipids. Stain Technol. 33, 155 (1958).Google Scholar
  4. Harms, H.: Handbuch der Farbstoffe für die Mikroskopie. Lintford: Staufen 1965.Google Scholar
  5. Holczinger, L., and S. Balint: Experimental data to the theory of fat staining. Histochemie 2, 389–392 (1962).Google Scholar
  6. Jencks, W. P., and E. L. Durrum: Paper electrophoresis as quantitative method: the staining of serum lipoproteins. J. clin. Invest. 34, 1437 (1955).Google Scholar
  7. Kay, W. W., and R. Whitehead: The role of impurities and mixtures of isomers in the staining of fat by commercial Sudans. J. Path. Bact. 53, 279 (1941).Google Scholar
  8. Kutt, H., D. Lockwood, and F. McDowell: Decompositions of Sudan Black B by ultraviolett light and gases; its lipid and protein staining properties. Stain Technol. 34, 203 (1959).Google Scholar
  9. Lansink, A. G. W.: will be published (1969).Google Scholar
  10. Meier, W.: Untersuchungen zur Theorie der Fettfärbungen. Z. wiss. Mikr. 64, 193–208 (1959).Google Scholar
  11. Pearse, A. G. E.: Histochemistry, theoretical and applied. London: Churchill 1960.Google Scholar
  12. Randerath, K.: Dünnschicht-Chromatographie. Weinheim: Verlag Chemie 1962.Google Scholar
  13. Rao, C. N. R.: Chemical applications of infrared spectroscopy. New York: Academic Press 1963.Google Scholar
  14. Schott, H. J.: Zur Frage der histochemischen Fettspezifität des Sudanschwarz B und zur Methodik der Auftrennung seiner Fraktionen. Histochemie 3, 467–476 (1964).Google Scholar
  15. —, u. W. Schoner: Beitrag zur Fettspezifität des Sudanschwarz B und anderer roter Sudan-farbstoffen bei Reinsubstanzen. Histochemie 5, 154–162 (1965).Google Scholar
  16. Stahl, E.: Dünnschicht-Chromatographie, ein Laboratoriumshandbuch, 2. Aufl. Berlin-Heidelberg-New York: Springer 1967.Google Scholar
  17. Terner, J. Y.: Stable Sudanophilia: Contributions to the histochemistry of the Sudan dyes. Lab. Invest. 12, 405–414 (1963).Google Scholar
  18. Trubey, R. H., and J. F. Christman: Chromatography and biological stains. II. Column Chromatographie separation of the coloured materials in commercial Sudan III. Stain Technol. 27, 87–92 (1952).Google Scholar
  19. Werner, H. J., and J. F. Christman: Chromatography and biological stains. III. Comparison of the fat staining efficiency of fractions of commercial Sudan III separated by column Chromatography. Stain Technol. 27, 93–96 (1952).Google Scholar

Copyright information

© Springer-Verlag 1968

Authors and Affiliations

  • A. G. W. Lansink
    • 1
  1. 1.Institute of Pathology of the Catholic UniversityNijmegenThe Netherlands

Personalised recommendations