An Improved Method for the Purification of Chlorophylls A and B; Quantitative Measurement of Their Absorption Spectra; Evidence for the Existence of a Third Component of Chlorophyll

Abstract

1. An improved method for the isolation of chlorophyll components a and b has been developed. The methods of WILLSTATTER and STOLL and of TSWETT were combined and modified. The final purification processes involve differential adsorption on talc and fractional precipitation from petroleum ether, for components a and b respectively. The steps of the procedure are presented in detail. 2. The absorption spectra of chlorophylls a and b were measured accurately from λ 3950-7800 Å. by a photoelectric method. Absorption coefficients at the maxima and minima were determined with an accuracy of 1.0%. The visible spectrum of each component consists of seven bands. 3. Spectral data indicate that the components prepared by WILLSTATTER and STOLL and by TSWETT were not separated completely from one another. The spectral method for detection of impurities is discussed. 4. A comparative study was made of the colors of a and b in various solvents and of certain chlorophyll derivatives. 5. Different tests of purity are discussed and it is concluded that a critical examination of the absorption spectrum is the most reliable and sensitive test. 6. Sources of leaf material and component yields are presented. 7. The improved preparation procedure is critically compared with previous methods. 8. It is suggested that there is at least one component other than chlorophylls a and b in the chlorophyll complex. Possible sources of error in the purification procedure are discussed. 9. Attempts to isolate the third "component c" were unsuccessful. Its properties are intermediate between those of components a and b. 10. Quantitative analytical data affirm the existence of a third component c.