Partial Separation of the Plastid Pigments by Paper Chromatography

Abstract

A method for separation of small quantities of plastid pigments was desired, and paper chromatography seemed to be the best prospect among the available techniques. A search of the literature revealed only one direct mention of the application of paper chromatography to the separation of the plastid pigments. STRAIN (3), in a review article, stated that chlorophylls and xanthophylls can be separated by paper chromatography using aqueous ethanol as the developing solvent. In a private communication Dr. S. Aronoff has informed us that he obtained a separation of the plastid pig-ments by repeated development of the paper chromatogram in Skelly-B (petroleum ether). When tested in this laboratory neither of these two methods gave a complete separation of all visibly present fractions.2 In these investigations plastid pigments were extracted from soybean and other leaves with acetone in a Waring Blendor and transferred to Skelly-B in a separatory funnel. The Skelly-B solution was washed thoroughly with water to remove acetone and acetone-soluble impurities, then concentrated under vacuum. One tenth of a milliliter of the Skelly-B solution was placed at the origin (fig. 1) on a square of Whatman no. 1 filter paper (23 cm. x 23 cm.) which previously had been washed with Skelly-B and air dried. The filter paper was stapled together to form a cylinder which was placed in a tightly covered widemouth gallon jar containing ca. 100 ml. of developing solvent. The paper was air dried after development in each of the succes-sive solvents. Among the solvents and solvent combinations tested the following sequence gave the best results: FIRST DIMENSION.-(a) Acetone: All the pigments moved with the sol-vent front and the development was stopped when the solvent reached the top of the original drop. This procedure served to collect the pigments from the original circular spot in a line front from which they could be more easily separated in the later steps. (b) Skelly-B: The carotenes followed 1 Journal paper no. J-2128 of the Iowa Agricultural Experiment Station, Ames, Iowa. Project no. 1139. 2 paper by Bauer (BAUER, L. Trennung der Karotinoide und Chlorophylle mit Hilfe der Papierchromatographie. Naturwiss. 39(4): 88. 1952.) became available after this paper was submitted for publication. Bauer separated the plastid pigments into nine fractions by two-dimensional paper chromatography, using petroleum solvents which are not readily available in this country. 325 PLANT PHYSIOLOGY the solvent front while the other visible pigments moved only slightly, if at all, from the origin. The development was stopped when the solvent was ca. 20 cm. from the bottom of the paper. This step brought the carotenes so far ahead of the other pigments that they did not interfere with further separations. (c) One volume per cent. n-propanol in Skelly-B: All pigments moved, but this treatment separated the remaining pigments into the follow-ing groups (top to bottom): Chlorophyll a overlapping the xanthophylls, chlorophyll b, and an unknown yellow pigment which appeared below and well separated from chlorophyll b in the chromatogram. The development SECOND