Chlorophyll paper chromatography

Sestak, Z., Paper chromatography of chloroplast pigments (chlorophylls and carotenoids), Part 3, Photosynthetica, 14, 239, 1980. 

Crude pigments were transfered in to the petrol ether and aceton was washed with water. Ascendent paper chromatography was used for pigment separation in system petrol ethar metanol (30 1) (Marcus, 1952). Absorbtion spectra were obtained by the spectrophotometar Aminco DW-2A operated at split beam mode. Chlorophyll concentration was determined according to Vernon (1960) and carotenoid content according to Holme (1954). 

Absorption spectra of pigments of box eledr separated by the paper chromatography (Fig 1) indicate that chlorophyls in diferent tissues are the same, when carotenoids seam to be different. The shifts of absorbtion maxima of carotenoids and xanthophylls could be either caused by different chemical forms or by different concentrations of pigments. Similar results were obtained with pigments of Chlorophytum (Fig. 2). Further more in leaf of this plant (Fig. 3) the distinct pigment with a new absorption maxima is detected. 

Immediately cork the tube filled with nitrogen and then add a drop or two of dichloromethane to dissolve the pigments for TLC analysis. Carry out the analysis without delay by spotting the mixture on a TLC plate about 1 cm from the bottom and 8 mm from the edge. Make one spot concentrated by repeatedly touching the plate, but ensure that the spot is as small as possible—less than 1.0 mm in diameter. The other spot can be of lower concentration. Develop the plate with 70 30 hexane acetone. With other plates try cyclohexane and toluene as eluents and also hexane/ethanol mixtures of various compositions. The container in which the chromatography is carried out should be lined with filter paper that is wet with the solvent so the atmosphere in the container will be saturated with solvent vapor. On completion of elution, mark the solvent front with a pencil and outline the colored spots. Examine the plate under the uv light. Are any new spots seen Report colors and i /values for all of your spots, and identify each as lycopene, carotene, chlorophyll, or xanthophyll. 

High-performance liquid chromatography (h.p.l.c.) is now used extensively to analyse the pigment compositions (carotenoids and chlorophylls) of plants and algae [ ] In spite of the great interest in bacterial photosynthesis and the pigments of photosynthetic bacteria, no widely applicable h.p.l.c. procedures have yet been reported. In this paper, we describe the development and... 

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