[Extraction and quantitative determination of carotenoids and chlorophylls of leaves, algae and isolated chloroplasts with the aid of thin-layer chromatography]

Abstract

Methods for a rapid quantitative determination of chlorophylls and carotenoids are decribed.The extraction of pigments was carried out with different kinds of plant material, such as algae, leaves and chloroplasts. The separation of the carotenoids from these extracts was succeeded by an adsorptionchromatographic process in which the thin-layer consists of anorganic adsorbents (CaCO3, MgO, Ca(OH)2). The basicity of the layer is further increased by the addition of KOH; thereby the chlorophylls are retained at the starting line and the overlapping of chlorophylls and xanthophylls on the chromatogram can be avoided.With this method even those carotenoids can be separated which differ only in the position of a double bond, as for instance α- and β-carotene, and lutein and zeaxanthin. Thus the separation of all the principal carotenoids on a single chromatogram is possible, for example from a Chlorella extract (in order of decreasing Rf-value): α-carotene, β-carotene, lutein-5,6-epoxide (traces), violaxathin, lutein, antheraxanthin, neoxanthin neo A, neoxanthin and zeaxanthin. Furthermore the chromatographic behaviour of the carotenoids ζ-carotene, γ-carotene, lycopene and rhodoxanthin, which are found only rarely, is described.The chlorophylls a and b are separated by a partition chromatographic process on the second thin-layer; this layer consists of silica gel mixed with ascorbic acid as an antioxidant. An apparatus for an equal spreading of defined quantities of the extract on the starting line and new methods for a rapid quantitative elution of the pigments from the adsorbent are described.The specific extinction coefficients E 1 cm (1 %) for antheraxanthin in ethanol and for α-carotene in chloroform, which were needed for the calculation of the pigment quantity were determined.