An Improved Method for the Extraction and Thin-Layer Chromatography of Chlorophyll a and b from Spinach

A direct method of extracting plant pigments from spinach leaves into a dry organic solvent is presented. A powder is obtained by grinding a mixture of raw spinach, drying agent, and sand (1:1:2) that can be extracted directly into acetone in 10 minutes to provide a sample suitable for chromatographic analysis. This method avoids liquid–liquid extractions and subsequent drying as is found in previously reported methods. Removal of the magnesium ion from chlorophyll pigments was observed by treating the acetone extract with a strong ion-exchange resin for three minutes. The loss of magnesium ion is often observed as a degradation product of chlorophyll samples. An improved solvent system consisting of petroleum ether, cyclohexane, ethyl acetate, acetone, and methanol in 60%, 16%, 10%, 10%, and 4% quantities, respectively, is also reported. TLC separation using this extraction method and solvent system allows for the clear separation of chlorophyll a and b from all other extracted pigments.