In the Laboratory www.JCE.DivCHED.org • Vol. 84 No. 9 September 2007 • Journal of Chemical Education 1505 The chemistry of pigments in green plants has been stud- ied since the first half of the 19th century, in particular with investigations by Berzelius (1). These compounds are biologi- cally important, in particular for their role in photosynthesis (2). In food, they have organoleptic and nutritious proper- ties (3). The main pigments in green beans are chlorophylls (Chl) a and b and carotenoids (Car) (4). When beans are blanched (prior to freezing) or stored in poor conditions, pig- ments include certain chlorophyll derivatives: pheophytins (Pheo) and allomers from the a and b forms. The group of carotenoids can be divided into (i) hydrocarbon carotenoids, β-carotene and α-carotene (β-Car and α-Car) (5), and (ii) oxidized carotenoids or xanthophylls, lutein (Lut), violaxanthin (Vio), and neoxanthin (Neo) (5). In this article, we propose to study pigments from green beans but our method can be applied to other plant tissues. Chromatographic separation of plant pigments was proposed by Tswett (6) and variations of this initial method have been studied (5, 6). Thin-layer chromatography (TLC) is fast, easy and inexpensive and thus useful for chemical studies (5, 7– 9). We propose a direct quantification of all the pigments through TLC separation and quantification of the TLC plates using a flatbed scanner and processing the digitized chromato- gram. Experimental Procedure Extraction of Pigments from Green Beans Fresh green beans were purchased from a local market and frozen green beans were obtained from a local store. The method used for the extraction was modified from a previ- ously published extraction method (7). Under subdued light (to prevent pigment degradation), green beans were combined with anhydrous sodium sulfate and sea sand and ground in a mortar with pestle until a light green powder was obtained. The powder was transferred to a test tube containing acetone. The test tube was agitated and the green acetone solution was removed and centrifuged. The extract was concentrated with a liquid–liquid partition with cyclohexane (10). A small quantity of the supernatant was taken in a capillary tube, weighed before and after filling, to assess the quantity being analyzed. The plant extract was immediately spotted on the TLC sheet. Separation of Pigments Using TLC The developing solvent system was cyclohexane–ac- etone–diethylamine (10:4:1, vvv) as in ref 8, but cyclo- hexane, a chemically pure substance, was substituted for the petroleum ether mixture. Preparation of Calibration Standards Standards of Chl a, Chl b, β-Car, and Lut were prepared from commercially available products. If these products are not available (e.g., due to their price), preparative layer chro- matography can be used. Solutions of these pigments with concentrations from 50 mgL to 1000 mgL in cyclohexane were used as test samples to prepare calibration plots. The Pheo a and b samples were prepared from Chl a and b, re- spectively, by addition of acid (HCl 25%) (11). As Vio and Neo have UV–vis spectra similar to Lut, their quantification was carried out using the Lut calibration curve. All pigments were spotted at different concentrations on the same plate. The TLC plates were always scanned under the same condi- tions. The digitized images were treated with image treatment software. Scanning The digitization of the TLC plates was done on a Canon CanoScan 5200F flatbed scanner (Figure 1). The image was scanned and registered in JPEG compressed files. Quantitative Determination of Photosynthetic Pigments in Green Beans Using Thin-Layer Chromatography and a Flatbed Scanner as Densitometer W Juan Valverde* and Hervé This** Equipe INRA de Gastronomie Moléculaire, UMR 214 (INRA/AgroParisTech) Laboratoire de Chimie Analytique 16, rue Claude Bernard, 75005, Paris, France; *juan.valverde@agroparistech.fr; ** herve.this@paris.inra.fr Marc Vignolle Marie Groupe UNIQ, 13-15 Pont des Halles, 94526, Rungis, France Figure 1. Scanned TLC plate where the photosynthetic pigments were developed in cyclohexane–acetone–diethylamine (10:4:1; v/ v/v): (1) neoxantin, (2) violaxanthin, (3) 5,6-epoxy lutein, (4) lutein, (5) chlorophyll b, (6) chlorophyll a, (7) pheophytin a, (8) α- and β- carotenes, and (x) origin.