In the Laboratory An Improved Method for the Extraction and Thin Layer Chromatography of Chlorophyll a and b from Spinach W Hao T Quach Robert L Steeper and G William Griffin Science Department Bunker Hill Community College Boston MA 02129 ggriffin bhcc mass edu The chromatographic separation of plant pigments has both historical 1 and practical 2 importance and as a result has remained a popular and well studied experiment in the chemical laboratory curriculum Moreover this experiment remains popular because students can easily relate to the materials used and the pigments involved including chlorophyll a and b as well as carotene that are easily observed without the need for UV lamps or post chromatographic chemical or physical treatment of the TLC plates Several improvements have been reported in the literature that address difficulties with both the sample preparation 3 5 and the chromatographic conditions 6 8 used For example the extraction of relatively nonpolar pigments into a suitable organic solvent is complicated by the presence of water in the raw spinach leaf or other pigment source Also many of the chromatographic solvent systems reported for this separation do not adequately resolve chlorophyll a and chlorophyll b from each other or from other pigments present in the sample A further complication exists in that the pigments to be isolated are susceptible to degradation 7 11 resulting in the observation of materials that are not representative of the pigments in the plant We present significant improvements in both the sample preparation and the mobile phase for the extraction and separation of plant pigments This simple and fast method clearly resolves chlorophyll a and b from spinach leaves on analytical TLC plates while minimizing the appearance of chlorophyll degradation products Experimental Procedure General Solvents and reference samples were purchased from Aldrich Chemical Company and were used without additional purification The TLC plates were also purchased from Aldrich and these experiments were optimized using silica gel plates on a polyester support containing a fluorescent indicator Comparable results were obtained on TLC plates without the fluorescent indicator The aluminum backed Merck TLC plates can be substituted but give Rf values approximately 15 lower than the corresponding polyester backed plates The sand used was obtained from Ward s Ottawa Sand and is much larger in particle size than sand commonly used for chromatographic applications A full description of the catalog numbers and CAS registry numbers of all materials used as well as the laboratory handout used by our students is available in the Supplemental Material W Sample Preparation Fresh or frozen spinach 0 5 grams was combined with 0 5 grams of anhydrous magnesium sulfate and 1 0 gram of sand The mixture was ground in a mortar and pestle until a light green powder was obtained 5 10 minutes The light www JCE DivCHED org green solid was transferred to a small test tube containing 2 0 mL of acetone This heterogeneous mixture was agitated to ensure complete mixing of the acetone and the solid This mixture was allowed to stand for 10 minutes and the green acetone solution was removed by pipette and transferred to a microcentrifuge tube The acetone extract was kept sealed when not being actively used Alternately a coffee grinder was used to prepare larger quantities of solid material for subsequent acetone extraction In this case 5 0 grams of fresh or frozen spinach was combined with 5 0 grams of anhydrous magnesium sulfate and 10 grams of sand in a commercial coffee mill and ground for 5 seconds The leaf pulp from the top and sides of the mill container was scraped back to the bottom of the mill and ground for an additional 30 seconds The resulting powder was extracted as described above 2 0 grams of solid with 2 0 mL of acetone TLC Separation TLC plates 3 5 cm 9 0 cm were cut from the commercially available sheets The acetone extract was transferred in the standard manner and the plates were eluted in a closed chamber with the following mobile phase 60 petroleum ether bp 35 60 C 16 cyclohexane 10 ethyl acetate 10 acetone 4 methanol The elution order using this elution solvent system was carotene Rf 0 95 chlorophyll a Rf 0 44 chlorophyll b Rf 0 32 and xanthophyll Rf 0 16 Commercial purified samples of carotene chlorophyll a and chlorophyll b were used as controls Demetalation of the Spinach Extract Dowex 50WX8 H resin 100 mg was combined with 100 L of the acetone spinach extract described above This mixture was allowed to stand for 3 minutes and a small sample was removed using a TLC applicator This sample was chromatographed as previously described This short treatment with ion exchange resin resulted in the complete loss of the chlorophyll a band and the appearance of both pheophytin a Rf 0 60 and pheophytin b Rf 0 49 Hazards There are no unusual hazards associated with this experiment The proper handling and disposal of the solvents used should be observed The organic solvents used in this experiment are flammable and should be isolated from ignition sources such as the coffee grinder described in the experiment Results and Discussion The extraction and separation of plant pigments is a common introduction to chromatography and is found in many laboratory manuals 12 14 The direct extraction of Vol 81 No 3 March 2004 Journal of Chemical Education 385 In the Laboratory Figure 1 TLC plates of spinach extracts A fresh spinach B frozen spinach C extract treated with ion exchange resin xan is xanthophyll chl is chlorophyll c is carotene phe is pheophytin and sf is solvent front Figure 2 Comparison of solvent systems used on the TLC plates A five solvent system B hexane acetone 70 30 14 C i octane acetone ether 60 20 20 11 D cyclohexane acetone ether 50 25 25 6 the plant pigments from spinach is complicated by the presence of water Since the chromatographic separation will be affected by even small quantities of water a bulk separation of the extract using a separatory funnel or comparable microscale techniques followed by drying of the organic layer with an appropriate drying agent is often required 2 3 6 11 14 A further complication arises in this separation owing to the well documented limited stability of the chlorophyll pigments 7 10 This can be confusing to the student since the sample can produce both the expected pigments and colored degradation products with similar Rf values One of