Separação de pigmentos do espinafre- carotenos e clorofila

Extract the nonpolar pigments from spinach with a mixture of solvents and separate them using a chromatographic column. Materials and Reagents: Funnel, mortar and pestle, petroleum ether or hexane, beaker, acetone, chromatographic column, spinach leaves, graduated cylinder, alumina or silica gel, heating plate, pipette Sample Preparation: Weigh 50 g of spinach leaves into a beaker, add 100 mL of distilled water and boil for 1-2 minutes to remove the surface layer of the leaves. Decant the liquid and dry the leaves with absorbent paper. Transfer the material to a mortar and pour in 10 mL of a mixture of petroleum ether and acetone (8:2), grinding to obtain a green solution. Column Preparation To determine the amount of alumina (or silica) used, fill the column to 2/3 of its capacity with alumina, then transfer this material to a 100 ml beaker and add petroleum ether up to the 20 ml mark. Transfer this suspension to the column using a spatula and open the stopcock to allow the alumina to pack. Add more solvent to transfer the solid. Note that the eluent level must always be kept above the level of the stationary phase, and to do this, adjust the stopcock and the solvent addition. Pigment Separation Allow the eluent to drain to the level of the stationary phase and add about 2 mL of the pigment solution with a pipette, allowing the top layer of the stationary phase to enter the column. Place 1 cm of alumina (or silica, or even dry sand) on top of the extract layer. Start the elution with a mixture of petroleum ether:acetone 40:60, adding the eluent gradually. Collect the first fractions in an organic waste container – a yellow ring (carotenes) will immediately appear descending along the column. At the top, there is a green ring, mainly composed of chlorophyll. Collect the yellow band in a test tube. When the entire yellow band has exited the column, begin adding acetone from the top of the column. Observe that the green band moves and, as it exits the column, collect it in another test tube. Waste Organic solvent waste should be disposed of in non-chlorinated organic waste and separated from aqueous waste.