"Introductory experiment in thin layer chromatography"
TLC Lab course Grundlagen der Chromatographie und chromatographischen Methoden VAK 02-03-5-AnC2-2 Introductory experiment in thin layer chromatography January 29, 2008 Equipment Mortar and pestle, sintered glass ﬁlter, suction ﬂask plus test tubes, water jet pump, separation funnel, 10 mL lid glasses, 20 x 20 cm TLC disks (self-prepared), chromatography containers, test tubes, 1.5 mL Eppendorf cups, spectrophotometer, cuvettes, paper ﬁlters, glass plates Substances Green leaves (please bring them along!) Acetone Calcium carbonate Sodium sulfate Methyl-tert-butylether Sodium chloride Sand Kieselguhr Silica gel Sodium isoascorbate Calcium hydroxide Light petroleum (100-140 ◦ C boiling point range) Isopropanol Demineralized water Methanol Oil and fat for impregnation Qualitative elucidation of the pigment pattern in green leaves by thin layer chro- matography Preparation of TLC-disks 36 g of Kieselgur G, 9 g of silica gel 0.08 mm, 9 g of calcium carbonate, 0.06 g of calcium hydroxide, and 220 mg of sodium isoascorbate are weighed into a 500 mL glass and mixed. 125 mL of water is added. The preparation is quickly mixed, transferred to the appropriate container and applied by a spatula 0.25 mm thick onto a glass plate. The plates have been previously wiped with methanol. The plates are dried overnight. Some of the plates are impregnated after drying. A solution of 3.5 g of LIVIO oil and 3.5 g of PALMIN in 150 mL of light petroleum is poured into a chromatographic chamber and the plates are placed into this solution for impregnation. Impregnation is stopped 3 cm short of the upper edge and the plates are dried at room temperature for 24 hours. Pigment extraction Caution: The pigments are unstable when exposed to light and are sensitive towards oxygen. Do not work in bright light and avoid total evaporation of solvent and drying of pigments! Roughly 1 g of the green leaves is ground with a mortar and pestle with calcium carbonate and sodium sulphate (one spatula tip each) plus 1 g of sea sand. 5 mL of acetone are added and grinding is continued. The acetone extract is sucked off using a sintered glass ﬁlter. If necessary, more acetone can be added until it is nearly colourless. The extracts are 1 TLC Lab course Grundlagen der Chromatographie und chromatographischen Methoden VAK 02-03-5-AnC2-2 combined in a small separation funnel and 2 mL of methyl-tert-butylether, 5 mL of a 10% (w/w) sodium chloride solution are added. After careful shaking and slight opening of the separation funnel the water-like, clear lower phase containing acetone is separated off. During shaking, be sure to vent the funnel regularly. The dark green ether phase is washed twice, each with 3 mL of water. A spatula tip of dry sodium sulphate is added to the extract. Thin layer chromatography The experiment requires impregnated and non-impregnated plates. The chromatographic chambers have to be saturated prior to the experiment. The eluent system for the non-impregnated plates consists of • 100 mL of petrol ether (boiling point range of 100-140◦ C), • 12 mL of 2-propanol and • 0.25 mL of water. The solvent system for impregnated plates contains • 120 mL of methanol, • 24 mL of acetone and • 18 mL of water. The mixture must be saturated with lipoids. Hence 3 g of LIVIO oil plus 3 g of PALMIN are molten in a water bath and added to the solvent mixture. After thorough mixing (shaking), settling and decanting, the upper phase is transferred into the chromatographic chamber. The non-impregnated plates are activated at 80◦ for 30 minutes prior to applying the extracts. Impregnated and non- impregnated plates are positioned horizontally and the concentrated extract is carefully applied as a 2-4 mm band in a 2.5 cm distance from the lower edge of the plate by means of a pipette. Keep off the edges by about 2 cm. Optionally you may apply again after the solvent has evaporated. In the case of the unimpregnated plates apply on the upper, non- impregnated edge, which is now to become the lower edge. Be careful not to damage the coating of the plates which is not as hard as the commercially available material. After application, the solvent system in the chambers is thoroughly mixed once again and the plates are put into the chambers. Make sure the lid ﬁts tight and keep the chambers in the dark. Development time is between 30 and 90 min. Isolation and identiﬁcation of individual components in the pigment extracts: With a spatula the carotene, chlorophyll and xanthophyll bands are carefully removed from the moist plate and are trans- ferred to the test tubes. 3 to 5 mL solvent (see below) is added and the mixture is suspended. After settling of the silica gel, about 1 mL of the supernatant is carefully transferred to a 1.5 mL Eppendorf cup and put into the centrifuge. Absorption spectra are to be taken from all extracts. Solvents for extraction from the silica gel: plate type band solvent non-impregnated all bands acetone impregnated chlorophyll acetone impregnated carotene petrol ether impregnated xanthophyll ethanol 2 TLC Lab course Grundlagen der Chromatographie und chromatographischen Methoden VAK 02-03-5-AnC2-2 Reporting the results 1. Chromatograms are documented on transparent paper, technical data is noted (layer, layer thickness, solvent system, start, solvent front, duration, colour of the bands). 2. The results of both chromatographic techniques (with and without lipid impregnation) are discussed. Rp-values are correlated with the chemical structure of the pigment. 3. The characteristics of absorption curves are discussed. 3