Thin layer chromatography experiments

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!)
Calcium carbonate
Methyl-tert-butylether
Sand
Silica gel
Calcium hydroxide
Isopropanol
Methanol
Acetone
Sodium sulfate
Sodium chloride
Kieselguhr
Sodium isoascorbate
Light petroleum (100-140 ◦ C boiling point range)
Demineralized water
Oil and fat for impregnation
Qualitative elucidation of the pigment pattern in green leaves by thin layer chromatography
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
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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 nonimpregnated
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, nonimpregnated
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 transferred
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
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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.
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