What is sheet chromatography

Chromatography experiments


Simple chromatography experiments

Experiment 1: Blackboard chalk


Small Petri dishes or jam jars, dropping pipettes, blackboard chalk (dried if possible), parsley or other fresh green leaves, fiber pens (not permanent, brown and black), denatured alcohol or ethanol, mineral spirits or petroleum ether, mortar with pestle, quartz sand

Leaf pigments:

By rubbing the leaves with sand and a few milliliters of ethanol (denatured alcohol), an intensely dark green colored extract of the leaf pigments is produced. After the sand has settled, the supernatant solution is decanted or pipetted into a Petri dish. The chalk is placed vertically in the extract and the solution is allowed to rise about 1 cm. Then the piece of chalk is placed in a Petri dish (jam jar), which is about 5 mm high filled with petroleum ether (cleaning benzine). When the solvent has risen about 5 cm, the chalk is removed from the vessel. One can clearly distinguish between a yellow (carotene) and a green (chlorophyll) color zone.

Felt pen colors:

The piece of chalk is marked all around at a height of about 1 cm with a clearly visible line and then placed in a vessel with pure ethanol (denatured alcohol), pure water or a mixture of both. Different mixing ratios should be tried out and their effect on color separation observed. Alternatively, you can let a black ink rise about 1 cm into the chalk as described in part a) and carry out the corresponding experiments.

Experiment 2: Playing with colors - "Runge" pictures


Round filter (9 cm in diameter) or chromatography paper (10 x 10 cm), jam jars or Petri dishes, dropper bottles (e.g. empty eye drop bottles) or dropper pipettes, water, denatured alcohol or ethanol, saline solution (prepare saline by dissolving a spatula tip in approx. 10 ml of water), hair dryer , colored inks, food colors (e.g. from McCormick or Brauns Heitmann, available in drugstores)


The paper is placed on an open jam jar or a petri dish. So that the paper is a little hollow, it is slightly pressed in the middle. Put a drop of food coloring (e.g. green) in the middle of the paper and wait for the color to spread. A blob of paint about 2 cm in size forms. Then put five drops of water one after the other onto this blob with a dropper. After each addition you have to wait until all the water has been absorbed by the paper. The dye spreads further due to the capillary force, with color gradations resulting from the center towards the edge.

In another experiment, a saline solution is used instead of water. The difference in the pictures can be seen in the color ramifications and especially in the outermost edge zone, which no longer appears smooth but strongly fissured. In addition, when the image is “developed” with the saline solution, it can be seen that a colorless aqueous front is moving in front of the dye. If water is used, however, the dye migrates with the front.
Further play options result from the use of different food and ink colors. A certain system can lead from the game to a targeted (scientific) experiment, e.g .:

1st picture: with 6 drops of saline solution
2nd picture: first 5 drops of saline solution, then 1 drop of water
3rd picture: 4 drops of saline solution, 2 drops of water, etc. ......to

7th picture: 6 drops of water

Further variation possibilities exist through:
· Reduction of the drop size of the dye
· Dripping of colors other than the original color during development
· Trying out other aqueous solutions (sugar, baking soda, vinegar ...)
The use of filter papers that have been impregnated by immersion in 3% metal salt solutions (e.g. copper (II) sulfate, iron (III) nitrate) and subsequent drying
· Obstruction of the material transport through targeted cuts in the paper
· Experiments with fiber pens (not permanent). Different mixtures of aqueous alcohol solutions should be used for development.

Examples of different Runge pictures:

The possibility of variation mentioned last can easily be extended to a real chromatographic experiment (paper chromatography according to the "round filter method"). To do this, the filter paper is pierced in the middle with a pointed object, so that a hole about 2-3 mm in size is created. Around this hole, the paper is colored circularly with a black or other colored felt pen. An approx. 2x10 cm strip of filter paper is rolled into a wick and pushed into the hole of the round filter. The filter with the wick is placed on the upturned lid of a preserving jar (Petri dish) in such a way that the wick protrudes clearly into the previously filled "solvent". Different mixtures of water and denatured alcohol (ethanol) serve as such.

Examples of the round filter method:

Experiment 3: Separation of synthetic dyes by thin-layer chromatography


TLC ready-made silica gel 60 plates, separation chamber (jar with lid), microcapillaries, inks, ballpoint pen refills, beaker (small), sturdy scissors

Eluent: Well suited are water-saturated butanol or butanol / glacial acetic acid / water (8: 1: 1)


Ballpoint pen refills must be crushed with a suitable tool (e.g. scissors). The pieces are placed in a small beaker and the dyes are dissolved out with a little ethanol. The inks are also diluted with ethanol in a 1: 1 ratio. The dye solutions are applied in dots or lines by means of capillaries at a distance of approx. 1 cm from the lower edge of a TLC plate (mark the start line beforehand with a thin pencil line). The application of felt-tip pen colors can be done directly with the pen. The application dots (lines) should be as small (narrow) as possible. The running distance should be at least about 5-6 cm. The result of the separation could then look like this:

different ballpoint pen refills

different felt pens
Image source: Schwedt, Georg; On the trail of dyes, kosmos experiment book

Experiment 4: Isolation and thin-layer chromatographic separation of food colors in sugar confectionery (smarties, gummy bears)

Preliminary remark: This experiment requires some effort and some experimental experience with DC. The greater effort is explained by the fact that the dyes of interest must first be isolated from the sample matrix, purified and brought into a phase capable of chromatography.

a) Isolation and purification of the dyes


Smarties or gummy bears, beakers (100 ml), dist. Water, tweezers, acetic acid (c = 2 mol / l), colorless sheep wool degreased with petroleum ether, ammonia solution (5%), heating plate


6 - 10 coated tablets or gummy bears of the same color are mixed with a little hot distilled water. Covered with water in a beaker, the dyes dissolved by swirling and the discolored residues removed immediately. 2 ml of acetic acid and a defatted wool thread are added to the solution, then it is briefly heated to boiling on a hot plate until the wool thread is strongly colored. The wool thread is washed thoroughly under running water and boiled again in a beaker with about 5 ml of ammonia solution until most of the dye has detached from the thread. The threads are removed and the colored solutions are concentrated to about 0.5 ml each.

b) Separation by thin layer chromatography

Equipment and chemicals:

Dye solutions from test part a), food colors (drugstore) for comparison, capillaries for application, TLC separating chamber (or jar with lid), TLC cellulose films, solvent mixture: n-propanol / ethyl acetate / water (6: 3: 1)


Depending on the chromatography vessel used, the TLC foil is cut to size and the starting line is carefully drawn with a pencil in the usual way and without damaging the layer at a distance of about 1.5 cm from the lower edge. The starting points are then marked at a distance of 1 cm according to the number of dye solutions present. The test and reference solutions are applied point-wise with capillaries. The substance spots should be colored as small as possible and clearly visible. After the starting spots have dried, the film is placed in the separating vessel with the specified solvent, and this is closed again. After a distance of about 8-10 cm, the TLC foil can be removed and dried. The result could look like this:

The separation can also be carried out by paper chromatography. A solution of 1 g tri-sodium citrate x 2 H2O in 50 ml ammonia solution (5%) is suitable as the mobile phase. However, the dye spots on the developed chromatogram are not as sharply demarcated as when using cellulose layers.

Experiment 5: column chromatography


Chromatography column, glass wool, aluminum oxide (dried at 110 ° C), ethanol (96%), acetic acid, color mixture of equal proportions of ethanolic solutions (1%) of methyl red and methylene blue, beakers, dropping pipette, glass funnel, solvent mixture (196 ml ethanol + 4 ml acetic acid)


The lower end of the chromatography tube is plugged with a little glass wool and about halfway filled with the specified solvent mixture. In a beaker, dried aluminum oxide is slurried with the same solvent mixture and carefully filled into the column through a small glass funnel. The filling should be as bubble-free as possible; light knocking on the column during the filling process is often helpful. Excess solvent mixture is allowed to run off by opening the tap until the meniscus is just above the aluminum oxide. About 2 ml of the dye mixture are now carefully placed on the top of the column using a pipette and the stopcock is opened very slightly. When the dye solution has just disappeared in the aluminum oxide, additional mobile phase is carefully added from a trot funnel attached above the tube or with the aid of a dropper pipette. It is important to ensure that the column filling is covered with the solvent for the entire duration of the experiment! The two dyes are separated from each other after a short time and can be collected when leaving the column.

Experiment 6: Demonstration of the principle of gas chromatography (GC)


Glass tube (d = 8 - 10 mm), gas bottle with hydrogen, glass wool, 2 washing bottles, sulfuric acid, aluminum oxide, copper wire, syringe with metal needle (1 ml, available in pharmacies), rubber tube, test mixture of equal parts pentane, dichloroethane and toluene


An approx. 1 m long piece of glass tube is filled with dried aluminum oxide, the filling is fixed between two glass wool stoppers (separation column with stationary phase). The gas bottle containing the hydrogen carrier gas is connected to the column with pieces of tubing via two washing bottles. The wash bottle behind the gas bottle is switched as a safety bottle, the second in front of the column serves as a bubble counter and is provided with colored sulfuric acid. At the end of the column, a glass tube bent at right angles and drawn out to a point is attached by means of a hose. A piece of copper wire, bent in a spiral shape, is attached over the tip. A moderate gas flow is regulated by carefully opening the valve on the gas cylinder. When all the air has been displaced from the system by the hydrogen (oxyhydrogen sample!), It is ignited at the glass tip. The hydrogen flame should be clearly visible (approx. 5 - 8 mm high) and serve as a detector with the copper wire protruding into it. A small amount (0.1-0.2 ml) of the specified test mixture is then injected with a small syringe through the hose attached in front of the column (dosing system). The separate exit of the individual components of the mixture at the end of the separation column is clearly indicated by the changes in the hydrogen flame.

Annotation: In addition to aluminum oxide, other solids are also suitable as the stationary phase. Powdered washing powder, for example, can also be used successfully as a sorbent.