How does solubility affect paper chromatography?
4 Important separation processes for mixtures of substances
Separating a mixture of substances by using the different properties of the components:
- Solubility in water, solvents, gases
- Molecular size
- Adsorption properties on surfaces
Illustration of the principle using the example of the different solubility of substances in water and organic solvents:
A stationary solvent (phase; in the example water) is adsorbed on the surface of a carrier (here a capillary). The second, mobile solvent (here an organic one) rises along the carrier and takes the substance mixture with it. Depending on the solubility of the components in the two solvents, their components migrate with the mobile phase at different speeds. Substances that are more soluble in water stay longer in the stationary wall layer, so to speak.
The stationary phase (water) is adsorbed as a very thin layer on the cellulose fibers of paper.
- The mixture of substances is applied in dots to the lower edge of a sheet of paper (e.g. 50 × 50 cm) and dried, in our example a mixture of 7 anthocyanin pigments (dyes of most red and blue fruits, fig.)
- The lower edge of the sheet of paper is then immersed in a solvent (mixture). This rises up in the paper by capillary action and takes the substances upwards with it at different speeds (Fig.).
- If the solvent has risen enough after a few hours, the paper is dried and the separated substances are identified with suitable reagents if they do not have their own color (Fig.).
Paper as a carrier is largely out of date (but is still used in riser technology or similar for the biodynamic assessment of food quality). Today it is cheap and up-to-date ...
Thin layer chromatography
An absorbent layer (silica gel, cellulose powder, aluminum oxide powder) is applied to a carrier plate (glass, aluminum foil) with a binding agent. This is thinner and finer structured than the paper.
The separation is sharper, the plate can be much smaller (a few cm edge length), the separation is much faster (20 - 60 min) and you can get by with smaller amounts of substance because the spots remain smaller
In the case of substance mixtures with many components (as in our example), these can usually not be completely separated in one go.
- The separation can be improved if the plate is not treated with reagent after the first run and drying, but rotated by 90 ° and the now lower edge is immersed in another solvent mixture (Fig.).
- The substances in it have different running properties and so the stains that converge the first time are separated (Fig.).
- With suitable combinations of superplasticizers, a complete separation is obtained (Fig.).
- A practical application is e.g. the comparison of the ingredients of different agricultural products (Fig.).
High pressure liquid chromatography (HPLC)
HPLC = high pressure liquid chromatography.
In processes in which the flow agent is driven by natural capillary forces, the separation distance is limited to a few cm for reasons of time.
- This can be circumvented if the stationary phase is accommodated in a capillary through which the mobile phase is pumped through at high pressure. This means that separating distances of a few meters can be traversed in a few minutes.
- The substance mixture is introduced at one end of the capillary and pumped through. The substances are decelerated to different degrees by the interaction with the stationary phase and leave the end of the capillary at different times. There they are verified by different procedures.
The procedure is expensive (devices cost from a few tens of thousands)
Gas chromatography (GC)
The mobile phase is a gas. The stationary phase is housed in a very long capillary.
Only volatile substances (e.g. aroma components) can be separated with this. The capillary is therefore placed in an oven so that substances that are not volatile at room temperature can also be separated. There is also the possibility of preparing volatile derivatives of substances by treatment with reagents, which otherwise could not be separated in the GC.
- The detection of the substances takes place when they leave the capillary after different times (retention times). In the case of fragrant aroma components, the detection can be made by sniffing persons (Fig.).
- The GC is able to separate mixtures of substances with hundreds of components (Fig.).
- Sometimes the GC is more sensitive than the human sense of smell (Fig.).
Molecular Sieve Process
A tube is filled with a solvent (e.g. buffer solution) and a powder suspended in it. Its grains consist of a porous substance whose pore size depends on the molecular size of the substances (e.g. proteins) that you want to separate.
- The mixture of substances is added at the top (Fig.).
- At the lower end of the tube (chromatography column) the solvent slowly drips out, at the top it runs accordingly and the substance mixture applied moves with it (Fig.).
- A stationary phase of the solvent forms in the pores and only migrates very slowly. The small molecular components can temporarily penetrate the pores and therefore migrate more slowly, the larger molecules migrate relatively faster through between the porous grains and this leads to a separation (Fig.).
Separating charged particles in an electric field
So only ions can be separated or particles that carry ionizable groups (typically proteins or nucleic acids). In these, the charge changes with the pH value of the solution, i.e. this determines how fast and where the particles migrate.
In general, the substances to be separated are dissolved in buffer and also migrate in buffer. This buffer solution must be stabilized so that it does not move during the electrophoresis (e.g. swirled by heating);
for this purpose, different absorbent carrier materials are used, which have a sponge-like or fine-meshed structure, e.g.
the pores correspond to plant cells (cotton hair)
Cellulose acetate (
spongy, very fine-pored
Acrylamide gel (
Gel made of crosslinked chain molecules, the mesh size of which depends on the concentration and degree of crosslinking of the molecules
Gel made from the well-known helically structured starch molecules, mesh size only depends on the starch concentration
Separation criteria: The pores of the carrier are partly much larger than the molecules to be separated (such as in paper or cellulose acetate) and hardly affect their migration; then it is only separated according to charge. If the mesh sizes are in the size range of the molecules (in the case of gels), molecular sieving processes also occur (figure series).
The electrical voltages that occur with the E. are mostly in the range between 50 and 2000 volts, so in a very dangerous range, especially when you consider that wet fingers often occur!
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