How does chromatography separate molecules

After a time the paper is taken out and dried: the substances can be seen at once if coloured, or located by treating with a suitable locating agent. The distance a substance travels depends upon the resultant between propelling and retarding forces.

Propellors a Solvent flow Usually the more soluble a substance is in the solvent, the more rapidly it will move along the paper. Solvents are chosen for the greatest differential solubilities of the substances concerned. Retarders a Adsorption Adsorption is reversible and cellulose gradually releases most substances into the solvent as it flows over the spot.

Adsorption is differential like solubility: some substances are more strongly adsorbed than others. When a substance which is soluble in the two non-mixing solvents is exposed simultaneously to both, it will partition itself between them.

The amount found in each solvent will depend upon the relative solubility of the solute in each. The degree of partition at equilibrium is known as the partition coefficient.

In fact the water forms the stationary phase and the solvent a moving phase. The water can be thought of as trapped in lots of little tubes over the tops of which the solvent is passing. When a drop is spotted on paper the solute dissolves in the water of the tubes. Its opposite, cation exchange chromatography, uses anionic resins to retain cationic molecules.

Here, the ions compete with your molecule for binding with the matrix, displacing it to the elution stream. The other approach is to change the pH of your buffer to alter the net charges and thus, the ionic binding. Affinity chromatography separates molecules based on their binding interactions with a specific small molecule that is covalently attached to a stationary matrix.

Think of chromatography by affinity as a lock-and-key mechanism — the small molecule on the matrix is the lock and your molecule is the key. Your crude sample is a set of keys, but your analyte is the only key with the complementary shape interaction that matches the lock on the affinity medium. Having your molecule fixed on the matrix allows you to flush out the unwanted stuff. To apply this method you must use a molecule with well-defined lock-and-key binding properties.

Examples of specific binding are those found between an enzyme and substrate, antigen and antibody, and receptor and ligand. Size-exclusion chromatography separates molecules by their size. This method, also known as gel permeation chromatography , is unlike those described above because it exploits a physical characteristic instead of chemical interactions. The stationary phase is a resin of porous beads that traps small molecules but not large ones.

Suppose you load a polymer sample onto the column. During the run, your polymer freely moves around and between the beads while the small impurities constantly enter and exit beads. So the travel is longer for small particles, but your large analyte moves with the elution stream, exiting the column first.

Have you used other column chromatography methods? Please add to the list by commenting below! Has this helped you? A simple demonstration of chromatography uses coffee filters and a variety of marker pens. If the pens use water-soluble inks, the solvent used is water. If the markers use permanent ink, isopropyl alcohol often works as a solvent. Begin by flattening out a coffee filter.

Place the coffee filter on a disposable plate or other material to prevent staining underlying surfaces. Use a variety of pens to make dots around the center portion of the filter. Add water or alcohol to the center of the coffee filter. A teaspoon works well for this. Do not add enough liquid to create a puddle; the water or alcohol should expand out from the center.

As the liquid moves out from the center, the inks will dissolve and move outward from center. Different pigments in the inks will separate, carried out from initial ink spot and deposited in rows based on the pigment molecules. A little more complicated but equally interesting chromatography project separates the chlorophyll found in leaves.

Chlorophyll occurs in the leaves of plants. Although chlorophyll is green, most leaves also contain additional pigments such as carotenoids, which create the red and orange colors you see in autumn. These carotenoids and other pigments are revealed as the green chlorophyll degrades, which is why deciduous plant leaves show different colors in the fall. Begin by selecting several green leaves.

Crush the leaves and soak the pieces in isopropyl alcohol or acetone also called propanone. The chlorophyll will leach out of the leaves and turn the liquid green. Isopropyl alcohol and acetone are both flammable. Do not place these or use these near or with flames or a heat source. To separate the pigments, cut about an inch-wide strip from the center of a flattened coffee filter or use chromatography paper.