Chromatography

Chromatography

1. Principles of chromatography

   • ~the aim is to separate a mixture into its constituents: in this case biological molecules ->are 2 key components, known as the stationary phase & mobile phase.

2. Stationary phase

   • ~is either the chromatography paper or thin-layer chromatography (TLC) plate. -paper is made of cellulose, the TLC plate is often a sheet of plastic, coated with a thin layer of silica gel or aluminium hydroxide. ->in each case there are free -OH groups pointing outwards, in contact with the mobile phase.

3. Mobile phase

   • ~is the solvent for biological molecule, at a simple level, we can use water or ethanol. ->mobile phase flows through & across the stationary phase, carrying the biological molecule with it

4. What happens?

   • ~as the solvent travels up the paper or plate, the components of the solution mixture travel with it, in the example in fig2 you can see they travel at different speeds. ->by the time the solvent has reached the top of the plate, some are travelling slowly & some quickly =diff positions on the plate.
   • ~can use relative distance travelled to help identify the pigments ->calculate the Rf value by measuring the distance from the pencil line to the centre of a spot of pigment (x), & the distance from the pencil line to the solvent font (y) -> Rf = x/y
   • ~Sometimes with colourless molecules, you cannot see where they finish. using thin-layer chromatography, there are solutions:
     • 1.Ultraviolet light =thin layer chromatography plates have a chemical fluoresces under UV light. ->if you look at the plate under UV light, most of it will glow, except those places where the spots have travelled to.
     • 2.Ninhydrin =to see amino acids, allow the plate to dry, & then spray it with ninhydrin =binds to the amino acids which are then visible as brown or purple spots
     • 3.Iodine =allow plate to dry, & place in an enclosed container with a few iodine crystals -iodine forms a gas, which then binds to the molecules in each of the spots

5. How does it work?

   • ~speed at which molecules move along the paper or TLC plate depends on their solubility in the solvent & their polarity. ->in case of paper chromatography, it may also depend on their size
     • ~Exposed -OH groups make the surface of the paper or plate very polar, & allow it to form hydrogen bonds with the molecules, alongside other dipole interactions. ->a highly polar solute will tend to stick to the surface & hence move more slowly. -> a non-polar solute will travel v. quickly up the plate.
       • ->if 2 moles travel at exactly same speed it will be difficult to separate them =use different solvent or change pH

6. How is it used?

   • ~thin layer chromatography is commonly used to monitor the progress of reactions, as it works relatively quickly. ->also used for urine testing of athletes for illegal drugs, analysing drugs for purity of componets & analysis of foods to determine presence of contaminats