2.2.14 Practical biochemistry 3: Chromatography

2.2.14 Practical biochemistry 3: Chromatography

1. Principles of chromatography

   • Aim= separate mixtures into its constituents (biological molecules)
   • 2 Key components:
     • Stationary phase
     • Mobile phase

2. Stationary phase

   • Either chromatography paper or thin layer chromatography plate
   • Paper is made of cellulose
   • The TLC plate is often a sheet of plastic coated with silica gel or aluminium hydroxide
   • In both cases there are free -OH groups pointing out towards the mobile phase

3. Mobile phase

   • This is the solvent for biological molecules
   • At a simple level= we use water or ethanol (polar and non-polar molecules)
   • The mobile phase flows through an across the stationary phase carrying the biological molecules with it

4. What happens?

   • As the solvent travels up the plate/paper the components of the mixture travel with it
   • The components travel at diff. speeds and are at different positions on the plate/paper
   • You can use the relative distance travelled to identify the pigments
     • Calculate the Rf value by measuring the distance from the pencil line to the pigment (x) and the distance from the pencil line to the solvent front (y)
       • Rf = x/y
     • If you repeat the investigation under the same conditions, the pigment will always have the sae Rf value
   • Sometimes with colourless molecules you can't see where they finish, Using TLC there are solutions:
     • UV light
       • TLC plates have a chemical which fluoresces under UV light, the places where the sports are don't glow, they mask the plate from UV light
     • Ninhydrin
       • To see amino acids, allow the plate to fry and then spray with ninhydrin = it binds to amino acids which are then visible as brown or purple spots
     • Iodine
       • Allow the plate to dry and place in an enclosed container with a few iodine crystals = Iodine forms a has which binds the molecules in the spots

5. How does it work?

   • The speed at which molecules move along the paper or plate depends on their solubility in the solvent and their polarity
     • In paper chromatography it can also depend on size
   • Exposed -OH groups make the surface of the paper or plate v. polar and allow it to form H bonds with molecules (along with other dipole interactions)
     • A highly polar solute will stick to the surface and is absorbed = moves more slowly
     • A non polar solute will travel v. quickly
   • If 2 molecules travel at exactly the same speed it is difficult to separate them, you could try using a different solvent or changing pH

6. How is chromatography used?

   • TLC us used to monitor the progress of reactions as it works fast
   • Also used for urine testing of athletes for illegal drugs
   • Analysing drugs for purity
   • Analysis of foods to determine presence of contaminants

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