(c) Separation techniques

• Use of centrifuge to separate substances of differing density

  • More dense components settle in the pellet; less dense components remain in the supernatant.

• Paper and thin layer chromatography can be used for separating different substances such as amino acids and sugars

  • The speed that each solute travels along the chromatogram depends on its differing solubility in the solvent used.

• Principle of affinity chromatography and its use in separating proteins

  • A solid matrix or gel column is created with specific molecules bound to the matrix or gel. 

  • Soluble, target proteins in a mixture, with a high affinity for these molecules, become attached to them as the mixture passes down the column. 

  • Other non-target molecules with a weaker affinity are washed out.

• Principle of gel electrophoresis and its use in separating proteins and nucleic acids

  • Charged macromolecules move through an electric field applied to a gel matrix.

• Native gels separate proteins by their shape, size and charge

  • Native gels do not denature the molecule so that separation is by shape, size and charge.

• SDS–PAGE separates proteins by size alone

  • SDS–PAGE gives all the molecules an equally negative charge and denatures them, separating proteins by size alone.

• Proteins can be separated from a mixture using their isoelectric points (IEPs)

  • IEP is the pH at which a soluble protein has no net charge and will precipitate out of solution.

• If the solution is buffered to a specific pH, only the protein(s) that have an IEP of that pH will precipitate

• Proteins can also be separated using their IEPs in electrophoresis

  • Soluble proteins can be separated using an electric field and a pH gradient. 

  • A protein stops migrating through the gel at its IEP in the pH gradient because it has no net charge.