1.1c separation techniques Flashcards
How does a centrifuge work?
separates compounds by differing density
- more dense compounds settle in a pellet
- less dense compounds remain in the supernatant
What is paper and thin layer chromatography used for?
separating different substances such as amino acids and sugars
What does the speed that each solute travels along the chromatogram depend on?
Its differing solubility in the solvent used
How does affinity chromatography work?
- 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 washes down the column
- other non-target molecules with a weaker affinity are washed out
What happens during electrophoresis?
charged macromolecules move through an electric field applied to a gel matrix
How are proteins separated during electrophoresis?
native gels separate proteins by their shape, size and charge
native gels do not denature the molecule so that the separation is by shape, size and charge
How does an SDS-PAGE separate proteins?
by size alone
it gives all the molecules an equally negative charge and denatures then, separating proteins by size alone
How can proteins be separated from a mixture?
using their isoelectric points
What is the isoelectric point?
the pH at which a soluble protein has no net charge and will precipitate out of solution
If a solution is buffered to a specific pH what will happen?
only the protein(s) that have an isoelectric point of that pH will precipitate
Explain how proteins are separated using their isoelectric points in electrophoresis
- soluble proteins can be separated using an electric field and a pH gradient
- a protein stops migrating through the gel at its isoelectric point in the pH gradient because it has not net charge
