1c- Separation Techniques Flashcards
What is a centrifuge and what does it separate mixtures based on?
A piece of equipment that spins a sample at high speeds and separates based on density
What are the two parts of a centrifuged mixture?
The pellet is composed of the dense components and the supernatant is composed of the less dense substances
What kind of substances can paper and thin-layer chromatography be used to separate?
Amino acids and sugars
What does the speed that each solute travels along the chromatogram in paper and thin-layer chromatography depend on?
Each solutes differing solubility in the solvent used
What is affinity chromatography used to seperate?
Proteins
How can affinity chromatography be used to separate 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
What can gel electrophoresis be used to separate?
Proteins and nucleic acids
What can gel electrophoresis separate based on?
Charge, shape and size
What happens during gel electrophoresis?
Charged molecules move through an electric field
How does SDS-PAGE separate proteins by size alone?
All molecules are given an equally negative charge and denatured, this separates proteins by size alone
What is a proteins IEP (isoelectric point)?
The pH at which a soluble protein has no net charge and will precipitate out of solution
How can soluble proteins be separated using an electric field and a pH gradient?
If the solution is buffered to a specific pH, only the protein(s) that have an IEP of that pH will precipitate
Why will a protein stop migrating through the gel at its IEP?
A protein has no net charge at its IEP
