Unit 1.1c Flashcards
What are the 4 separation techniques?
Centrifugation
Chromatography
Gel Electrophoresis
Iso-electric point
What are the 4 features that molecules posses that allows us to separate them?
Size
Charge
Density
Solubility
What does centrifugation do?
It separates the solution into a pellet and supernatant of different densities
How does the centrifuge work?
It spins a high speeds to seperate the components
What are the two phases of solubility?
Mobile and stationary
Which of the two phases is the one in which mixture is dissolved in?
The mobile phase
Which of the two phases is the one in which the mixture passes and is separated?
paper chromatography
The stationary phase
What are the 3 types of chromatography?
Paper
Thin-layer
Affinity
How does paper chromatography work?
Stationary phase I’d a polar strip of cellulose fibre paper
So, non-polar solvent is used
Dot of concentrated mixture used on bottom of paper
Place bottom of paper but not mixture dot into solvent
Solvent will move through the paper at different rates depending on solubility
How does thin layer chromatography work?
Uses and absorbent layer of silica gel or cellulose
This medium is thinly placed on glass, plastic or metal
The rest is the same as paper
Why is thin layer chromatography a more advantageous technique?
- only small volume of solvent required = less fumes
- development phase quicker
- separated pigments more defined and visible
How does affinity chromatography work?
Relies on binding of proteins and ligand bound to a matrix or gel
Specific molecule passed through and inert (unreactive) support in a column
Protein mixture passed through the column
Complementary target protein binds to specific molecule binds in the column
Other components wash away
Target protein stripped from support
Separation and purification from original sample
How does gel electrophoresis work?
A current is passed through a buffer to separate proteins
In gel electrophoresis what two features separate the molecules?
Charge and size
In gel electrophoresis which travels further, large or small proteins?
Small proteins
What is electrophoresis gel made of?
Argarose
Which direction does the solution/DNA move in during gel electrophoresis?
Towards the positive terminal
Why do smaller molecules move further in gel electrophoresis?
Move through the gels pores easily
What gives a protein it’s overall charge?
The amino acids that make up the protein with + - and neutral charges
What does the term iso-electric mean?
The point were proteins have a neutral charge . If positive the pH is below this point, if negative the pH is above
What happens to a protein at its iso-electric point?
Protein becomes a precipitate and can become solid in liquid suspension
