1.3 Separation Techniques

Question 1

What does a centrifuge separate a substance by?

Answer 1

Separates substances of different density.

Question 2

When separating a substance using a centrifuge, more dense substances settle in the what?

Answer 2

Pellet

Question 3

When separating a substance using a centrifuge, less dense substances settle in the what?

Answer 3

Supernatant

Question 4

What types of substances can be separated by paper and thin layer chromatography?

Answer 4

Amino acids and sugars

Question 5

The speed that each solute travels along the chromatogram depends on what?

Answer 5

Its differing solubility in the solvent used.

Question 6

What is affinity chromatography

Answer 6

When 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.

Question 7

What type of molecules are separated by affinity chromatography?

Answer 7

Proteins

Question 8

What type of molecules are separated by gel electrophoresis?

Answer 8

Proteins and nucleic acids

Question 9

What is the principle of gel electrophoresis?

Answer 9

To separate proteins and nucleic acids

Question 10

What are the two types of gel electrophoresis?

Answer 10

SDS- PAGE and native gels

Question 11

How do native gels separate proteins?

Answer 11

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

Question 12

What does SDS- PAGE do to molecules before separation?

Answer 12

Gives molecules a negative charge and denatured them

Question 13

How does SDS- PAGE separate molecules?

Answer 13

Size alone. Since all molecules are given a negative charge they all move to the positive electrode

Question 14

In what state can proteins be analysed by native gels?

Answer 14

In their folded state since native gels do not denature the molecule

Question 15

What does IEP stand for?

Answer 15

Isoelectric point

Question 16

What is a substances IEP?

Answer 16

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

Question 17

What happens a solution is buffered to a specific pH?

Answer 17

Only the protein(s) that have an IEP of that pH will precipitate

Question 18

What can be separated using IEP?

Answer 18

Proteins from other proteins and proteins from a mixture

Question 19

How can soluble proteins be separated?

Answer 19

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.