1.13 Separation Techniques

Question 1

Substances can be separated according to ___, ___, ___, and ____.

Answer 1

size, shape, charge and solubility

Question 2

Centrifuges can separate substances according to ___, with the ___ materials forming a pellet at the bottom. The liquid which remains above is called the ___.

Answer 2

density, densest.
supernatant

Question 3

Gel electrophoresis can separate ___ and ___ acids.

Answer 3

proteins, nucleic acids

Question 4

Gel electrophoresis separates macromolecules according to their ___, ___, and ___. This is possible because the molecules move at different ____ through the gel matrix, which has an ___ field applied to it.

Answer 4

size, shape, charge.
speeds, electric

Question 5

Gel electrophoresis relies on an ___ ___ flowing through a buffer, to ___ proteins. The gel acts as a ___.

Answer 5

electric current, separate.
Seive

Question 6

True or False?
Smaller proteins will travel further through the gel than larger proteins.

Answer 6

TRUE

Question 7

What are the two types of protein gel electrophoresis?

Answer 7

SDS-PAGE and Native

Question 8

Native gel electrophoresis does not ___ its proteins, and so is able to separate different proteins according to ___, ___, and ___.

Answer 8

denature, size, shape, charge

Question 9

SDS-PAGE electrophoresis separates proteins based on ___ alone since the (positive/negative) charge it applies to the proteins causes them to ___.

Answer 9

size, (negative), denature

Question 10

Name the three types of chromatography.

Answer 10

Affinity
Paper
Thin Layer (TLC)

Question 11

‘Chromatography’ refers to a set of techniques that separates the ___ of a ___.

Answer 11

components of a mixture

Question 12

Chromatography can be used to separate ___ and ___ ___.

Answer 12

Sugars, amino acids

Question 13

In paper chromatography, a sample of the mixture to be ___ is placed in a dot or a line near the ___ of the paper, which is placed in a ___. The components of the mixture will travel at different ___ based on their ___ in the ___.

Answer 13

separated, bottom, solvent.
speeds, solubility, solvent

Question 14

Thin Layer Chromatography (TLC) uses a strip of absorbent material, such as ___ ___, on a non-___ backing. It’s the same principle as Paper Chromatography, where different substances travel at different ___ based on their different ___ in the solvent.

Answer 14

silica gel, reactive.
rates/speeds, solubilities

Question 15

Affinity chromatography relies on the ___ ___ between a protein and a ___.

Answer 15

binding interactions, ligand

Question 16

In affinity chromatography a ligand is immobilised on a ___, and a ___ mixture is passed through.
Target molecules with a high ___ for the ligand become ___ to them, whilst the others are washed out.
The target molecule can then be ___.

Answer 16

column, protein.
affinity, attached.
isolated.

Question 17

Proteins can be separated from their mixtures according to their ___.

Answer 17

IEPs (Iso-Electric Points)

Question 18

What is the IEP of a protein?

Answer 18

The pH at which it (a soluble protein) has no charge, and will therefore precipitate (solidify) out of the solution

Question 19

If a solution is buffered to a specific pH, only proteins with the same ___ will ___.
How can this information be used?

Answer 19

IEP, precipitate (solidify).
In IEP electrophoresis!

Question 20

A variety of unknown, soluble proteins can be separated and identified using an electric ___, and a pH ___.
Each protein will stop migrating through the gel at its ___, and precipitate.

Answer 20

field, gradient.
IEP

Question 21

Why do proteins precipitate once they reach their IEPs?

Answer 21

They have no charge and so won’t be attracted to the electric field - they stop moving