Separation Techniques

Question 1

What is centrifugation and what is it used for

Answer 1

Method for separating materials in suspension. It separates by mass/ density
Material is rotated in a centrifuge tube at between 2000-120000 revolutions per minute.
Heavier the component the lower it will go in the tube
Most dense- the pellet
Less dense- supernatant

E.g extracting enzymes from tissue.

Question 2

Types of chromatography

Answer 2

Paper chromatography
Affinity chromatography
Gas-liquid chromatography

Question 3

Describe chromatography

Answer 3

Uses 2 phases: mobile phase and stationary phase
Each substance in the mixture has different level of solubility in the , mobile phase as it moves through the stationary phase
Each compound is dropped out at a characteristic point allowing you to analyse the mixture

Question 4

Describe affinity chromatography

Answer 4

Technique for the separation of one specific soluble protein from a mixture.
Stationary phase chosen to bind the substance required by: charge, chemical association or antibiotics binding
Mixture is passed over stationary phase and substance is trapped while the rest is discarded
Required substance is removed from stationary phase (elution)

Question 5

Describe gel electrophoresis

Answer 5

Uses an agarose gel which various biological molecules pass through according to size and charge. Such as nucleic acids, proteins, amino acids

Question 6

Describe iso-electric points

Answer 6

The iso-electric point is the pH at which a molecule has not net charge
Uses a pH gradient (range of charges) set up in polyacrylamide gel
Compounds will migrate until they reach their IEP then drop out of the solution
Very useful technique in protein separation since the amino acids determine the overall charge