Chromatography

Question 1

Chromatography definition

Answer 1

A technique for analysing or separating mixtures of gases, liquids or dissolved substances

Question 2

Stationary phase

Answer 2

The solid compound

Question 3

Mobile phase

Answer 3

Usually a liquid, but can be a gas

Question 4

Partitioning

Answer 4

The principle of chromatography is that partitioning between the two phases is used to separate compounds

Partitioning describes the relative affinity that a compound has for the mobile phase vs. the stationary phase

Question 5

Protein purification

Answer 5

Chromatography can be used to purify proteins

• allows characterisation of proteins
• use in industry
• use clinically

Question 6

Separation principles

Answer 6

Compounds can be separated based on several characteristics

• size
• hydrophobicity
• charge
• biorecognition
• isoelectric point

Question 7

Column chromatography

Answer 7

Uses a column to separate a mixture

The column is coated with solid phase, which the compounds bind to

A liquid phase is then used to elute the compounds from the solid phase

The sample can then be separated into fractions

Question 8

Extraction - preparing for protein chromatography

Answer 8

The proteins that will undergo chromatography must first be extracted from their source

• homogenisation
• sonication
• freeze-thaw cycles
• organic solvents

Question 9

Concentration - preparing for protein chromatography

Answer 9

The protein can be further concentrated based on its chemical properties

• salting out
• detergents
• ultracentrifugation

Question 10

Conditions for protein chromatography

Answer 10

Protein chromatography must occur at:

• low pressure
• low flow rate
• low temperature
• large volumes

This ensures the protein is not denatured

Question 11

Size-exclusion chromatography

Answer 11

Also called gel-filtration chromatography

The solid phase contains different sized pores

The size of the compound determines whether it can enter the pore

Hence smaller molecules are able to enter more pores, travelling the greatest distance and eluting from the column last

Question 12

Ion-exchange chromatography

Answer 12

Can use either positively (anion exchange) or negatively (cation exchange) charged matrices

The binding of the protein to the charged matrix is then used

The ionic strength of the buffer is steadily increased, which displaces the protein, depending on binding strength

Question 13

Isoelectric point

Answer 13

The isoelectric point is the pH at which a compound has neutral charge

Therefore, the isoelectric point may influence ion-exchange chromatography

Question 14

Cation exchange

Answer 14

Occurs below the isoelectric point

Question 15

Anion exchange

Answer 15

Occurs above the isoelectric point

Question 16

Gradient elution

Answer 16

A technique that allows separation of proteins, this usually involves salt concentration

• step gradient
• pH gradient