Separating Molecules

Question 1

What is chromatography used for

Answer 1

To separate biological molecule in a mixture, to individually identify them

eg. can be used to separate out & identify biological molecules, sa amino acids, carbohydrates, vitamins & nucleic acids

Question 2

Different types of chromatography

Answer 2

• Paper chromatography
• Thin-layer chromatography

Question 3

Method of Paper chromatography
(identifying amino acids in a mixture - method varies slightly for other biological molecule, principles are same)

Answer 3

1) Draw a pencil line near bottom of piece of chromatography paper & put a concentrated spot of the mixture of amino acids on it
2) Add small amount of prepared solvent (mixture of butan-1-ol, glacial ethanoic acid & water used for amino acids) to a beaker & dip bottom of the paper into it (not the spot). This should be done in a fume cupboard. Cover w a lid to stop solvent evaporating
3) As solvent spreads up paper, the diff amino acids (solutes) move w it, but at diff rates, so they separate out
4) When these solvent’s nearly at top, take paper out & mark the solvent front with a pencil. Then, leave paper out to dry
5) Amino acids aren’t coloured, meaning you wont be able to see them on the paper. Before analysing, spray paper w ninhydrin solution to turn amino acids purple (in fume cupboard, w gloves)
6) Use Rf values to identify the separated molecules. You could also compare your chromatogram to chromatogram of a known mixture & identify components that way - if 2 solutes have traveled same distance in solvent, they will be the same molecule

Question 4

What is an Rf value

Answer 4

The ratio of the distance travelled by the solute to the distance travelled by the solvent

Question 5

Formula for Rf value

Answer 5

Rf value = distance travelled by solute / distance travelled by solvent

Question 6

How to calculate Rf value

Answer 6

• When measuring how far a solute has travelled, measure from the point of origin to the vertical centre of the spot
• Use the formula
• You can work out what was in the mixture by calculating an Rf value for each solute & looking each value up in a database/table of known values

Question 7

The 2 phases of paper chromatography

Answer 7

• Mobile phase
• Stationary phase

Question 8

What is the mobile phase

Answer 8

When the molecules can move
- In both paper & thin-layer chromatography, the mobile phase is a liquid solvent, sa ethanol or water

Question 9

What is the stationary phase

Answer 9

Where the molecules cant move
- In paper chromatography, the stationary phase is a piece of chromatography paper
- In thin-layer chromatography, the stationary phase is a thing layer of solid (eg, silica gel) on a glass or plastic plate

Question 10

Basic principle of chromatography

Answer 10

• The mobile phase moves through or over the stationary phase
• The components in the mixture spend diff amounts of time in the mobile phase & the stationary phase
• The components that spend longer in the mobile phase travel faster or further
• The time spent in the diff phases is what separates out the components of the mixture