chromatography

Question 1

What is the stationary phase for TLC chromatography?

Answer 1

Stationary phase : silica (silicon dioxide) or alumina (aluminium oxide) fixed to a glass or metal plate

Question 2

What are the advantages of TLC over paper chromatography?

Answer 2

• it runs faster
• smaller amounts of mixtures can be separated
• the spots usually spread out less
• the plates are more robust than paper

Question 3

How do you carry out TLC chromatography?

Answer 3

1. Draw a line in pencil near the bottom of the TLC plate (the baseline) and put a very small drop of each mixture to be separated on the line.
2. Allow the spots on the plate to dry.
3. Place the plate in a beaker with a small volume of solvent (this is the mobile phase).
   The solvent level must be below the baseline, so it doesn’t dissolve your samples away.
4. The solvent will start to move up the plate. As it moves, the solvent carries the substances in the mixture with it
5. Leave the beaker until the solvent has moved almost to the top of the plate.
6. Then remove the plate from the beaker. Before it evaporates, use a pencil to mark how far the solvent travelled up the plate (this line is called the solvent front).
7. Place the plate in a fume cupboard and leave it to dry. The fume cupboard will prevent any toxic or flammable fumes from escaping into the room.

Question 4

How colourless chemicals revealed in TLC chromatography?

Answer 4

1. special fluorescent dye added to the silica or alumina layer that glows when UV light shines on it. Put the plate under a UV lamp and draw around the dark patches to show where the spots of chemical are.
2. Expose the chromatogram to iodine vapour. lodine vapour is a locating agent - it sticks to the chemicals on the plate and they’ll show up as brown/purple spots.

Question 5

What is the stationary and mobile phase for column chromatography?

Answer 5

A buretter or a glass column packed with silica or aluminia

• Stationary phase– aglass column/ burettepacked with thepowdered stationary phase silica or aluminium oxide.
• Mobile phase– A solvent, called theeluent, is added to thetop of the columnand allowed torun downthe glass column.

Question 6

Why is column chromatography sometimes used over TLC?

Answer 6

it can separate larger volumes

Question 7

How do you carry out column chromatography?

Answer 7

• A liquid solvent phase,mobile phase, is added into the column until it is saturated with solvent
• The sample mixture is dissolved in the solvent and introduced at the top of the column
• Once the sample has been added, more solvent (eluent) is added on top of the sample
• As the solvent runs through, fresh solvent is added to the top of the column so that it does not dry out
• The sample flows through the column via gravity
• The component with the greatest attraction / affinity to the stationary phase takes the longest time to flow through the column
• If the components are coloured, then they can be identified using the Rvalue
• If the components are colourless, then other techniques such as fluorescence under UV light can be used to show their position in the column

Question 8

What is the mobile and stationary phase for gas chromatography?

Answer 8

• Stationary phase– asolid or a powderthat has been coated with a liquid such as oil (usually aviscous liquidis used). This is coated onto the inside of a long capillary tube
• Mobile phase– The mobile phase is anunreactive gas, usuallynitrogen or heliumis used.

Question 9

How do you carry out gas chromatography?

Answer 9

• The sample is injected into the machine and carried by an inert gas (e.g. nitrogen) which is the mobile phase.
• Each substance takes a different amount of time to travel through the column and reach the detector. The length of time it takes is called the retention time
• The time it takes for the sample to travel through varies as some molecules spend more time stuck to the stationary phase and some spend more time travelling in the mobile phase.

Question 10

What type of molecules have greater affinity for the stationary phase?

Answer 10

polar molecules

Question 11

Outline the steps needed to locate the positions of the amino acids on the TLC plate and to determine their Rf values.

Answer 11

• Spray with developing agent or use UV
• Measure distances from initial pencil line to the spots (x)
• Measure distance from initial pencil line to solvent front line (y)
• Rf value = x / y

Question 12

Explain why different amino acids have different Rf values.

Answer 12

• Amino acids have different polarities
• different solubility in the developing solvent