Chromatography

What is chromatography used for?

To separate and identify the components in a mixture

Phase

A state such as solid, liquid or gas.

Moving (or mobile) phase

The phase that moves

Stationary phase

The phase that doesn’t move

What does separation of a mixtures components in chromatography depend on when the stationary phase is solid?

• The balance between the solubility in the moving phase and retention by the solid stationary phase (the retention is by adsorption)

• The stronger the retention to the solid stationary phase, the slower the component moves with the mobile phase

What does separation of a mixtures components in chromatography depend on when the stationary phase is liquid?

• The balance between the solubility in the moving phase and retention by the liquid stationary phase (the retention is by solubility)

• The stronger the retention to the liquid stationary phase, the slower the component moves with the mobile phase

What are the 2 measures that can be used to record rate of movement of a component?

• Rf value

• Retention time

What property affects the strength of adsorption and relative solubility of a molecule?

Polarity

What are the 2 uses of Thin Layer Chromatography (TLC)?

• Checking purity of compounds

• Identifying components in a mixture

What is the stationary phase used in Thin Layer Chromatography (TLC)?

• A thin layer of solid SiO₂ or Al₂O₃ on inert support (e.g: glass or plastic)

What is the mobile phase used in Thin Layer Chromatography (TLC)?

A liquid solvent which moves up the plate

Why is a lid used in thin layer chromatography?

To ensure the atmosphere inside the beaker is fully saturated with solvent vapors

Outline the process of thin layer chromatography (TLC) (8 steps)

1. Dissolve a small sample of the mixture in the solvent

2. Draw a pencil line a short distance from the bottom of the TLC plate

3. Place a small spot of the sample on the pencil line and allow to dry

4. Place the TLC plate in a beaker containing a small amount of the solvent (The bottom of the plate must be in the solvent BUT the solvent must be below the pencil line)

5. Seal the beaker with a lid

6. Allow the solvent to rise up the TLC plate

7. Once the solvent has risen to almost the top of the TLC plate, remove the plate and mark the solvent front

8. Use a locating agent to make the compounds visible

What are 3 example of locating agent that can be used for thin layer chromatography?

• Ninhydrin (good for amino acids)

• U.V light

• Iodine

Rf value

Distance moved by spot / Distance moved by solvent

What can Rf values be between?

0 and 1

What is Column Chromatography (CC) used for?

Separation and collection of larger amounts of material

What is the stationary phase used in Column Chromatography (CC)?

A glass column packed with asolid such as SiO₂ or Al₂O₃

What is the mobile phase used in Column Chromatography (CC)?

A liquid solvent (called elvent) which moves down the column

Outline the process of column chromatography (CC) (7 steps)

1. Fill glass tube with the stationary phase held in place by a filter or mineral wool plug

2. Cover all the powder in solvent

3. Dissolve the mixture to be sampled in the minimum amount of solvent

4. Place the mixture on toр of the solid phase

5. Run the mixture through the column by opening the tap and adding solvent at the top

6. The time taken for each component to reach the end of the column is recorded

7. This is known as the retention time

What is an advantage of column chromatography (CC)?

Fairly large amounts of material can be separated and collected

What is gas chromatography (GC) used for?

Separating volatile (low boiling point) compounds

What are the 2 possible stationary phases in gas chromatography (GC)?

• Thin layer of liquid on an inert solid support inside column

• A column packed with a solid

What is the mobile phase in gas chromatography (GC)?

Inert gas (E.g: He or N₂) which moves under pressure through the column

Outline the process of gas chromatography (GC) (4 steps)

1. The mixture is injected into the gas chromatograph where it vaporizes

2. The carrier gas (moving phase) flushed the mixture through the column

3. Components slow down as they interact with the stationary phase

4. Each leaves the column at a different time (Its retention time) and is detected

Retention time

The time taken for a component to pass from the column inlet to the detector

The longer the retention time is…

• The greater the component’s retention by the stationary phase

• The less soluble the component is in the moving phase

The shorter the retention time is…

• The lower the component’s retention by the stationary phase

• The more soluble the component is in the moving phase

What does the number of peaks on a gas chromatogram equal?

Minimum number of components in the mixture (A large peak may hide a smaller one)

What is the area under the peak on a gas chromatogram proportional to?

The amount of component in the mixture

What is gas Chromatography- Mass Spectroscopy (GCMS) used for?

• Gas chromatography is used to separate components

• Mass spectrometry is used to analyse the separated components

Outline the stages of Gas Chromatography- Mass Spectrometry (GCMS) (4 stages)

1. GC is first used to separate the components in the mixture

2. Each separated component is directed to the mass spectrometer in turn

3. Each mass spectrum can be analysed or compared with a spectral database, so enabling each component to be identified

4. The quantity of each component can also be determined

Give 4 uses of Gas Chromatography- Mass Spectrometry

• Forensic and drug analysis

• Environmental analysis

• Airport security

• Space probes