Separation by chromatography

What is chromatography?

• Chromatography is a separation technique first used to separate pigments in plant leaves.

• There are 2 types of chromatography that you will study:

1)  Thin Layer Chromatography - TLC

2)  Gas Chromatography - GC

• The plate is placed in a tank with the solvent below the sample.

• The tank is covered and allowed to rise near the top of the plate.

• The plate is removed, the solvent front marked and the solvent allowed to evaporate.

• The data is analysed.

• Chromatography is used in analysis but is more widely used as a method of separation.

How does chromatography work?

• Chromatography works on the basis that components (molecules) have a different affinities for a stationary phase and for a mobile phase

Phases:

A)  Stationary phase - is in a fixed place (paper in paper chromatography)

• The molecules interact with the stationary phase slowing down their movement - ADSORPTION

B)  Mobile phase - moved in a definite direction (water rises up in paper chromatography)

• The molecules interact with the mobile phase speeding up their movement - SOLUBILITY

OVERALL - DIFFERENT MOLECULES MOVE AT DIFFERENT SPEEDS DUE TO THEIR DIFFERING INTERACTIONS WITH THE 2 PHASES

1)  Thin Layer Chromatography - TLC

• Solid stationary phase and liquid mobile phase

2)  Gas Chromatography - GC

• Liquid on solid support stationary phase and a gas mobile phase

Separation:

• Is by adsorption or solubility depending on whether the stationary phase is a liquid or a solid:

Qu 1,2  P77

Thin - Layer Chromatography - TLC

• Is used to check purity / monitor the extent of a reaction.

Phases:

A)  Stationary phase - Silica gel, SiO₂ or Aluminium oxide, Al₂O₃

B)  Mobile phase - Solvent

Producing the chromatogram:

1)  Dissolve sample.

2)  Draw a pencil line and spot sample using a capillary tube, allow to dry.

3)  Place plate in a tank of solvent - solvent must be below line, seal the tank.

4)  Separation is by adsorption - allow solvent to almost reach the top, draw a line here - solvent front.

5)  Each separated component is a spot, if colourless use a UV lamp:

R_f values:

• An R_f value shows how far a component has travelled compared with the solvent front:

		Rf	=	Distance moved by component
				Distance moved by solvent front
		Rf Green	=	1.65	0.34
				4.85
		Rf Pink	=	2.15	0.44
				4.85
		Rf Blue	=	4.30	0.89
				4.85

• R_f is a ratio of the distance of the component moved : solvent front.

• This means that the R_f values in this solvent will always be the same:

• Comparisons can also be made against pure components.

Limitations:

• Similar compounds often have too similar R_f values.

• Unknown compounds have no R_f value for comparison.

• It is hard to find a solvent that will have the carrect amount of solubility - Goldilocks!!

Qu 1,2   P79

Gas Chromatography - GC

• Is used to separate volatile compounds (gases) in a mixture.

• The compounds, being volatile will have low boiling points (to evaporate easily

• This process takes place in a gas chromatograph:

Producing the chromatograph:

• The mixture is injected into the chromatograph, it is vaporised and the mixture is carried through the column by the mobile carrier gas.

• As the mixture flows through the capillary column, the components are slowed down by the stationary phase lining the column:

The stationary phase: The stationary phase is a liquid or solid lining of the capillary tube. This tubing is called the chromatography column. A suitable liquid lining for the stationary phase is usually a long chain alkane (high boiling point) - solubility A suitable solid lining for the stationary phase is usually a silicone polymer - dsorption Depending on what is separated depends on whether you use a liquid or solid stationary phase. The mobile phase: Is an inert carrier gas such as helium or nitrogen.

• Different components are slowed down by different amounts - separation:

Separation can be improved by using different flow rates and oven temperatures. Each component leaves the column at a different time and is detected as it leaves the column. The time taken for a component to leave the column is called the retention time:

Retention time:

• Is the time taken for a component to pass from inlet to detector.

• Just like R_f values the retention time can be used to identify a component.

• Known compounds will have known retention times at the same temperature, carrier gas and stationary phase.

• The area under each peak (component) is equivalent to the amount of that component in the sample:

This is the gas chromatogram of blood / alcohol. The relative concentrations of ach component can be estimated by comparing peak areas. Limitations of gas chromatography: Thousands of chemicals have similar retention times, peak shapes.  This means that most compounds cannot be positively identified. Not all substances can be separated.  Some substances can 'hide' under others.  This can give a higher concentration of the other. Unknown compounds have no reference retention times.  Analysts need to know what is expected. Due to the limitations, gas chromatography is usually used in conjunction with spectroscopy.

Qu 1,2   P81

Gas Chromatography - Mass Spectroscopy - GC-MS

Combining gas chromatography with mass spectroscopy:

• This is 2 techniques combined to provide a powerful analysis tool.

• Each component is separated using GC then analysed using MS.

Summary:

Uses for GC-MS 1)  Forensics - scenes of crime 2)  Environmental analysis - air pollutants, waste water, pesticides in food. 3)  Airport security - explosives in luggage / airport security 4)  Space probes - planetary atmospheres

Qu 1-3   P83        Qu 1,2  P105        Qu 1  P107