⚪️ Topic 19 - Modern Analytical Techniques II

Question 1

What is a good method to check whether a synthesised organic compound is pure or not, and if not, to tell what impurities are present?

Answer 1

Chromatography - paper chromatography, thin layer chromatography (TLC), column chromatography, high performance liquid chromatography (HPLC), gas chromatography (GC)

Question 2

Why should the container with the solvent and chromatography paper in, in paper chromatography, have a lid on?

Answer 2

To prevent the evaporation of the solvent

Question 3

What is a mobile phase? Give an example.

Answer 3

The liquid that moves through the stationary phase and transports the components.
In paper chromatography the solvent is the mobile phase.

Question 4

What is a stationary phase? Give an example.

Answer 4

The liquid/solid that does not move.
In paper chromatography, the stationary phase is the water trapped in the fibres of the chromatography paper

Question 5

How does chromatography separate components of a mixture?

Answer 5

It separates them between a mobile phase and a stationary phase - each component in the mixture is attracted to both phases, but more strongly to one than the other

Question 6

Describe what happens to each component when separated by chromatography.

Answer 6

• the more polar component (remember to say why it is more polar eg. can hydrogen bond/amino acid which has multiple NH2 groups [which form NH3+ in acidic conditions] so has multiple positive charges) is strongly attracted to the (polar) stationary phase but weakly attracted to the mobile phase, so will not travel very far up the paper/ travel very slowly through the column (therefore smaller Rf value/longer retention time)
• the less polar component is weakly attracted to the stationary phase but strongly attracted to the mobile phase, so will travel a long way up the paper/travel quickly through the column (therefore larger Rf value/shorter retention time)

Question 7

Organic compounds are mostly colourless. How is this problem overcome to see the components on the chromatogram?

Answer 7

They must be visualised using ultraviolet radiation or by spraying with a chemical (developing) reagent (such as ninhydrin - which is toxic) that will react with the component to form a coloured product.

Question 8

What is thin layer chromatography (TLC)?

Answer 8

Very similar apparatus and method as that of paper chromatography, except that instead of paper, a sheet of glass or plastic coated in a thin layer of a solid such as silica or alumina (the stationary phase) is used

Question 9

Describe the process of column chromatography, stating what the mobile and stationary phases are.

Answer 9

• the stationary phase is alumina or silica packed into a tube such as a burette
• the mobile phase is the solvent that this is soaked in
• the mixture to be separated is placed on top of this, and more solvent is added
• when the tap of the burette is opened, the solvent drips through the tip and the components of the mixture begin to move down the tube and separate (due to greater attraction to mobile/stationary phase)
• more solvent is added and eventually one component of the mixture leaves the column and can be collected

Question 10

What is the advantage of column chromatography over paper chromatography?

Answer 10

Much larger quantities of material can be separated

Question 11

What are the names of some refinements of column chromatography?

Answer 11

• High performance liquid chromatography (HPLC)
• Gas chromatography (GC)

Question 12

What are the main differences between high performance liquid chromatography (HPLC) and column chromatography?

Answer 12

In HPLC:
- the solvent is forced through a metal tube under high pressure, rather than being allowed to pass through by gravity
- the particle size of the stationary phase is much smaller, which leads to better separation of the components
- the sample is injected into the column
- the components are detected after passing through the column, usually by their absorption of ultraviolet radiation
- the whole process is automated and the results are quickly available on a computer display (as a graph [absorption-time] that shows the concentrations of the different components - the areas under the peaks represent the relative concentrations of the components)

Question 13

What is retention time, and what does it mean?

Answer 13

The time taken from injection to detection of a component of a sample in HPLC/GC - these values can be important in identifying the components (similar to the retention factor (Rf) values obtained from paper and thin layer chromatography)
In HPLC/GC the components will move at different speeds depending on how strongly they are attracted to each phase. Those with weaker attractions to the stationary phase move more quickly and have shorter retention times.

Question 14

What do retention times depend on?

Answer 14

• the nature of the solvent
• the pressure used
• the temperature inside the column

Question 15

How do you calculate the retention factor (Rf value)?

Answer 15

Rf = distance travelled by component / distance travelled by solvent

(Measured from base line, no units, will always be <1, the Rf value of a component will depend on the solvent used and other factors)

Question 16

What are the main differences between gas chromatography (GC) and column chromatography?

Answer 16

In GC:
- the metal tube can be several metres long and is coiled to save space
- the stationary phase is a solid or liquid coated on the inside of the tube
- the mobile phase is an inert carrier gas (often nitrogen or helium)
- the sample is injected into the column, as in HPLC
- the components passing through the column are detected
- the whole process is automated and the results are quickly available on a computer display (as a graph [absorption-time] that shows the concentrations of the different components - the areas under the peaks represent the relative concentrations of the components)
- the coiled column is in an oven

Question 17

In gas chromatography, why is the coiled column in an oven?

Answer 17

So that the components of the sample vaporise once injected, and move through the coiled tube with the carrier gas

Question 18

Retention times depend on how strongly the components are attracted to each phase. What else do retention times depend upon in gas chromatography?

Answer 18

The boiling temperature of the compound - the higher the boiling temperature, the less time the compound spends in the gas phase (mobile phase) so have longer retention times

Question 19

What advantage does column chromatography have over HPLC and GC?

Answer 19

Reasonable amounts of the components can be collected (in GC, the components are often detected in a flame, so they are destroyed during the detection process)

Question 20

HPLC and GC are very useful for separating small quantities of components in a mixture, but they are not very good at positively identifying them. Why?

Answer 20

• partly because of the difficultly in controlling all of the variables (such as solvent, pressure and temperature)
• partly because different substances may have the same retention times
• also a component may have a retention time for which there is no reference to identify it from

Question 21

When do the results of chromatography (HPLC/GC) need to be exactly accurate?
(Made accurate by combining with a different technique)

Answer 21

• in providing forensic evidence (for use in a court law)
• in detecting banned drugs in sportsmen, women and racehorses

Question 22

How are HPLC and GC made more useful (and accurate) in identifying components?

Answer 22

They are often combined with mass spectrometry (which cannot separate a mixture but can provide information about the structures of compounds) - the technique used is therefore often abbreviated to HPLC-MS or GC-MS
(The mixture is injected into the column and due to the different retention times of each component, they emerge at different times, so one at a time each component enters the mass spectrometer where the m/z values and relative abundances of components (fragmentation) within this component are recorded and the component identified)

Question 23

Why do different amino acids have different Rf values?

Answer 23

Different amino acids have different solubilities in the stationary phase AND different solubilities in the mobile phase.
(Remember the components of a mixture are attracted to BOTH the mobile and stationary phase so the attraction to both the phases will affect the Rf values)