4: Chromatography

Question 1

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

Answer 1

A thin layer of solid (e.g. silicia gel, SiO₂) mounted on a piece of glass, metal or plastic

Question 2

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

Answer 2

An organic solvent

Question 3

what does adsorb mean

Answer 3

Stick on surface

Question 4

What does the speed at which a component moves up the TLC plate depend on

Answer 4

How strongly the component is adsorbed to the stationary phase. The stronger the adsorption, the slower the migration up the plate

Question 5

What does the strength of adsorption depend on

Answer 5

The attractive forces between the component and the stationary phase.
The solubility of the components in the mobile phase

Question 6

how to calculate Retardation factor Rf

Answer 6

Rf = distance travelled by the component / distance travelled by the solvent

Question 7

Do polar substances tend to have a low or high Rf value with SiO₂ stationary phase

Answer 7

SiO₂ is quite polar so polar substances will tend to adsorb more strongly hence lower Rf values

Question 8

Advantages of TLC

Answer 8

It is quick and the equipment is inexpensive

Question 9

Limitations of TLC

Answer 9

Very limited resolution- so compounds with similar Rf values will not be clearly separated.

Some spots may contain more than one compound

Question 10

Why is TLC not very good for separating molecules of similar sizes and functional groups

Answer 10

They might be poorly separated as they are likely to adsorb to the stationary phase with similar strength

Question 11

What is the stationary phase in Gas Chromatography (GC)

Answer 11

A liquid adsorbed onto an inert solid support

Question 12

What is the mobile phase in Gas Chromatography (GC)

Answer 12

A gas (unreactive - most commonly helium)

Question 13

Why is GC done in an oven

Answer 13

So that the sample is vapourised

Question 14

What is retention time

Answer 14

the time taken for each component of the sample to travel through the tube

Question 15

how does solubility affect the retention time in GC

Answer 15

As the stationary phase is a liquid, movement of components is slowed down by dissolving in the stationary phase. So the more soluble a component is in the stationary phase the longer the retention time

Question 16

What does solubility in the stationary phase depend on in GC

Answer 16

What sort of intermolecular interactions are are possible between the substances being analysed and the stationary phase.
i.e. if the stationary phase is polar, polar substances will tend to be more soluble as they can form permanent dipole-dipole forces which are stronger than induced dipole-dipole forces

Question 17

If the substances separated and stationary phase are non-polar what will affect the retention time

Answer 17

The surface area of the molecule

larger surface area = more induced dipole-dipole forces - more bonds needed to be broken - longer retention time

Question 18

What is the area of a peak on a Gas Chromatogram proportional to

Answer 18

the amount of the substance

Question 19

How do you find the actual amount of a substance from a gas chromatogram

Answer 19

You have to calibrate the GC system

Question 20

How do you calibrate a GC system

Answer 20

• Inject a series of samples containing different, known amounts of a substance.
• Measuring the peak area for each of these samples.
• Plotting a calibration curve, showing peak area as a function of amount of substance.
• The unknown sample is then run and the peak area for the sample of interest compared with the calibration curve. The concentration of that substance can thus be estimated.
• For greatest accuracy, the machine should be calibrated with the actual substance of interest. However some kinds of detector give virtual identical responses to any type of molecule, so for example, 1 micromole of methylbenzene will give a virtually identical peak area to 1 micromole of testosterone. In this case the machine can be calibrated with one substance and used to determine the molar amounts of any other substance.
• Calibration “curves” are usually (but not always) linear

Question 21

How does GC-MS work (combining chromatography and mass spectrometry)

Answer 21

The components are sent to a mass spectrometer after GC so that they can be compared with spectral databases to identify the components

Question 22

Advantages of GC-MS (Gas chromatography and mass spectrometry)

Answer 22

Very good resolution

Can operate with very tiny amounts of sample mixture

A very large database of mass spectra is available, so there is a good chance of positive identification of individual components

Question 23

Limitations of GC-MS

Answer 23

• Similar compounds often have similar retention times so may be difficult to separate and identify
• Previously unknown compounds in a mixture have no reference retention times of mass spectra
• Equipment is relatively sophisticated and expensive

Question 24

Uses of GC-MS

Answer 24

• Forensic science
• Testing for organic pollutants
• In airport security, for detecting explosives
• On space missions