4: Chromatography Flashcards
What is the stationary phase in Thin Layer Chromatography (TLC)
A thin layer of solid (e.g. silicia gel, SiO₂) mounted on a piece of glass, metal or plastic
What is the mobile phase in Thin Layer Chromatography (TLC)
An organic solvent
what does adsorb mean
Stick on surface
What does the speed at which a component moves up the TLC plate depend on
How strongly the component is adsorbed to the stationary phase. The stronger the adsorption, the slower the migration up the plate
What does the strength of adsorption depend on
The attractive forces between the component and the stationary phase.
The solubility of the components in the mobile phase
how to calculate Retardation factor Rf
Rf = distance travelled by the component / distance travelled by the solvent
Do polar substances tend to have a low or high Rf value with SiO₂ stationary phase
SiO₂ is quite polar so polar substances will tend to adsorb more strongly hence lower Rf values
Advantages of TLC
It is quick and the equipment is inexpensive
Limitations of TLC
Very limited resolution- so compounds with similar Rf values will not be clearly separated.
Some spots may contain more than one compound
Why is TLC not very good for separating molecules of similar sizes and functional groups
They might be poorly separated as they are likely to adsorb to the stationary phase with similar strength
What is the stationary phase in Gas Chromatography (GC)
A liquid adsorbed onto an inert solid support
What is the mobile phase in Gas Chromatography (GC)
A gas (unreactive - most commonly helium)
Why is GC done in an oven
So that the sample is vapourised
What is retention time
the time taken for each component of the sample to travel through the tube
how does solubility affect the retention time in GC
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
What does solubility in the stationary phase depend on in GC
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
If the substances separated and stationary phase are non-polar what will affect the retention time
The surface area of the molecule
larger surface area = more induced dipole-dipole forces - more bonds needed to be broken - longer retention time
What is the area of a peak on a Gas Chromatogram proportional to
the amount of the substance
How do you find the actual amount of a substance from a gas chromatogram
You have to calibrate the GC system
How do you calibrate a GC system
- 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
How does GC-MS work (combining chromatography and mass spectrometry)
The components are sent to a mass spectrometer after GC so that they can be compared with spectral databases to identify the components
Advantages of GC-MS (Gas chromatography and mass spectrometry)
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
Limitations of GC-MS
- 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
Uses of GC-MS
- Forensic science
- Testing for organic pollutants
- In airport security, for detecting explosives
- On space missions
