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Flashcards in Chromatography Deck (30):

What is the stationary phase?

The phase where molecules can’t move


What is the mobile phase?

The phase where molecules can move


What is adsorption?

The process by which a solid holds molecules onto its surface


What are the Uses of TLC

Checking the purity of a compound ie the experimental chromatogram is compared to a standard one


What are the mobile and stationary phases for TLC chromatography

Stationary phase is a thin layer of an adsorbent chemical such as al2O3 or SiO2 on glass
The mobile phase is the liquid solvent which moves vertically up the plate( I.e ethanol)


Describe the procedure of TLC

Dissolve a small sample of mixture in the solvent
Draw a pencil line a short distance from the bottom of the TLC plate
Place a small spot on the sample on the pencil line and allow to dry ( use a hairdryer to accelerate this process)
The bottom of the plate must be in the solvent but the solvent must be below the pencil line
Seal the beaker with a lid to prevent any solvent evaporating
As it moves up the plate, the solvent will carry the substances in the mixture with it
Separation occurs
Once the solvent has almost risen to the top remove the plate and mark the solvent front
Use a locating agent to make the compounds visible(uv light or ninhydrin)


How does separation occur in solid stationary phases?

If the stationary phase is solid separation occurs by adsorption
The stronger the adsorption to the solid stationary phase, the slower the component move through the mobile phase


How does separation occur in liquid stationary phases with a solid support?

If the stationary phase is a liquid then separation occurs by relative solubility
The greater the solubility in the stationary liquid phase , the slower the component moves through the mobile phase
The components are separated as they travel different distances


Which factors can affect the relative solubility/strength of adsorption of a molecule?



General separation theory ?

Separation depends on the balance between solubility in the moving phase and retention in the stationary phase( ie separation by adsorption or relative solubilities)


What are the advantages of TLC chromatography compared to paper chromatography?

Faster - mobile phase moves faster through/ over the stationary phase faster
Requires smaller amounts of mixtures
Better resolution


Why is a lid used in TLC

Solvents with rapidly evaporate from the plate


What are the limitations of TLC plating?

Similar compounds have similar Rf values
Unknown compounds have no Rf values for reference
Can be difficult to find a solvent to separate all the components in a mixture


How do you calculate the Rf value?

Distance moved by spot/distance moved by solvent


What are the uses of column chromatography?

Separation and collection of larger amounts of material e.g by separating a mixture of amino acids
Or purifying an organic product


What is the moving phase in CC

A liquid solvent - this is called the eluent which is added at the top of the column


What is the stationary phase for CC?

A solid such as SILICA(SiO2) or alumina(Al2O3) or a synthetic resin . This is packed in a glass column.


What is the procedure for column chromatography?

Fill the glass tube with a stationary phase( adsorbent material like alumina or silica ). This is held in place by a filter or mineral wool plug.
Cover all the powder in solvent( ie water)
Gather a beaker and dissolve the mixture to be sampled in the minimum amount of solvent
Place the dissolved mixture( that contains the solvent) ( eluent) on top of the solid phase.
Run/drain the mixture( ie of amino acids) through the column by opening the tap and adding more solvent on top( solvent is the eluent) - the mixture forced down by the eluent and the mixture separates into different components as they all have different attractions to the solid stationary phase.
Eventually each different component can reach the end of column and it can be collected in a beaker( this is the eluate)
The time taken for each component to reach the end of the column is recorded. This is known as the retention time.


What are the advantages of CC

Fairly large amounts of material can be separated and collected


What are the uses of gas chromatography?

To test a very wide range of compounds particularly low boiling point ones. It separates a mixture of volatile liquids( ones that turn into gases easily)


What is the moving phase in gas chromatography?

Inert carrier gas such as He or N2 at high pressure and high temperatures


What is the stationary phase of gas chromatography?

A solid or liquid coating the long capillary tube
The stationary phase coating the long, coiled capillary tube is usually a hydrocarbon/oil. This usually has a high boiling point as we tend to heat the mixture -


What are the experimental details for gas chromatography?

The mixture is injected into the gas chromatograph( which is the gas chromatography machine) via the injector port . The mixture is vaporised due to the high temperatures of the oven
The carrier gas( the mobile phase) such as Nitrogen or Helium carries/moves the mixtures( and the components within the mixture) through the column
Components slow down as they interact with the stationary phase
Each leave the column at different times
The separation time depends on solubility in the mobile phase and retention in the stationary phase( I.e adsorption or relative solubility)
Components reach a detector (once they leave the column) which produces a gas chromatogram. This gives us the information about the retention time, i.e retention to stationary phase( solubility or adsorption)


What is the retention time?Why are these useful? What is the relationship between retention time and a components solubility/ adsorption to the stationary phase

The retention time is the time taken for a component to pass from the column inlet to the detector
The longer the retention time(higher time value), the greater the components solubility/ adsorption to the liquid/solid stationary phase. It therefore moves slower through the mobile phase
Different components have different retention time values therefore these can be used for identification
A retention time is compared with those of known compounds to help identify the unknown


What are the features of a gas chromatogram?

Number of peaks= Number of components in the mixture
The area under each peak is proportional to the amount of a component in the mixture


What are the limitations of gas chromatography?

Many thousands of compounds have the same retention time and peak shape
Unknowns have no reference
Peaks for components present in high concentrations can hide smaller peaks with the same retention time


What is GC- MS and why is it used ?

Gas Chromatography-Mass Spectroscopy
Gas chromatography is used to separate components but it is not good at identifying compounds
Therefore mass spectroscopy is used to analyse the separated components( using M+ (molecular ion peak) fragmentation patterns and this is all seen on a mass spectrum


How does GC MS work?

GC is first used to separate components in a mixture
Each separated component is directed to the mass spectrometer in turn. A mass spectrum is plotted for each component
Each mass spectrum can be analysed or compared with a spectral database, so enabling each component to be identified
The quantity of each component can be determined


What are the features of a mass spectrum?

A mass spectrum is a plot of relative abundance/intensity against mass to charge ratio
It consists of a series of peaks at different masses corresponding to the mass the the whole molecule ( the molecular ion) and the masses of the fragment ions



What is the main molecular ion peak in a mass spectrum?

The peak with the highest mass to charge ratio will be the molecular ion