Chromatography (Add to when doing PPQs)

What is chromatography?

A technique to both separate and identify the components in a sample

What does the separation of compounds depend on?

• The solubility of compounds in the mobile phase

• The adsorption in the stationary phase

What is the stationary phase?

• This phase doesn’t move

• Compounds in the mixture are attracted (adsorbed) to it and slowed own

• Can be a solid or a liquid

What is the mobile phase?

• This phase moves

• The more soluble compounds in the mixture are carried faster as the mobile phase moves

• Can be a liquid or gas

Why are different compounds in a mixture able to be separated?

• Components will have different affinities for the mobile and stationary phases

• This means they will have different Rf values/retention times

What does components adsorbing to the surface of the stationary phase mean?

Components will form weak intermolecular forces with the surface of the stationary phase

What is the solvent front?

The distance the solvent moves up the TLC plate

What is used as the TLC plate stationary phase?

• Aluminium oxide/alumina gel

• Silicon dioxide/silica gel

Why is the line of a TLC plate drawn in pencil?

If drawn i ink, the ink would separate as it is a mixture of dyes and mix with the components

Why are spots not put too close together on the pencil line?

Spots may merge as they travel up the plate

Why is the solvent level below the pencil line?

The samples would dissolve in the solvent before they could travel up the plate

Why must you use only very small spots of sample?

If the spots are too large they cannot be carried by the solvent leading to elongated smudges

Why is the TLC plate removed before the solvent reaches the top?

• To allow us to have a clear reference to calculate Rf values

• If the solvent reaches the top, it can’t go any further so we don’t actually know how far the solvent front would have reaches and therefore cannot calculate an accurate Rf value

Why does the TLC chamber/jar need a lid?

• To have a saturated atmosphere of solvent vapour

• This prevents the TLC plat drying as the solvents normally used in TLC are volatile and evaporate easily

Why should we wear gloves when handling a TLC plate?

To prevent contamination of the plate with chemicals from our hands

How can we visualise invisible spots on TLC plates?

• We can use UV light

• For amino acids, we can use ninhydrin which stains them purple

Why is ninhydrin dangerous to use as a stain?

It’s carcinogenic

How is the affinity of a component to stationary and mobile phases determined?

• Determine by the intermolecular forces

• A compound that has permanent dipole dipole forces (or even better, hydrogen bonding) between molecules will be more strongly adsorbed to a stationary phase with these same IMFs more than a compounds with only van der Waals forces

What does a component being more soluble in the mobile phase mean?

• Components that are more soluble in the mobile phase will have a greater Rf value and less retention

• This means the components will move further up the plate

What does a component being more strongly adsorbed to the stationary phase mean?

• Components with a greater adsorption to the stationary phase don’t travel far from the pencil line

• They will have a smaller Rf value and will have a greater retention

What is two direction chromatography?

• You separate the components the same way as in normal chromatography in one solvent

• You then turn the TLC plate 90^o clockwise or anticlockwise and separate the components again with a different solvent

Why is two direction chromatography useful?

• Two components in a mixture may not separate in one solvent because they have similar Rf values and therefore they have the same solubility in the mobile phase

• Using two way chromatography allows us to be able to separate these components by using a different solvent that a mixture of components will not have the same solubility in

What is column chromatography?

• Used to purify, separate and collect individual chemical compounds from mixtures of compounds

What is another name for the stationary phase is column chromatpgraphy?

Adsorbent

What compound is the stationary phase in column chromatography?

Silica gel, aluminium oxide or zeolite

What is the glass wool at the bottom of the chromatography column for?

Keeps the stationary phase in place

What is the setup for column chromatography and how does it work?

• The stationary phase and mixture of compounds are added to the column

• The mobile phase is then added on top

• As the mobile phase moves down, components in the mixture that are more soluble in the mobile phase/less absorbed to the stationary phase will move down the column further and faster than components less soluble/more adsorbed

• We can then collect the samples from the column separately

How can we analyse our sample collected from column chromatography if the components are colourless?

• A detector can be used

• At the baseline, the graph produced by the detector is flat

  • When detecting a component, a peak is produced and it flattens out again when all of that component is collected

• Separate sample have different retention times so we can use this to detect the samples separately as they produce distinct peaks

What happens to components that are more soluble in the mobile phase in column chromatography?

• More soluble components will elute (exit) the column faster

• They will have a shorter retention time

What happens to components more strongly adsorbed to the stationary phase in column chromatography?

• They elute (exit) the column the column later

• They have a longer retention time

What is gas chromatography useful for?

Volatile mixtures

What happens in gas chromatography?

• A column is packed with a solid or a solid coated by a liquid

  • This packed column is the stationary phase

• An inert carrier gas like argon (or another noble gas) is passed through the column pressure at high temperature

  • The gas is the mobile phase

• There is then a detector which will produce peaks for each sample

What is retention time of components affected by is gas chromatography?

• The greater the retention by the stationary phase the longer the retention time

• The more soluble the component is in the mobile phase (carrier gas) the shorter the retention time

How can we identify chemical components of a mixture in gas chromatography?

The retention time of compounds separated can be compared to a database of known retention times

What do the peaks of a detector in gas chromatography tell us?

• The quantity of each sample (given by the peak area)

• The number of components in the sample (number of peaks)

How can gas chromatography be combined with mass spectrometry?

Mass spectra of separated components can be compared to the spectra of pure samples and give the m/z ratio (an indication of Mr)

Why should we use a fume cupboard during TLC chromatography?

The volatile solvent is toxic