Chromatography Flashcards
What is chromatography
An analytical technique that separates components in a mixture between a mobile phase and a stationary phase
what are the types of chromatography
- thin layer chromatography ( TLC) - a plate is coated with a solid and a solvent moves up the plate
- Column chromatography (CC) - a column is packed with a solid and a solvent moves down the column
- gas chromatography ( GC) - a column is packed with a solid or with a solid coated by a liquid , a gas is passed through the column under pressure at high temperature
what is the mobile phase
may be a liquid or a gas
the part where the molecules can move through
what is the stationary phase
may be a solid or either a like or solid on a solid support
This is the part where molecules can’t move
in gas liquid chromatography ( GC) what is the mobile phase
An inert gas such as nitrogen , helium , argon
in gas liquid chromatography what is the stationary phase
a liquid on an inert solid
what does the separation by column chromatography depends on
the balance between solubility in the moving phase and retention in the stationary phase
what is a solid stationary phase separated by
adsorption
what is a liquid stationary phase separated by
relative solubility
what happens if the stationary phase was polar and the mobile phase was non polar
non polar compounds would pass through the column more quickly than polar compounds as they would have a greater solubility in the non polar mobile phase
what’s the method for TLC
- wear gloves , draw a pencil line 1cm above the bottom of a TLC plate and mark spots for each sample , equally spaced along the line
- Use a capillary tube to add a tiny drop of each solution to a different spot and allow the plate to air dry
- add solvent to a chamber or large beaker with a lid so that it’s no more than 1cm in depth
- place the TLC plate into the chamber , making sure that the level of the solvent is below the pencil line . Replace the lid to get a tight seal .
- When the level of the solvent reaches about 1cm from the top of the plate , remove the plate and mark the solvent with a pencil . Allow the plate to dry in the fume cupboard .
- Place the plate under a UV lamp in order to see the spots
Draw around them lightly in pencil - calculate Rf values of the observed spots
why do we use a pencil line for TLC
Will not dissolve in the solvent
why do we use a tiny drop of each solution in TLC
too big a drop will cause different spots to merge
why don’t we use too deep of a solvent in TLC
If the solvent is too deep it will dissolve the sample spots from the plate
Why do we use a lid in TLC
to prevent evaporation of toxic solvent
why do we dry in a fume cupboard in TLC
as the solvent is toxic
why do we use a UV lamp in TLC
used if the spots are colourless and not visible
what are some preventions of risks in TLX
Wear plastic gloves to prevent contamination from the hands to the plate
how do we calculate the rf value
distance moved by amino acid / distance moved by the solvent
what’s the method for column chromatography
- a glass tube is filled with the stationary phase usually silica or alumina in powder form to increase the surface area
- a filter or plug is used to retain the solid in the tube
- solvent is added to cover all the powder
- the mixture to be analysed is dissolved in a minimum of a solvent and added to the column
- a solvent or mixture of solvent is then run through the column
- the time for each component in the mixture to reach the end of the column is recorded ( retention time )
what is the mobile phase in column chromatography
solvent
( eluent )
what is the stationary phase in column chromatography
aluminium oxide / silicon oxide in powder form to increase surface area
what is HPLC and what does it stand for
High performance liquid chromatography
- it’s a type of column chromatography used in industry
what’s the mobile phase for HPLC
a liquid
what’s the ststionary phase for HPLC
solid silica
what is gas liquid chromatography used for
to separate mixtures of volatile liquids
what is the retention time in GC
the time taken for a particular compound to travel from the injection of the sample to where it leaves the column to the detector .
It can be used to identify a substance
what happens if some compounds have similar retention times
Will not be distinguished
what does GC tell us
•how many components there are in the mixture by the number of peaks .
• the abundance of each peak
- the area under each peak will be proportional to the abundance of that component
what is gas chromatography mass spec used in (GC-MS)
analysis , in forensics , environmental analysis , airport security and space probes
EQ : suggest why it was necessary to use two different solvents ( TLC) (1)
Some of the amino acids did not dissolve / separate with the first / either solvent
• Some amino acids have the same Rf values or have the same affinity with first / either solvent
suggest a suitable reagent for the hydrolysis of a protein (1)
conc HCl
EQ : suggest how the positions of the amino acids on the TLC plate were located (1)
Using ninhydrin / UV lamp
EQ : state in general terms what determines the distance travelled by a spot in TLC (1)
The affinity for the mobile phase and the stationary phase
EQ :To obtain the chromatogram , the TLC plate was held by the edges and placed in the solvent in the beaker in the fume cupboard . The lid was then replaced on the beaker
Give one other practical requirement when placing the plate in the beaker (1)
solvent depth must be below start line
What do you measure for stationary phase
Affinity for stationary phase
what do you measure for moving / mobile phase
Solubility in moving phase
EQ : suggest why propanone was present in the samples of the eluent collected first ( those with shorter retention times , ) whereas samples containing propan-2-ol were collected later
propanone is less polar than propan-2-ol
Propan-2-ol has a greater affinity for the stationary phase
OR propanone is more soluble in the moving phase
EQ : Outline briefly why chromatography is able to separate a mixture of compounds . Practical details are not required (3)
- solvent is mobile
- stationary phase
- Separation depends on balance between solubility or affinity of compounds in each phase
OR different rf values
