3.3.16 Chromatography Flashcards
What is chromatography?
a method for seperating mixtures that allows for the possibility of identifying the components of that mixture.
What does all chromatography comprise of:
- a mobile phase (this is always a liquid or gas)
- a stationary phase (this is a solid or a liquid on solid support)
What are the three types of chromatography?
- TLC (RP12)
- Coloumn chromatography (primarily a seperation technique)
- Gas chromatography
why do we use a lid in TLC
- prevents the evaporation of the solvent
- means that the atmosphere in beaker is saturated with solvent vapour
What is an eluent?
the solvent added to coloumn chromatography
formula for Rf value
Rf= distance travelled by the substance/ distance travelled by the solvent front
what is Rf
retention - compares distance moved by each component of the solute with the distance moved by the solvent during the experiment. (TLC)
what is the equivalent for Rf in gas chromatography?and how does it work
retention time
this is time measured from the time at which the sample is injected to the point at which the display shows a maximum peak height for that compound. Different compounds have different retention times.
How does chromatography work (generally)
it works as a seperation technique because each component of a mixture will interact differently with the two phases.
Substances are seperated according to relative affinity (attraction) to the stationary and mobile phases.
What does it mean if a substance moves quickly
they have a stronger affinity for the mobile phase
What does it mean if a substance moves slowly
they have a strong affinity for the stationary phase
link together ‘solubility’ ‘affinity’ and ‘adsorbance’ with speed.
components that are more soluble (have a greater afffinity with the mobile phase) will travel further (faster) than components that are more strongly adsorbed (stuck/attached) to the stationary phase.
what are some examples of substances used for the stationary phase
- SiO2 (silica)
- Al2O3 (alumina)
regarded as polar due to surface structure nature
what are some examples of mobile phases
- alkane (non-polar)
- polar solvant (water, alcohols)
if a component has a strong affinity for the stationary phase what does this mean
it adsorbs well
if a component has a strong affinity for the mobile phase what does this mean
it is very soluble
How come amino acids can have different solubilities in the same solvent (and thus can be seperated using chromatography)
the different ‘R’ groups
Are amino acids coloured
no
How can we make amino acids visible so we can read the chromatogram?
- adding a flourescent dye to the silica or alumina layer that glows when UV light shines on it
- spraying ninhydrin solution onto the plate- this causes the amino acids to turn purple.
What do you do if there are components with very similar Rf values
two-way chromatography
–> two solvent fronts (two different solvents at 90 degrees to each other.)
Suggest a suitable reagent for the hydrolysis of a protein
conc HCl
