Chromatography Flashcards
(25 cards)
Point of chromatography
Separates components of a mixture between mobile and stationary phase
To identify components
Mobile phase
Where the molecules can move (always liquid or gas)
Stationary phase
Where the molecules can’t move (solid/liquid on a solid support)
Basic principle of chromatography
Mobile phase moves through/over stationary phase
Distance each substance moves depends on solubility in mobile phase and retention/adsorption to stationary phase
So components more soluble = travels further/faster
Differences allow separation of substances
Types of chromatography
Paper chromatography
HPLC (high performance liquid chromatography)
Gas chromatography
Paper chromatography outline
Solvent (mobile phase) moves over paper (stationary phase)
Separates substances in a mixture based on solubility in solvent (more soluble travels further up paper)
Caclulate Rf value = identify
Solvent front in paper chromatography
End of where solvent travelled to up the paper
Baseline in paper chromatography
A few cm of bottom of paper where mixture being separated was put
Rf value formula
Distance travelled by spot (from baseline)
divided by
Distance travelled by solvent (solvent front subtract baseline)
Are Rf values always be the same for a given compound ?
Provided that same paper material, solvent used and temp is same
Then no matter the size of the paper or time it is run the Rf should be the same since it is a proportion of distance spot moves to distance travelled by solvent
Paper chromatography to identify unknown amino acids set up
Chromatography paper
Line draw in pencil near bottom of the paper with conc spot of mixture of AA
Suspend paper in beaker containing small amount of solvent at bottom so solvent touches tip of paper
Watch glass on beaker
End of paper chromatography
Remove paper and mark line in paper the solvent travelled up towards top of paper= solvent front
Visualise amino acids spots
How to make amino acid spots visible in paper chromatography
Spray ninhydrin solution = developing agent = spots become purple
Dip paper into jar with crystals of iodine = sublimes to idoine gas = spots turn purple
Goal of paper chromatography of AA mixture
COmplete mixture alongside spots of known aa to see if the spots in mixture have same RF value = mixture contains same AA
Compare RF to table of known AA to identify AA present
TLC
Same as paper chromatography but silica SiO2 is used or alumina Al2O3 plate is used instead of paper
HPLC outline
Small solid particles eg silica bonded to hydrocarbons is stationary phase in column
liquid Solvent eg water and methanol dissolve sample so is passed through column in solution
Mixture separated due to different attraction of different amounts to solid silica + hydrocarbon
So takes longer for some substances in mixture to pass through column thus detected
Detection of HPLC
liquid leaves column (with dissolved mixture)
UV light passed through
UV absorbed by parts of mixture so UV detector measures UV absorbed by mixture parts
= production of a chromatogram
WHat do chromatograms show?
Retention times of components in a mixture
aka time taken for substance to pass through column and detected by absorbing UV light
Those that take longer have greater retention to solid stationary phase
Identification using HPLC
Compare retention times of substance with data booklet value of retention times
Mobile phase in HPLC
Liquid water/methanol
Dissolves mixture in soution
GC outline
Column contains solid/oil (stationary phase)
Where mixture injected into stream of gas (mobile phase) into column
Mixture separated based on mixture’s retention to stationary phase (adsorption to solid/dissolving in oil)
Evaporates into gas then detected = chromatogram
Mobile phase in GC
Gas
Similarities between HPLC and GC
Column chromatography
Stationary phase is solid in column
Why do we combine HPLC/GC with mass spectroscopy>
To provide a useful analytical tool
Chromatography can separate a mixture into components then immediately fed into a mass spectrum to identify what original sample consisted of