RP6 Chromatography Flashcards
what are the basic principles of all kinds of chromatography?
a family of separation techniques that depend on the principle that a mixture is separated if it is dissolved in a solvent and this mobile phase is passed over a solid (the stationary phase).
what is the mobile phase?
carries the soluble components of the mixture
what relationship between a sample and the mobile phase makes the sample move faster?
more soluble components/components with more affinity to the solvent move faster
what does the stationary phase do?
holds back components of the mixture that are attracted to it.
what is the relationship between a sample and the stationary phase that make the sample move slower? what kind of bonding does this often involve?
more affinity for the stationary phase means that a component moves slower; often attracted by hydrogen bonding
how are substances separated by chromatography?
if suitable stationary/mobile phases are chosen, the balance between affinity for the mobile phase and affinity for the stationary phase is different for each component of the mixture. thus, they move at different rates and are separated over time.
why will different substances show different Rf values?
they are bonded differently and have different polarities - more polar bonds mean longer retention time or smaller Rf value, since hydrogen bonding/dipoles are attracted more strongly to the stationary phase
what are the advantages of TLC over paper chromatography?
runs faster
smaller amounts of a mixture can be separated
TLC plates are more robust that paper
what does TLC stand for?
thin layer chromatography
what is the stationary phase in TLC?
plastic/glass/metal sheet or “plate” coated in silica (SiCO2) or alumina (Al2O3)
how can you observe colourless spots?
shine UV lights on them.
or spray with a developing agent (e.g. ninhydrin turns amino acid spots from colourless to purple, so they can be seen) (heating needed with ninhydrin)
what is column chromatography?
column packed with silica, alumina or resin has solvent run through it downwards
how do you calculate the Rf value?
measure the distance from the initial line (that the mixture was spotted onto) to the solvent front, and the distance from the initial line to the spot.
calculate Rf using: Rf = distance moved by spot / distance moved by solvent front
what does Rf value stand for?
retention factor; a measure of the rate of movement of a component through the chromatography apparatus; a ratio between the rate of movement of the solvent and that component
how could you confirm the identity of a substance from its Rf value?
compare your Rf calue to accepted values Rf for that substance run in the same solvent and set-up; if they match, then identity is confirmed
