2.2.14 practical biochem 3: chromotography Flashcards
aim of chromatography
separate mixture into constituents
2 components of chromatography
- stationary phase
- mobile phase
stationary phase
= chromatography paper or thin-layer chromatography (TLC) plate
- paper made of cellulose
- TLC plate often plastic sheet coated w/ thin layer of silica gel or aluminium hydroxide
- free OH- groups pointing out, in contact w/ mobile phase
mobile phase
= solvent for bio molecules
- water (polar molecules) or ethanol (non-polar molecules)
- flows through/across stationary phase, carrying bio molecules with it
(if ink) how can the pigments be determined
- each travel at different speeds
- use relative distance travelled to help identify pigments
calculate Rf value
measure distance from pencil line to centre of pigment spot & distance from pencil line to solvent front
solvent front
distance travelled by solvent
3 solutions to identify colourless molecules
- UV light (TLC plate)
- ninhydrin - too see amino acids, let plate dry & spray
- iodine - allow plate to dry & place in enclosed container w/ few iodine crystals
what does the speed at which molecules move depend on
- solubility in solvent
- polarity
- size (paper chromatography)
how does chromatography work
- exposed -OH groups make surface very polar
- allows surface to form hydrogen bonds with molecules alongside other dipole interactions
highly polar solute
tend to stick to surface (absorbed) & move more slowly
non-polar solute
travel very quickly
what should you do if 2 molecules travel at similar speeds (difficult to separate)
try using different solvent or changing pH
how is thin-layer chromatography used (TLC)
- monitor progress of reactions (works quickly)
- urine testing of athletes for illegal drugs
- analysing drugs for purity of components
- analysis of foods to determine presence of contaminants
