Chromatography Flashcards
(30 cards)
What is chromatography?
an analytical technique used for separating a mixture of chemical substances into its individual components, so the individual components can be thoroughly analyzed
What is the mobile phase/carrier in chromatography?
solvent moving through the column
- moves through stationary, carrying components of mixture with it
What is the stationary phase/adsorbent in chromatography?
substance that stays fixed inside column
- can be solid or liquid that is supported on a solid
What is an eluent in chromatography?
fluid entering column
- solvent or buffer acting as mobile phase, moving through stationary and facilitating separation of mixture’s components
What is an eluate in chromatography?
fluid exiting column (that is collected in flasks)
- fluid that exits chromatography through column, containing separated components of mixture
What is elution in chromatography?
process of washing out a compound through a column using a suitable solvent
What is analyte in chromatography?
mixture whose individual components have to be separated and analyzed
What is the principle of separation of different components in chromatography?
differential affinities (strength of cohesion) of various components of analyte towards stationary and mobile phase results in differential separation of components
What are the two components that affinity is dictated by?
Adsorption and solubility
What is adsorption?
how well a component of the mixture sticks to the stationary phase
What is solubility?
how well a component of the mixture dissolves in the mobile phase
What happens to the movement of the molecules in high adsorption?
the molecules will move more slowly through the column
What happens to the movement of the molecules in high solubility?
the molecules will move more faster through the column
What is normal phase chromatography?
implies that stationary phase is polar (hydrophilic) and mobile phase is nonpolar(hydrophobic)
What is reverse-phase chromatography?
the scenario is reversed from normal phase, where stationary phase in nonpolar and mobile phase is polar
What is paper chromatography?
compound is spotted directly on cellulose paper
- stationary is a solid (cellulose), and mobile is a liquid
- basis of separation: polarity of molecules
What is Thin Layer chromatography (TLC)?
glass coated with thin layer of silica on which is spotted the compound
- stationary is a solid (silica or alumina) and mobile is a liquid
- basis of separation: polarity of molecules
What is Liquid Column chromatography?
glass column packed with slurry of silica
- stationary is solid (silica or alumina) and mobile is a liquid
- basis of separation: polarity of molecules
What is Size Exclusion chromatography (SEC)?
small molecules are trapped in pores of stationary phase and large molecules flow through gaps between beads and have very low retention times, so the larger molecules come out first
- no interaction (chemical or physical) between analyte and stationary phase
- stationary is a solid (microporous beads of silica) and mobile phase is a liquid
- basis of separation: size of molecule
What is Ion-Exchange chromatography (IEX)?
molecules with opposite charge as resin will bind tightly to resin, and molecules with same charge as resin will flow through column and elute out first
- stationary is a solid (cationic/anionic resin) and mobile is a liquid
- basis of separation: charge of molecules
What is Affinity Chromatography?
if molecule is a substrate for an enzyme, it’ll bind tightly to enzyme, and unbound analytes will pass through in mobile phase and elute out of column, leaving substrate bound to enzyme, which can be detached from stationary and eluded out of column with an appropriate solvent
- stationary is a solid (agarose or porous glass beads on to immobilized molecules like enzymes and antibodies) and mobile is a liquid
- basis of separation: binding affinity of analyte molecules to molecules immobilized on stationary phase
What is Gas chromatography (GC)?
samples are volatized, and molecules with the lowest boiling point comes out column first, meaning a high boiling point will make a molecule come out last
- Stationary is a liquid or solid support and mobile is a gas (argon or helium)
- basis of separation: boiling point of molecules
What is a retention factor (Rf)?
distance travelled by the individual component divided by total distance travelled by solvent?
What is the component’s polarity of there is a high retention factor?
the component will be highly polar
