Chromatrography Flashcards
What is chromatrogrphy
Is a technique used to separate and identify the components of a mixture.
Chromatography is a series of analytical techniques that can be used to separate mixtures of compounds for further use or for analysis
How does it work
Works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile medium.
What does chromatrography consist of and what are they
stationary phase – this phase does not move. Compounds in the mixture are attracted to it (adsorbed) and slowed down. Either a solid or a liquid.
mobile phase – this phase moves.
The more soluble compounds in the mixture are carried faster as the mobile phase moves. Either a liquid or a gas.
What is the moobile phase ? give some exampls
This is the solvent for the biological molecules
Water can be used for polar molecules or ethanol for non polar molecules
The mobile phase flows through and across the stationary phase, carrying the biological molecules with it
What happens
The solvent travels up the paper or plate and the components travel at different speeds with it.
They end up at different positions on the plate.
How does it work?
The speed at which molecules move along the paper or TLC plate depends on
Solubility in the solvent
Polarity
Size of the molecules (in the case of paper chromatography)
How do you calculate rf
Rf = distance moved by substance / distance moved by solvent front
What substances have an rf value of 1
For substances that are very soluble in the liquid Rf will be close to 1
What substances have an rf value of 0
For substances that are rather insoluble in the liquid Rf will be close to 0
How may you imporve chromotgraphy
Ultraviolet Light: Thin Layer Chromatography plates have a chemical which floureses under UV light. If you look at the plate under UV light most of it will glow, except those places where the spots have travelled to. They mask the plate from the UV light.
Ninhydrin: To see aa, allow the plate to dry, and then spray it with ninhydrin. This binds to the aa which are then visible as brown or purple spots.
Iodine: allow the plate to dry, and place in an enclosed container with a few iodine crystals. The iodine forms a gas, which then binds to the molecules in each of the spots.
What are the advantages of chromotagraphy
It can separate complex mixtures with great precision
Even very similar components, such as proteins that may only vary by a single amino acid, can be separated with chromatograph
Used to separate delicate products since the conditions under which it is performed are not typically severe
