3.3.16 chromatography

what is chromatography used to do?

to separate mixtures of substances into their individual components

all forms of chromatography work on what?

on the same principle

what needs to happen before chromatography can be performed?

the mixture must be dissolved in a solvent (or carried by a gas in gas-liquid chromatography) → this is called the mobile phase

what are the two parts a chromatography run is made up of?

• a stationary phase

• a mobile phase

outline the stationary phase

• a solid (TLC + column chromatography) or liquid (gas-liquid chromatography) that the mixture can travel across

• the stationary phase stays still whilst the mobile phase travels across it

what is the movement of the mixture in the stationary phase related to?

the more attracted a portion of a mixture is to the stationary phase, then the slower it moves

(retention can also be used to describe attraction)

outline what the mobile phase is + what happens

• a liquid (TLC + column chromatography) or gas (gas-liquid chromatography)

• with liquid, the mixture is dissolved in a solvent → this is called an eluent

• with gas, the flow of gas carries the mixture

• the mobile phase flows across the stationary phase + carries the components of the mixture with it

what is the movement of the mixture affected by?

the more soluble a component of the mixture is in the solvent, then the faster it moves with the solvent

different components of the mixture travel at ______ rates across the ______ phase, what does this allow?

• different

• stationary

• this allows the mixture to separate out into its different parts

outline the two factors the separation of the mixture depends on

1. the attraction of each mixture component to the mobile phase

   • the more soluble components will travel more quickly with the solvent as it moves across the stationary phase

   • the less soluble components will travel but not as quickly

2. the attraction of each mixture component to the stationary phase (retention)

   • if a component has a very strong attraction to the stationary phase, it will move slowly across the phase with the solvent

   • if there is a weaker/no attraction to the stationary phase, the component will travel faster with the solvent

so why will the components separate?

because they have a different balance between their solubility in the mobile phase and their attraction to the stationary phase

what will the attraction to the stationary phase + solubility in the solvent depend on?

it depends on polarity

describe how polarity affects attraction to the stationary phase + solubility in mobile phase

• polar substances dissolve well in polar solvents/mobile phases

• non-polar substances dissolve well in non-polar solvents/mobile phases

• polar substances attract well to polar stationary phases

• non-polar substances attract well to non-polar stationary phases

*remember ‘like attracts like’

therefore one phase will be ____ and one phase will be ____

• polar

• non-polar

what are the 3 types of chromatography?

1. thin layer chromatography (TLC)

2. column chromatography

3. gas-liquid chromatography

what does thin layer chromatography involve?

it is done using a thin, uniform layer of silica gel or alumina coated onto a piece of rigid plastic

give the stationary and mobile phases of TLC + their polarity

stationary phase: usually silica gel (polar)

mobile phase: is a suitable liquid solvent eg cyclohexane (non-polar)

describe the step by step method of TLC

why is pencil used?

because pen would dissolve in the solvent

why is a watch glass used to cover the beaker in TLC?

• to stop solvent from evaporating

• also used to create a saturated atmosphere of the solvent in the beaker

how can you identify a component of the mixture?

• a retention factor or Rf can be calculated for each spot

• it can then be compared to known Rf values from a database

how are Rf values calculated?

value will always be between 0-1

• which component is the least polar + why?

• which component is the most polar + why?

• the top component is the least polar as it has better solubility in the non-polar solvent + less interactions with the polar silica

• the bottom component is the most polar as it interacts with the silica more

what is TLC most commonly used for?

to separate a mixture of amino acids

describe how the primary structure of a protein can be determined using TLC

why is two way paper chromatography used?

to get around the problem of separating out substances which have very similar Rf values

describe the method of two way paper chromatography

• a single spot of mixture is placed towards one end of the base line

• it is stood in a solvent as before + left until the solvent front gets close to the top of the paper

• wait for the paper to dry out completely + then rotate it through 90° + develop the chromatogram again in a different solvent

• it is very unlikely that the two similar spots will have the same Rf values in the second solvent as well as the first so the spots will move by a different amount

outline what column chromatography involves

• a thin, vertical column (similar to a burette) packed with silica + the sample solution is poured through it

• the solvent is added periodically from the top + is known as the eluent (mobile phase)

give the stationary and mobile phases + their polarities of column chromatography

stationary phase: silica gel (polar) within the column

mobile phase: is a suitable liquid solvent eg cyclohexane (non-polar)

describe the method of column chromatography

• the mixture moves down the column + the different components travel at different speeds according to the balance between their solubility in the eluent + their affinity/interaction with the silica powder

• this means with a beaker at the bottom we can collect each portion separately at the bottom

• if one component of the mixture does not move down the column then a different solvent can be used that may better match the polarity of the molecule

which compound is most polar + why?

the blue compound is the most polar as it interacts with the silica better

give the stationary + mobile phase of gas-liquid chromatography + their polarities

stationary phase: a thick viscous liquid, such as an oil (non-polar) or silica gel (polar) coated on the lining of the column

mobile phase: an inert gas eg nitrogen or helium (it carries the components through the column)

describe the method of gas-liquid chromatography

what is produced from gas-liquid chromatography?

a spectrum

which compound is most polar + why?

outline how you can interpret the spectrum produced from gas-liquid chromatography - ie what information can you gain from the spectrums

GLC is very ______ + is used to test for what?

• very accurate

• its used to test the urine of athletes for doping