Chromtaography Flashcards
What is the purpose of chromatography?
separate mixtures of substances into their individual components
What is the stationary stage?
a solid liquid or gas that the solvent can travel across
- the more attracted a portion of the mixture is to the stationary phase the slower it moves
What is the mobile phase?
a liquid or gas that carries the components of the mixture across the stationary phase
- with a liquid the mixture is dissolves in a solvent (eluent)
- with gas the flow of gas carried the mixture
How do the components of the mixture separate
different components travel at different rates
What factors do the separation of the mixture depend on?
- the attraction of each component to the mobile phase
- the attraction of each component to the stationary phase
How does the attraction of each component to the mobile phase affect the separation of the mixture?
- the more soluble components travel more quickly with the solvent
- less soluble components travel slower
How does the attraction of each component to the stationary phase affect the separation of the mixture?
- if a component has a strong attraction to the stationary phase it will move slowly with the solvent
- If there is a weaker attraction to the stationary phase the component will travel faster with the solvent
How do polar substances dissolve?
they dissolve well in polar solvents
- they attract well to polar stationary phases
How do non-polar substances dissolve?
they dissolve well in non-polar solvents
- they attract well to non-polar stationary phases
What is thin layer chromatography (TLC)?
uses a thin uniform layer of silica gel or alumina coated onto a piece of rigid plastic
stationary phase: silica gel (polar)
mobile phase: liquid solvent
Outline the procedure of TLC?
- draw a pencil line 1cm from the bottom of the paper
- place a small drop of the sample on the pencil line
- Stand the plate in a beaker containing the solvent, the solvent must be below the pencil line so it doesn’t dissolve immediately
- cover the beaker so the solvent doesn’t evaporate**
- let the solvent travel up the plate
- remove the plate when the solvent has travelled 1cm below the top and mark it
- let it dry and spray with ninhydrin (developing agent) or shine UV light
What is the equation for the Rf value?
distance travelled by spot / distance travelled by solvent
What is the Rf value used for?
to identify a components retention factor
What is the purpose of ninhydrin or UV light?
developing factor so the spots can be viewed
How can you determine the primary structure of a protein using TLC
- hydrolyse the protein using concentrated acid, adding more NH2 groups on an amino acid makes it more polar after acid hydrolysis as more NH3+ is made
- run a TLC plate
- calculate the Rf value and compare with know data
What is the purpose of two way chromatography
separates substances with similar Rf values using two different solvents
Outline the procedure of two-way chromatography
- after the first TLC allow the plate to dry completely
- rotate the plate anticlockwise 90 deg and develop the chromatogram in a different solvent
What is column chromatography
involves a thin vertical column packed with silica and the sample is poured thru
the solvent (eluent) is added periodically from the top
Stationary: silica
Mobile: liquid
How does column chromatography work?
- the mixture moves down the column and the different components travel at different speeds according to their solubility in the eluent and their affinity to the silica
- in the beaker at the bottom, we can collect each portion separately
- if one component doesn’t move down the column then a different solvent can be used to match the polarity of the molecule
Outline gas chromatography
- sample is injected thru port
- the mixture is carried through the column and the different components travel at different speeds due to their attraction to the stationary phase
- less polar components are carried by the gas quickly and emerge first others react with the polar lining and emerge later
- components are detected later as they leave the column, the time they exit is recorded at their Retention time
- a spectrum is produced
What is the stationary and mobile phase in gas chromatography?
stationary: thick viscous liquid e.g oil (non-polar) OR silica gel (polar) coated on the lining of the column
mobile: an inert gas e.g nitrogen/helium
How do you determine the number of components in gas chromatography?
the number of peaks
How do you identity a sample using chromatography
retention factor/value is compared with a known value
What equipment is used in gas chromatography?
a gas column instrument which is connected to a mass spectrometer
How do you calculate the amount of each component in gas chromatography?
The area under the peak is proportional to the concentration of a substance
What is an advantage of gas chromatography ?
it is very accurate
