Chromatography Flashcards
Describe how to carry out TLC
- on a plastic plate covered in SiO2 typically, draw a pencil line 1cm from bottom and add crosses for each sample
- add a drop of each sample using a glass capillary tube along the pencil line
- add filter paper in beaker
- place plate in beaker holding it by the edges
- add the solvent making sure the level is below the baseline then place a lid on top of the beaker
- wait until solvent travels almost the the top of the plate then take it out and carefully mark the edges of the solvent front
- spray on stain or use UV to visualise spots when dry
why is the baseline drawn in pencil?
- pencil doesn’t dissolve in solvent (mobile phase) so does not interefere with results
why must the solvent level be below the baseline?
- so that the sample spots do not dissolve directly into the mobile phase straight away before having a chance to travel up the TLC plate
why should you avoid spotting the sample on the TLC plate too many times?
to preserve the quality of separation ; to avoid tailing
why should you allow the solvent to reach almost to the top of the TLC plate?
to allow maximum separation of the components of the mixture, ensuring Rf value is accurate
if the solvent front runs over the edge of the plate, the spots may be lost
why should you add filter paper and a lid to the beaker ?
- to saturate the air inside the beaker with solvent vapours and to prevent the solvent from evaporating straight away
- this allows the solvent to move further up the plate allowing maximum separation
why should you wear gloves when handling the TLC plate?
- to prevent contamination of the TLC plate with the natural oils in you hands (they would also produce marks of the plate interfering with results)
Describe how column chromatography is carried out
- a column is a long wide tube with a tap at its bottom
- add the stationary phase (powdered silica typically) to the column. Place a filter at the bottom to keep solid in tube.
- add the mixture to be tested
- add enough solvent until powder is covered
- open the tap at the bottom and place a beaker underneath
- change the beaker at regular intervals
- as the eluent (solvent) is carried through the column, the components of the mixture are separated based on their relative affinites due to the MP and SP
- retention time = time taken for component to be collected in beaker
describe how gas chromatography is carried out
- stationary phase = capillary glass or metal tube filled with a powdered solid /liquid adsorbed onto a solid
- mobile phase = inert gas e.g He
- used to separate mixtures of volatile compounds
- carried out at high pressure and temperature
- mixture injected into inert gas which carries it through the tube
- based on relative affinities for the MP and SP, the components reach the detector at the end of the tube at different times (Retention time)
- detector produces a graph of the peaks of each components and their corresponding retention times
- greater area under peak= higher abundance of component
What are the advantages/disadvantages of TLC?
- Advantages;
- quick and simple method,
- cheap and easily accessible equipment
- very small volume of sample needed
- Disadvantages:
- cannot collect separated components
- chemically similar substances may have the same Rf value in a particular solvent (further tests needed to confirm identities)
- qualitative test (quanitity of components presence in a mixture cannot be deduced)
How do you separate components with the same Rf value using TLC?
- components may produce the same Rf value in a particular solvent
- when the plate is dry, rotate it 90º and repeat the experiment again using the same method but a different solvent (without adding more of sample) . Mark the new baseline and solvent front using pencil
- the components will have different affinities for the new solvent = different Rf values = components separated
What are the advantages/disadvantages of column chromatography?
Advantages:
- components of mixture can be collected
- produces a purer version of sample
Disadvantages:
- TLC may be needed to further identify the components
- Large volume of samples needed = more expensive
- time-consuming
What are the advantages/disadvantages of gas chromatography?
Advantages:
- rapid
- column can be reused
- widere range of samples can be analysed
- high degree of separation of components (2 components will not have the same retention time)
- small sample needed
- quantitative method (tells you both presence and quantity of components)
Disadvantages:
- expensive equipment
- components cannot be collected at the end (fractional distillation needed)
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