chromatography

• Define: chromatography

  A technique used to separate substances in a mixture. The mixture is dissolved in a mobile phase, which is then passed through a stationary phase. Substances are separated based on the balance between their affinity with the stationary phase compared to their solubility in the mobile phase.
• Define: mobile phase

  The solvent which will dissolve the mixture to be separated and pass over the stationary phase (can be a gas or liquid)
• Define: stationary phase

  The part of chromatography that remains stationary and separates the components of a mixture by interacting with them to different degrees (usually a solid, often in powder form).
• Define: adsorption


  The adhesion of molecules to a surface (The molecules that attach are called adsorbate and the surface is the adsorbent).
• What are the stationary and mobile phases in paper chromatography?

  Mobile phase: The solvent e.g. water/ ethanol Stationary phase: Chromatography paper
• Describe a method to separate and identify a mixture of food colourings/ inks using paper chromatography

  Draw a pencil line 2cm from the bottom of the chromatography paper Mark pencil spots along the horizontal line On each pencil mark, use a capillary tube to place a dot of a known food colouring On the final pencil mark place a dot of the unknown mixture (if any of the substances are solids you must dissolve a sample in the solvent first) Place the chromatography paper in a beaker containing water to a depth of 1cm Wait for the water to reach roughly 1cm from the top Remove the paper from the beaker, draw a line where the solvent reached and hang it up to dry Calculate Rf values and compare them.
• Why is pencil used to draw the line?

  Pencil is insoluble in the solvent
• Why should you remove the stationary phase when the solvent reaches roughly 1cm from the top?

  You will get more accurate Rf values the higher up the solvent travels (lower percentage uncertainty)
• What technique can be used to determine the different types of amino acids present in a protein?

  Hydrolysis followed by thin-layer chromatography (TLC)
• What is thin

  layer chromatography (TLC)? -A form of chromatography in which the stationary phase is a thin layer of adsorbent material attached to a plate of glass or plastic (TLC plate).
• What stationary phase might be used in TLC?

  Silica (SiO2) or aluminium oxide (Al2O3) coated onto a glass/plastic plate
• What is the mobile phase in TLC?

  The liquid solvent
• How can you locate the amino acids?

  Spray with developing agent e.g. ninhydrin (amino acids turn purple) Or use UV; silica will fluoresce and the amino acids will leave black spots.
• Why is it necessary to wear gloves in TLC?

  To prevent contamination from your hands
• Why do you use a capillary tube to add a small drop of the sample?

  To ensure the drop is small; if the drop is too large it might merge with the neighbouring drop.
• Why must the solvent initially be below the pencil line?

  So that the solvent doesn't dissolve the sample spots from the plate
• When would you need to let the plate dry in a fume cupboard?

  If the solvent produced toxic fumes
• Why do you use a lid?

  To prevent the evaporation of the (potentially toxic) solvent
• Define: retention factor

  Distance travelled by solute/Distance travelled by solvent front
• Why do different amino acids have different Rf values?

  Amino acids have different polarities due to different R groups Therefore, they have different solubilities in the mobile phase And different retention on the stationary phase Rf value depends on balance between the two
• How can you determine the Rf values of the amino acids?

  Locate the amino acid spots using a locating agent or UV Measure the distance from the initial pencil line to the centre of each spot (x) Measure the distance from the initial pencil line to the solvent front (y) Rf = x/y
• What is 2D TLC?

  Use a square TLC plate Spot the amino acids in a corner and run the TLC in solvent 1. Flip the TLC plate 90o and repeat the TLC in a different solvent
• Why is 2D TLC used?

  It gives 2 Rf values for each amino acid (for each solvent) therefore it is easier to identify them and distinguish them from each other
• What are the advantages of TLC over paper chromatography?

  Runs faster than paper chromatography Smaller amounts of mixtures can be separated spots spread out less The plates are more robust so more of the sample can be tested A wider variety of mixtures can be separated and identified by use of different combinations of adsorbent and solvent. Silica or alumina can be removed and replaced
• What is another word for the solvent (mobile phase)

  Eluent
• What is column chromatography?

  A type of chromatography which works on a larger scale by packing the stationary phase into a vertical column. It is used for large scale separation and collection.
• What are the stationary and mobile phases in column chromatography?

  Mobile phase: solvent Stationary phase: silica gel (SiO2) or alumina (Al2O3) (packed into the column)
• Why do you place cotton wool at the bottom of the column?

  To prevent the stationary phase from being washed out
• Why do you add the solvent until just above the stationary phase?

  So the stationary phase doesn't dry out
• How do you add the sample to a column?

  Evenly, using a pipette.
• How could you identify the fractions produced in column chromatography?

  If coloured: identify them directly by their retention time. If not coloured: collect fractions with different retention times, use a sample from each fraction and run them using TLC
• Explain how the choice of mobile and stationary phase affects retention time (for column chromatography).

  If the stationary phase is polar and the mobile phase was non-polar, non-polar substances would have a lower retention time as they have a great solubility in the mobile phase.
• How can you change the retention time of a substance?

  Change the solvent. e.g. if a substance is non polar, use a non polar solvent to decrease its retention time.
• How could you determine whether substance x is present in a mixture?


  Use chromatography to run a pure sample of substance x alongside the mixture. If the Rf value of one of the spots from the mixture matches the Rf value of substance x- it is likely to contain x.
• What is gas chromatography?

  A type of chromatography which uses a stream of gas (mobile phase) to carry a mixture of vapours through a tube packed with either a solid, or a solid coated by a liquid (stationary phase).
• What are the stationary and mobile phases in gas chromatography?

  Mobile phase: Inert gas e.g. Ar or N2 Stationary phase: silica (SiO2) or alumina (Al2O3) powder (sometimes coated with liquid)
• What is gas chromatography used for?

  It is used to identify and separate gases or volatile liquids.
• What is gas chromatography mass spectrometry (GCMS)? Describe the process.

  An analytical technique which combines gas chromatography and mass spectrometry. A mixture is fed into a column and components travel at different speeds (due to balance between their solubility in the mobile phase and retention by the stationary phase). The components emerge at different times, called their retention time, and they are fed directly into a mass spectrometer where their mass and abundance is measured.
• How are components identified in GCMS?

  By its retention time By comparing it to a database of known compounds. It will be identified by its molecular ion peak and fragmentation pattern.
• What can GCMS be used for?

  Chemical analysis in forensics, measuring water pollution, drug testing athletes & racehorses.