Chromatography

• What determines distance travelled by spot?

  -different solubility to solvent/mobile phase and retention time to stationary phase

  -different affinity/attraction to mobile and stationary phase
• How are different amino acid separated?

  -Amino acids have different polarities

  -different affinity to mobile and stationary phase

  -different solubility and retention times
• If the stationary phase is polar and mobile phase is non-polar, what is the polarity of the substance that travelled furthest on the TLC plate?

  -non-polar, high affinity/ attraction to the solvent
• Importance of wearing gloves

  prevent contamination from hands to plate
• Why use pencil line instead of ink?

  Pencil line will not be dissolved by solvent
• Why use small drops on starting line?

  Drops that are too big will merge
• Where should the depth of the solvent go up to?

  Below the starting line, so samples don't dissolve in the solvent
• Why is lid needed in TLC?

  prevent evaporation of toxic solvent
• Why should TLC plate be dried in the fume cupboards?

  solvent is toxic
• How to make the spots visible at the end of the practical?

  use UV light, locating agent
• Is it essential for solvent to travel all the way to the top?

  No, Rf value can still be calculated with solvent front, wherever
• How to find Rd value from TLC?

  -use UV light to make spots visible

  -measure distance from pencil line to spot

  -measure distance from pencil line to solvent front

  -use formula
• What is 2 directional chromatography?

  separate with 1 solvent then turn the TLC plate 90' and use second solvent
• Why might 2 directional chromatography be used?

  -some amino acid not separated by first solvent

  -some amino acids have same Rf values
• Mobile phase of TLC

  liquid or gas
• Stationary phase of TLC

  solid
• Mobile phase of column chromatography

  -liquid
• stationary phase of column chromatography

  solid silica
• Column chromatography

  -burette is filled with stationary phase

  -mixture dissolved in solvent added to column

  -solvent run through continuously

  -time for each component recorded
• Why might a filter or plug be used in CC?

  retain solid
• why might solvent be used in glass tube in CC?

  cover the powder/stationary phase
• What does separation of spots in GC depend on?

  different solubility to solvent and and retention time in stationary phase. Different compounds in mixture run through at different rates
• Retention time is used to identify a substance, compounds with similar retention time cannot be distinguised
• What does GC separate?

  mixture of volatile liquid
• What does CC separate?

  large volume of a mixture of liquids
• Gas chromatography

  sample injected into machine carried by inert gas, each substance take different retention time to travel through column and reach detector
• Mobile phase of GC

  inert gas
• Stationary phases of GC

  high boiling point liquid absorbed onto solid
• GCMS - shows how many components are in the substance
• What does the area under the peak represent?

  amount of substance
• What is the area under peak proportional to?

  abundance of the component
• Why is it important to use an inert gas?

  so it doesn't react with the component being separated
• Which factor change retention time?

  -temperature

  -column length

  -flow rate
• What does GCMS allow?

  identification of component
• why is gas chromatography combined to mass spectra?

  to generate a spectra which can be analysed or compare with data base by computer for positive identification of each component in the mixture
• Things that GCMS is used for

  -analysis in forensic science

  -environment analysis

  -airport security

  -space probes