Chromatography

• chromatography is a technique where soluble compounds csn be separated and identified.

  Using a moving solvent/ strips of filter paper.

  EG, used to separate plant pigment or amino acids
• Rf value: distance moved by compound from origin DIVIDED BY distance solvent front moved from origin
• TO PREPARE:

  1. handle paper with gloves (sweat has amines)
  2. Draw line of origin. Use pencil. 3cm long. Draw from bottom edge
  3. Mark origin for each amino acid and label them
  4. Spot a.a using FINE needle. make sure they are small.
  5. Concentrate the spot by repeating it 3 times.
  6. let them dry inbetween repeating
• line of origin should be equally spaced. Not too close to edge or it will run off.
• RUNNING THE CHROMATIGRAM:

  1. Add solvent to tank of approx 1cm.
  2. DO this early- keep lid on 2 allow atmosphere 2 become saturated with solvent vapour
  3. add c.paper to tank- dont let edges touch sides- ensure spots above solvent
  4. solvent will rise up paper (each has diff solubilities-travel diff parts)
• DEVELOPMENT OF CHROMATIGRAM:

  1. remove chg. from tank.
  2. mark solvent front in pencil
  3. Dry and spray ninhydrin- fume cupboard
  4. heat ninhyrdin- develops stain
  5. amino acids appear dark blue
• MEASUREMEANT OF RF VALUE:

  1. Distance from solvent is measured
  2. a.a spots ringed in pencil
  3. midpoint is marked
  4. work out Rf
  5. Identify using table