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School Science/Paper chromatography of amino acids

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This is a description of how to use paper chromatography of amino acids (to identify amino acids).

  • To demonstrate separation of amino acids by paper chromatography
  • To use chromatography to identify amino acids

Materials

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  • Chromatography tank and lid
  • Chromatography paper (or filter paper), to fit tank
  • Capillary tubes or pasteur pipettes
  • Amino acid samples in 1 molar hydrochloric acid (IRRITANT)
  • Unknown samples (e.g. 'unknown' mix or filtered fruit juice)
  • Solvent (BAW): Butan-1-ol : Acetic acid : Water 60 : 15 : 25 (FLAMMABLE, TOXIC)
  • Hairdryer or heating tray
  • Spray can of Indanetrione (Ninhydrin) 1% in butan-1-ol (FLAMMABLE, TOXIC)
  • Latex gloves
  • Access to fume cupboard
  • Drying oven at 100 °C

Methods

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BAW solvent should be made up fresh on the day

Using a pencil, lightly draw a line 1.5–2 cm above the bottom of the chromatography paper. Make small marks at 1.5 cm intervals along the line.

Fill a capillary tube or pipette by capillary action with your first sample. Touch the tip of the capillary to the first mark and pull it away. You should leave a small (>5 mm) wet spot. Let this dry and apply the sample again. Make a note of which sample is placed on the spot (you can lightly write on the paper beneath the mark, using a pencil). Repeat for each sample or mixture, on a different mark and using a fresh tube or pipette each time. Let the paper dry.

Put 1 cm or less of BAW into the chromatography tank. The sample spots should not dip into the solvent. Place the tank in a cool place out of direct sunlight. Stand the chromatography paper in the tank so that the bottom edge is in the solvent but the remaining paper does not touch the tank. Place the lid on the tank and leave for about 1 hour.

Wearing gloves, remove the damp paper and mark where the solvent has reached. Dry the paper in the fume cupboard. Still in the fume cupboard, spray the whole of the paper with ninhydrin in butanol. Dry the paper and transfer it to the drying oven for 1–2 min.

Mark the position of each spot that develops. Note the colour and Rf (or travel distance) of each standard and hence find the amino acid composition of the unknown samples.

Hazards

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  • Acetic acid
  • Irritant vapour. On skin; flood with water. Ventilate area.
  • Butan-1-ol
  • Highly flammable. Remove ignition sources
  • Harmful by inhalation. Irritant vapour. Use fume cupboard.
  • Hydrochloric acid 1 molar
  • Irritant, may cause burns. On skin; flood with water. Spills, neutralise with sodium bicarbonate.
  • Indanetrione
  • Irritant. Wear gloves. Indanetrione, spilt on the skin, is not immediately noticeable. It stains skin purple, slowly, and even seems to work through gloves. Wash frequently to remove it.
  • Hot Oven; also produces Butan-1-ol fumes.
  • Remove papers wearing gloves, or use forceps. Ventilate area
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