Chromatography

Chromatographic separation

  • Cut the chromatography sheet carefully to a convenient size (40x24cm).
  • Draw a line with pencil across the sheet about 5 cm away from one end.
  • Mark a number of points at intervals of 3 cm.
  • Apply a small volume (say, 25µl) of each amino acid as a separate small spot using a micro syringe.
  • Dry the spot using a stream of hot air from a hair - dryer.
  • Draw a line with pencil across the sheet about 5 cm away from one end.
  • Similarly spot different known aliquots of sample extract.
  • Apply a small volume (say, 25µl) of each sample as a separate small spot using a micro syringe. 
  • Descending chromatography: After spotting, place the sheet in a stainless steel trough in the chromatography chamber, firmly hold it by placing a long steel rod over the sheet.
  • The spot should be as small as possible for better resolution.
  • The spot-end of the sheet should be in the trough
  • Ascending chromatography: Roll the sheet as a cylinder, tie together with fine thread and place upright with the spots at the bottom in a large petridish for upward movement of solvent.
  • Add the organic (Mobile phase) solvent to the trough/petridish and close the chamber airtight.
  • Develop the chromatogram, preferably overnight or longer, until the solvent moves almost to the other end.
  • Note the solvent front and dry the chromatogram free of solvent in a fume chamber. 
Last modified: Monday, 19 March 2012, 6:35 AM