Column Chromatography
Paper Chromatography

Discover a New Antibiotic
Isolating Active Compounds

Christine L.Case

Materials

Mortar & pestle
Solvent
Gel filtration column
 
 

Column Chromatography

  1. Prepare plant tissue extracts by grinding the appropriate tissue in a few milliliters of solvent in a mortar and pestle. Possible solvents are sterile water; methyl alcohol, ethyl alcohol, or acetone.
  2. Use gel filtration to separate the extract into fractions. The column shown at the left contains Sephadex G-25 beads.
  3. Moisten sterile filter paper disks in each of the samples collected and perform the disk diffusion assay to determine which fraction contains the active ingredients.

Materials

Mortar & pestle
Extraction solvent
Hot plate
Isopropyl alcohol
Water
Chromatography solvent
Chromatography paper or TLC strips

 

Paper or Thin-layer Chromatography

  1. Remove chlorophyll and other pigments: a. Dip each tissue in boiling water for 30 sec to kill the protoplasm in the cells. b. Put each tissue in a beaker of alcohol and set the beaker on a hot plate until the tissue is colorless (approx. 30 sec). Be careful, alcohol is flammable.
  2. Prepare plant tissue extracts by grinding the appropriate tissue in a few milliliters of solvent in a mortar and pestle. Possible solvents are sterile water; methyl alcohol, ethyl alcohol, or acetone.
  3. Use paper chromatography or TLC to separate the extract into fractions. (Possible solvents: 1 part 2% NaOH: 2 parts isopropyl alcohol; or 9 Petroleum ether: 1 acetone.
  4. Use a hole punch to cut disks from the chromatogram and perform the disk diffusion assay to determine which fraction contains the active ingredients.