Column chromatography
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Column chromatography

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Column chromatography Presentation Transcript

  • 1. COLUMN CHROMATOGRAPH PROCEDURE Hussain Ali 11-ARID-441
  • 2. o Plug a Pasteur pipet with a small amount of cotton. o Use a wood applicator stick to tamp it down lightly. o Take care that you do not use either too much cotton or pack it too tightly. You just need enough to prevent the adsorbent from leaking out. o Add dry silica gel adsorbent, 230-400 mesh. o Usually the jar is labeled "for flash chromatography." o One way to fill the column is to invert it into the jar of silica gel and scoop it out... o Then tamp it down before scooping more out o Another way to fill the column is to pour the gel into the column using a 10 mL beaker Step 1: Prepare the column
  • 3. Step 1: Prepare the column
  • 4. Step 1: Prepare the column
  • 5. Step 1: Prepare the column When properly packed, the silica gel fills the column to just below the indent on the pipet. This leaves a space of 4-5 cm on top of the adsorbent for the addition of solvent. Clamp the filled column securely to a ring stand using a small three-pronged clamp.
  • 6. Step 2: Pre-elute the column  Add solvent to the top of the silica gel  The solvent flows slowly down the column  Monitor the solvent level, both as it flows through the silica gel and the level at the top  But make sure it does not go below the top of the silica  When the bottom solvent level is at the bottom of the column  The pre-elution process is completed and the column is ready to load
  • 7. Step 2: Pre-elute the column
  • 8. Step 3: Load the sample onto the silica gel column  The sample to be purified is dissolved in a small amount of solvent, such as hexanes, acetone, or other solvent.  This solution is loaded onto the column  In this case, if you use the wet method of column loading  It is critical that you only use a few drops of solvent to load the sample
  • 9. Step 3: Load the sample onto the silica gel column
  • 10. Step 4: Elute the column  Force the solvent through the column by pressing on the top of the Pasteur pipet with a pipet bulb  The colored bands will travel down the column as the compound is eluted  Only force the solvent to the very top of the silica  Do not let the silica go dry  Add fresh solvent as necessary.  The colored bands will travel down the column as the compound is eluted.
  • 11. Step 4: Elute the column
  • 12. Step 5: Elute the column with the second elution solvent  As soon as the colored compound begins to elute, the collection beaker is changed  The process is complicated if the compound is not colored  In such experiments, equal sized fractions are collected sequentially  carefully labelling is necessary for later analysis
  • 13. Step 5: Elute the column with the second elution solvent
  • 14. Step 6: Analyze the fractions If the fractions are colored then you can simply combine like-colored fractions,