Lecture Notes: Column Chromatography

Introduction to Column Chromatography

• Column chromatography is a powerful separation technique.
• Widely used in chemistry, biology, and pharmaceutics.
• Useful for separating compounds that cannot be purified by other methods like distillation, sublimation.

Basic Concepts

• Stationary Phase: Usually silica gel, where compounds are adsorbed.
• Mobile Phase: Solvent or solvent mixture used to separate the compounds.
• Adsorption: Phenomenon where compounds are physically or chemically attached to a material (e.g., silica gel).

Types of Chromatography

• Adsorption Chromatography is classified into:
  • Thin Layer Chromatography (TLC)
  • Column Chromatography

Performing Column Chromatography

• The mixture used contains a yellow compound (Component A) and a red compound (Component B).
• Involves a glass tube packed with silica gel (stationary phase).
• Compounds separated based on adsorption and polarity.

Preliminary Step: Thin Layer Chromatography (TLC)

• Purpose: To obtain a suitable mobile phase for separation.
• Suggested the use of an 80/20 mixture of hexane and ethyl acetate.

Experimental Procedure

1. Prepare the Column

   • Fix column to a stand.
   • Add cotton to the bottom to prevent silica gel from passing through.
   • Make a slurry of silica gel with hexanes.
   • Load slurry into the column using a funnel.
   • Wash sides of the column to remove stuck silica gel.

2. Load the Sample

   • Dissolve compounds in chloroform.
   • Add silica gel to form a fine powder.
   • Load this compound mixture into the column.
   • Add sand on top to prevent disturbance during mobile phase addition.

3. Elution Process

   • Use 80/20 hexane and ethyl acetate as the mobile phase.
   • Collect eluents in test tubes as compounds are separated.
   • First eluent: Yellow compound eluted faster indicating it is less polar.
   • Increase polarity to 50/50 to elute the red compound.

Observations

• Yellow compound (2-nitroaniline) moves faster, indicating less polarity compared to red (methyl red).
• Demonstrates separation based on polarity and adsorption.
• Column chromatography is quantitative, allowing separation of grams of compounds, unlike TLC which is qualitative and for milligram quantities.

Conclusion

• Column chromatography effectively separates compounds based on polarity and adsorption.
• Utilizes silica gel as a polar adsorbent.
• Differences observed in TLC are mirrored in column chromatography.

Next Steps

• Students are encouraged to practice the technique on new compounds.
• Upcoming lessons will cover other techniques.

End of Lecture