Paper, TLC, Gel Permeation and Ion Exchange Chromatography I B.Sc I M.Sc I CSIR NET I GATE Exams

Introduction

This tutorial provides a comprehensive overview of various chromatography techniques, essential for students preparing for the CSIR NET Life Science exam. Understanding these techniques is vital for analyzing biological samples and can significantly impact exam performance.

Step 1: Understanding Chromatography

• Chromatography is a separation technique used to analyze, identify, and purify compounds within a mixture.
• It relies on the differential adsorption of substances on a stationary phase while being carried by a mobile phase.
• Common types of chromatography include:
  • Paper chromatography
  • Thin-layer chromatography (TLC)
  • Gel permeation chromatography (GPC)
  • Ion exchange chromatography

Step 2: Paper Chromatography

• Used primarily for separating small amounts of substances, particularly dyes or pigments.
• Procedure:
  1. Draw a line on chromatography paper to mark the starting point.
  2. Apply small dots of the sample along the line.
  3. Immerse the edge of the paper in a solvent without submerging the sample spots.
  4. Allow the solvent to travel along the paper, carrying the samples with it.
  5. Once the solvent has traveled, remove and dry the paper.
• Tip: Ensure the solvent does not touch the sample spots initially to prevent smudging.

Step 3: Thin-Layer Chromatography (TLC)

• A more advanced technique than paper chromatography, suitable for separating a variety of compounds.
• Procedure:
  1. Coat a glass or plastic plate with a thin layer of adsorbent material (like silica gel).
  2. Apply spots of the sample onto the adsorbent layer.
  3. Place the plate in a developing chamber with a solvent.
  4. Allow the solvent to ascend the plate, separating the components.
  5. Visualize the separated components using UV light or staining.
• Common Pitfall: Overloading the sample can lead to poor resolution.

Step 4: Gel Permeation Chromatography (GPC)

• Also known as size-exclusion chromatography, this technique separates molecules based on size.
• Procedure:
  1. Prepare a column packed with porous beads.
  2. Inject the sample into the column.
  3. Smaller molecules enter the pores of the beads and take longer to travel through the column, while larger molecules pass quickly.
  4. Collect fractions as they elute.
• Real-World Application: GPC is commonly used in polymer science to determine molecular weight distributions.

Step 5: Ion Exchange Chromatography

• This technique separates ions and polar molecules based on their affinity to the ion exchanger.
• Procedure:
  1. Prepare a column with resin that has charged groups.
  2. Load the sample onto the column.
  3. Ions in the sample will displace ions bound to the resin, allowing for separation.
  4. Elute with a buffer solution to collect the separated ions.
• Practical Tip: Adjusting the pH of the buffer can significantly affect separation efficiency.

Conclusion

By mastering the principles and procedures of chromatography techniques, you will enhance your understanding of biochemical analysis relevant for the CSIR NET Life Science exam. Focus on the key aspects of each technique, practice sample separations, and review the practical applications to solidify your knowledge. Consider enrolling in preparatory courses for more in-depth learning and practice.