Introduction: What is Reverse Phase Chromatography?

Reverse phase chromatography (RPC) is one of the most widely used techniques in analytical and preparative separation science, especially for biomolecules, pharmaceuticals, and peptides. This technique is based primarily on hydrophobic interactions, and not molecular weight, which is a common misconception.

✅ Correct Principles of Reverse Phase Chromatography

Let’s take a closer look at each of the correct statements in the question:

(1) The solutes elute with decreasing order of polarity

Absolutely correct. In reverse phase chromatography:

• Polar molecules elute first because they have less interaction with the non-polar stationary phase.

• Non-polar molecules elute later due to stronger hydrophobic interactions with the stationary phase.

(2) The stationary phase surface covering the silica particles involves non-polar functional groups

Yes! Reverse phase chromatography typically uses silica beads modified with non-polar alkyl chains, such as:

• C18 (octadecyl) or C8 (octyl) groups

• These hydrophobic ligands interact with non-polar solutes more strongly.

(4) The pH of the mobile phase has a profound influence on retention, selectivity, and separation

Correct. The pH affects:

• Ionization states of solutes

• Charge-based interactions

• Therefore, pH adjustment is crucial to optimize separation and peak resolution in RPC.

❌ (3) INCORRECT: The solutes elute with increasing order of molecular weight

This is false because molecular weight is not the primary factor influencing elution in reverse phase chromatography.

• Polarity and hydrophobicity, not size, determine retention time.

• For example, a small hydrophobic molecule may elute after a larger, more polar molecule.

If size-based separation is the goal, techniques like gel filtration (size-exclusion chromatography) are more appropriate.

🔬 Applications of Reverse Phase Chromatography

• Peptide and protein purification

• Pharmaceutical compound analysis

• Quality control in food and cosmetics

• Environmental testing for organic pollutants

🧪 Summary

📌 Final Takeaway:

Reverse phase chromatography separates molecules based on polarity, not size. Always remember that molecular weight is irrelevant in RPC, making option (3) the incorrect statement in this context.

Would you like a diagram or infographic for this concept?