In Chapter 1, we learned how the instrument works. In Chapter 2, we will learn how to choose the chemistry. The “Separation Mode” determines what kind of column and solvents you use based on the properties of your sample.
The Golden Rule: “Like Dissolves Like.” Polar compounds prefer polar phases, and non-polar compounds prefer non-polar phases.
90% of all HPLC analysis is done using Reversed Phase Chromatography (RPC). It is critical to understand why.
“Non-Polar Stationary / Polar Mobile”
Non-polar (gray) gets stuck in the chains.
“Polar Stationary / Non-Polar Mobile”
Note: Rarely used today except for water-sensitive samples or isomers.
You are analyzing a highly polar drug (like Vitamin C) using a C18 column (Reversed Phase). When will it elute?
✓ Correct! Since the C18 column is Non-polar and Vitamin C is Polar, they repel each other. The Vitamin C stays in the water (mobile phase) and rushes out quickly.
✗ Incorrect. This would happen if the drug was non-polar.
Sometimes, polarity isn’t enough to separate complex mixtures like proteins or polymers. That’s when we use physical size or electric charge.
Separates based on molecular size (hydrodynamic volume). It works like a sieve.
Separates based on electric charge.
When you start a new method, look at the molecular weight and polarity of your sample.
| Sample Type | Recommended Mode | Typical Column |
|---|---|---|
| Neutral, Organic (Non-polar) e.g., Ibuprofen, Steroids |
Reversed Phase (RPC) | C18, C8 |
| Ionic / Salts e.g., Amino Acids, Proteins |
Ion Exchange (IEX) | SAX (Anion), SCX (Cation) |
| High Molecular Weight e.g., Plastics, Antibodies |
Size Exclusion (SEC) | Silica or Polymer Gel |
| Chiral Isomers e.g., R- vs S-Enantiomers |
Chiral Chromatography | Amylose/Cellulose based |
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