Overview
This lecture introduces ion pair chromatography in HPLC, covering its purpose, mechanisms, optimization strategies, appropriate mobile phases, and key limitations.
Introduction to Ion Pair Chromatography
- Ion pair chromatography is used in HPLC to separate highly polar or charged analytes that regular reverse phase cannot handle effectively.
- Suitable when analytes exist in ionic forms, such as amino acids with ionic side chains.
Comparison with Reverse Phase & pH Adjustment
- Reverse phase HPLC uses a non-polar stationary phase (e.g., C18 column) with a polar mobile phase.
- Adjusting pH can suppress ionization but is problematic with mixtures of acids and bases or when extreme pH is needed.
- Extreme pH can degrade HPLC columns, making pH adjustment risky for strongly ionized analytes.
How Ion Pair Chromatography Works
- An ion pairing reagent (with hydrophobic tail and ionic head) is added to the mobile phase.
- Common agents: alkyl sulfonic acid salts (for bases) and quaternary alkyl amine salts (for acids).
- The hydrophobic tail binds to the stationary phase; the ionic head pairs with analyte ions, aiding separation.
- In effect, the C18 column is modified to have ionic sites, increasing retention of charged analytes.
Optimizing Retention Time
- Increasing the hydrophobic chain length (e.g., using octane instead of hexane) increases retention time.
- Adjusting the concentration of the ion pairing reagent changes retention; more reagent increases retention until saturation.
- Over-saturating with reagent can neutralize analytes, preventing effective separation.
Choice of Mobile Phase
- Mobile phases should be polar (water, methanol) to ensure solubility of the ion pairing agent.
- Less polar solvents (e.g., acetonitrile, THF) decrease the solubility and effectiveness of the ion pairing reagent.
Limitations and Considerations
- Column equilibration with ion pair reagents is much slower than with reverse phase; requires 10-20x typical mobile phase volume.
- Once a column is used for ion pairing, it is difficult or impossible to fully clean, making the column dedicated to that method.
- Excessive ion pairing reagent or using for neutral analytes decreases separation efficiency.
Key Terms & Definitions
- Ion Pair Chromatography — HPLC method using an ion pairing reagent to improve retention/separation of charged analytes.
- Ion Pairing Reagent — Molecule with a hydrophobic tail and ionic head that forms temporary ion pairs with analytes.
- Retention Time — Time an analyte spends on the column before eluting, affected by ion pair reagent and chain length.
- Saturation Point — Maximum useful concentration of ion pairing reagent, beyond which separation declines.
Action Items / Next Steps
- Review previous lectures, especially on reverse phase chromatography.
- Ensure understanding of column sensitivity to pH and mobile phase choice.
- Prepare for the next lecture, potentially on normal phase chromatography.