1. HPLC Analysis of Ionic Compounds Nicholas H. Snow Seton Hall University

2. HPLC of Ionic Compounds Ion Suppression Ion Exchange Chromatography (IEX or IEC) Ion Pair Chromatography (IPC)

3. Weak Acid Equilibrium

4. LeChatelier Principle DO NOT chromatograph at pH = pKa !!

5. Ion Suppression Weak acid - Use pH 2-3 –acetic or phosphoric acid Weak base - Use pH 8 –ammonium hydroxide, sodium carbonate Ionization is suppressed Neutral molecules chromatographed by reversed phase LC

6. Ionization Suppression Effect

7. Strong Acids and Salts 100% Ionized

8. IEC Mechanism

9. Anion Exchange

10. Ion Exchange

12. Ion Exchange Parameters Increasing pH –Increases ionization of acids - retained longer –Decreases ionization of bases - elute faster Increasing buffer strength –More competition for exchange sites - decreased retention Increasing Temperature –Equilibrium favors mobile phase - retention decreases

13. Cation Exchange Resin

14. Anion Exchange Resin

15. Ion Exchange Resin Styrene-divinylbenzene

16. Ion Exchange Groups Strong anion exchanger – -NR 3 + Weak anion exchanger – -NR 2 H + Strong cation exchanger –-SO 3 - Weak cation exchanger –-CO 2 -

17. pH Effect

18. Ion Exchange Packings

19. Krebs Cycle Acids by Ion Exchange Chromatography

20. Ion Exchange Summary

21. Ion Chromatography Originally meant IEX + suppressor device + conductivity detector Inorganic cations and anions Organic acids, amines carbohydrates

22. Ion Chromatography Schematic

23. Inorganic Anions by Ion Chromatography

24. Limitations of Ion Exchange Less Efficient Columns Lot to lot column reproducibility poor Gradients require long equilibration Limited choice of packings Only ionic analytes

25. Ion Analysis

26. Ion Pair Chromatography Adjust pH to fully ionize analyte Add ion pair former of opposite charge in mobile phase –acids (anions): tetraalkyl ammonium salt –bases (cations): alkyl sulfonic acids Use standard reversed phase column

27. Reversed-phase

28. Ion Pair Mechanism

31. IPC Mechanism - Key

32. Optimizing IPC Separation

33. Effect of Organic Modifier

34. Effect of Ion Pair Reagent Concentration

35. Effect of Added Salt

36. Ion Pair Reagent Chain Length