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HPLC Analysis of Ionic Compounds

HPLC Analysis of Ionic Compounds. Nicholas H. Snow Seton Hall University. HPLC of Ionic Compounds. Ion Suppression Ion Exchange Chromatography (IEX or IEC) Ion Pair Chromatography (IPC). Weak Acid Equilibrium. LeChatelier Principle. DO NOT chromatograph at pH = pKa !!. Ion Suppression.

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HPLC Analysis of Ionic Compounds

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  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 Salts100% Ionized

  8. IEC Mechanism

  9. Anion Exchange

  10. Ion Exchange

  11. 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 ResinStyrene-divinylbenzene

  16. Ion Exchange Groups • Strong anion exchanger • -NR3+ • Weak anion exchanger • -NR2H+ • Strong cation exchanger • -SO3- • Weak cation exchanger • -CO2-

  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

  29. Ion Pair Mechanism

  30. 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

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