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 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
Ionization Suppression Effect
Strong Acids and Salts 100% Ionized
IEC Mechanism
Anion Exchange
Ion Exchange
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
Cation Exchange Resin
Anion Exchange Resin
Ion Exchange Resin Styrene-divinylbenzene
Ion Exchange Groups Strong anion exchanger – -NR 3 + Weak anion exchanger – -NR 2 H + Strong cation exchanger –-SO 3 - Weak cation exchanger –-CO 2 -
pH Effect
Ion Exchange Packings
Krebs Cycle Acids by Ion Exchange Chromatography
Ion Exchange Summary
Ion Chromatography Originally meant IEX + suppressor device + conductivity detector Inorganic cations and anions Organic acids, amines carbohydrates
Ion Chromatography Schematic
Inorganic Anions by Ion Chromatography
Limitations of Ion Exchange Less Efficient Columns Lot to lot column reproducibility poor Gradients require long equilibration Limited choice of packings Only ionic analytes
Ion Analysis
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
Reversed-phase
Ion Pair Mechanism
IPC Mechanism - Key
Optimizing IPC Separation
Effect of Organic Modifier
Effect of Ion Pair Reagent Concentration
Effect of Added Salt
Ion Pair Reagent Chain Length