HPLC Analysis of Ionic Compounds Nicholas H Snow

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

Le. Chatelier Principle DO NOT chromatograph at p. H = p. Ka !!

Ion Suppression • Weak acid - Use p. H 2 -3 – acetic or phosphoric acid • Weak base - Use p. H 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

Ion Exchange Parameters • Increasing p. H – 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 -

p. H 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 p. H 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

Ion Pair Mechanism

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