HPLC Analysis of Ionic Compounds

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 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 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 -NR3+

Weak anion exchanger -NR2H+

Strong cation exchanger -SO3- Weak cation exchanger

-CO2- 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

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

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