Use of Ion-Pairing Reagents

The addition of millimolar concentrations of most salts to the mobile phase has the effect of decreasing the retention time of a protein sample and improving peak shapes. This effect can be attributed to suppression of the interaction between polar groups on the solute and silanol groups. Other effects such as ion pairing can also operate and these will be described here. Higher salt concentrations may result in increased retention 

The concept of ion pairing refers to the ionic association between ions added to the mobile phase and oppositely charged ionized groups of the sample molecule, i.e.. 

This pictorial representation of a possible mechanism of action of a polar ion-pairing reagent has allowed prediction of what anions will be useful in decreasing the retention of a given protein. Alternatively, a nonpolar anion such as hexane sulfonate could be expected to associate with an ammonium ion present in the sample, with an increase in the nonpolar surface area and hence retention time. For example, apolipopro-tein C-III, which is readily eluted by an organic solvent gradient from a Cig column in the presence of phosphate anions, is retained indefinitely if butanesulfonate is used as the counterion. 

In Table IV, a list of ion-pairing reagents is shown and illustrates the variety of combinations which can be achieved. In addition to anionic 

Anionic and Cationic Counterions Useful in Reversed Phase HPLC 

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Ionic or partially ionic compounds can be chromatographed on reversed-phase columns through the use of ion-pairing reagents. These reagents are typically long-chain alkyl anions or cations that, in dilute concentrations, can increase the retention of analyte ions. For cationic compounds, C5 to CIO alkyl sulfonates are commonly used combinations may also be used... 

Since tetracyclines have several pKa values and can exist as zwitterions, one must always consider the control of the mobile-phase pH in the development of an analytical method. The property of tetracyclines to exist in the form of ions also makes the use of ion-pairing reagents attractive, and several methods have been developed that utilize this approach (289, 299, 303, 307). 

Highly concentrated salts (> 0.2 M) and caustic mobile phases can reduce pump seal efficiency. The lifetime of injector rotor seals also depends on mobile phase conditions, e.g., operation at high pH. In some cases, prolonged use of ion pair reagents has a lubricating effect on pump pistons that may produce small leaks at the piston seal. Some seals do not perform well with certain solvents. 

Eor the highly polar biomolecules, RP phases have several shortcomings, namely, low retention, often a need for derivatization, requirement for the use of ion-pairing reagents, analysis time too short, lack of robustness, and poor quantitation. More recently, systems have been developed to separate and quantify this class of compounds. 

In RPC, the retention mechanism is based on an apolar stationary phase and the relative hydrophobidty of the analyte. The main advantages are good reprodudbihty, a wide scope, effective separation at low buffer concentrations, high species resolution, and extended flexibility. The use of ion-pairing reagents extends the scope of reversed-phase chromatography to include ionic analytes [15]. 

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