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A major challenge in the development of a chromatographic method is the rational selection of experimental conditions that can provide adequate resolution in a reasonable run time.
To increase the retention of ionic and polar analytes with a reversed phase column, it is important to choose an oppositely charged ion-pair reagent (IPR) with an appropriate hydrophobic tail. As longer alkyl chains are retained better by the stationary phase, too strong of an interaction (> C12) may lead to unreasonable run times and long column equilibration periods.
How to choose the most suitable ion-pair reagent?
- Select a column such as commonly used endcapped octadecylsilyl (ODS).
- If a mixture of ionic and nonionic compounds is present, separate nonionic compounds from each other first.
- Choose an IPR that will selectively bind to the analyte of interest.
- Choose the alkyl chain length (through a process of elimination) that results in the best separation.
- Begin at the lowest effective mobile phase concentration of the IPR (ideally 0.005 M) and increase in small increments to enhance retention slightly and optimize separation.
- Note that high concentrations of (very) hydrophobic IPRs should not be used.
Choice of mobile phase composition and pH
Use only HPLC-grade water and chromatography-grade reagents (called organic modifiers) to mobilize the analyte from the stationary phase. A trade-off exists between eluent strength and the analyte’s solubility in the mobile phase. Higher concentrations of the organic modifier and polarity (acetonitrile > methanol > tetrahydrofuran) affect this trade-off.
The pH of the mobile phase is extremely important for modulating the extent of ionization of ionizable (ionogenic) analytes. To ensure that basic analytes remain positively charged, the pH should be at least two units below its pKa. For acidic analytes, the pH should be at least two units above its pKa. Aqueous buffers (commonly phosphate, acetate, or formate) in the mobile phase are used to regulate pH.
- Choose mobile phase compositions and concentrations that yield the best separation. Examples from the literature may help to gauge a starting point.
- Adjust the pH of the mobile phase in small increments to maximize resolution. Monitor carefully.
- Scan the mobile phase for turbidity or crystal formation, which may interfere with the analyte.
- Fine-tune the elution mode (isocratic vs. gradient) for optimal separation.
Column wash and storage
Rinse the column with an organic solvent such as acetonitrile to remove the IPR from the stationary phase as much as possible and to prevent agglomeration. It may be a good idea to dedicate columns to specific ion-pairing applications (i.e. strongly hydrophobic IPRs) to prevent problems with reproducibility.
- IPRs that weakly interact with the stationary phase are usually chosen for gradient runs, so that the eluent concentration can more easily modulate its stationary phase concentration. Moreover, the concentration of the IPR should be maintained consistent during the gradient run.
- A porous graphitized carbon (PGC) stationary phase can also be considered to optimize separation, gaining popularity because of its pH stability range of 1 – 13 and high density. 
- Temperature should also be monitored and analyzed as it may affect analyte retention, efficiency, and selectivity.
- Pereira, L. Porous Graphitic Carbon as a Stationary Phase in HPLC: Theory and Applications. Journal of Liquid Chromatography & Related Technologies 2008, 31(11-12), 1687-1731.