The use of ion-pair reagents can be a handy tool in improving retention and peak shape of ionizable compounds in HPLC (typically reversed phase). Here’s how:
Overview
Ion-pairing agents are compounds that contain both an ionic functional group and a hydrophobic portion, such as a hydrocarbon chain. The most common ion-pairing agents are sulfonic acid derivatives such as hexane-, heptane-, octane-sulfonic acids, quaternary ammonium salts such as tetramethyl- or tetrabutylammonium hydroxide, and volatile agents such as trifluoroacetic acid and triethylamine.
Interaction Mechanisms
As the name implies, ion-pairing agents may exert their principle effect by interacting with any counter-ions in solution, thus forming pseudo-neutral complexes:
Another possible type of interaction can relate to the hydrophobic portions of the ion-pairing agents partitioning into the alkyl bonded phase of the column, forming transitory ion-exchange sites:
Common Drawbacks
It is sometimes unclear which mechanism is the true or primary occurring, and in most cases it is likely a combination of both in various degrees. This could partly relate to the notorious reputation of some ion-pairing methods yielding inconsistent and irreproducible results. Lengthy equilibration and general mass spec incompatibility are among some of the other commonly noted drawbacks. The additional cost of the ion-pair reagent itself may also be a subtle disadvantage that is frequently overlooked.
New Technologies
Recent developments in column technology can in some cases overcome the common drawbacks of ion-pair reagents while meeting the chromatographic challenges:
- pH stable stationary phase technology can allow for improved retention and peak shape of basic analytes by running at basic mobile phase pHs controlled with buffers instead of utilizing ion-pair reagents.
- Hydrophilic Interaction Liquid Chromatography (HILIC) stationary phases are improved.
- HILIC method development techniques can allow for improved retention of polar analytes under mass spectrometry compatible conditions.
Ideal Ion-Pairing Scenarios
While some of the utility of ion-pairing solutions may be usurped by newer technologies and techniques, there still remain cases where ion-pairing remains the most useful:
- Samples with a wide range of polar, non-polar, and ionizable compounds that would not otherwise be characterized under the same conditions
- Ionized compounds lacking a chromophore for UV detection (some ion-pair conjugates absorb UV where the analyte itself would not)
- Method development familiarity and abundance of literature to reference and extrapolate.
Related resources:
• Ion-Pairing pH 8.3 LC/MS Oligonucleotides
• Trityl-on RNA and DNA Purification
• Basic Compounds by LC/MS Using a High pH Mobile Phase
• Low Molecular Weight Separation by GFC
• Synthetic Oligonucleotides by LC/MS
• pH LC Poster
• HPLC Column Care Guide