Types and Applications of Ion-Pair Reagents in Liquid Chromatography
In liquid chromatography, in order to achieve better retention of the target peak when analyzing ionic compound samples, ion-pair reagents (IPRs) are considered during method development to increase retention and improve peak shape. This is also known as ion-pair chromatography (IPC).
Types of Ion-Pair Reagents
Based on the historical status and physical & chemical properties, ion-pair reagents can be classified into the following types:
Traditional Ion-Pair Reagents
These are the earliest and still most commonly used types, including the following two categories:
• Cationic Ion-Pair Reagents: Commonly include organic amines or ammonium salts with varying alkyl chain lengths, such as triethylamine and tetrabutylammonium bromide. Adding these to the mobile phase can enhance the retention of anionic analytes.
• Anionic Ion-Pair Reagents: Common examples are alkyl sulfonates, such as sodium dodecyl sulfate and sodium heptanesulfonate. Adding these to the mobile phase can enhance the retention of cationic analytes.
Volatile Ion-Pair Reagents
As more laboratories are equipped with liquid chromatography-mass spectrometry (LC-MS) systems, traditional ion-pair reagents lack volatility and are incompatible with mass spectrometry (MS). Additionally, ion-pair reagents added to increase analyte retention may also affect the ionization of analytes, requiring the use of ion-pair reagents compatible with MS.
Small molecule organic acids, such as formic acid and acetic acid, are commonly used volatile ion-pair reagents in LC-MS analysis. They can influence the retention behavior of compounds sensitive to pH conditions, alter their charge states, and provide protonation effects. Meanwhile, these organic acids can suppress the activity of residual silanol groups on the chromatographic column, benefiting peak shape.
When analytes are unstable in acidic conditions or do not separate well under low pH conditions, more neutral conditions may be required. In such cases, volatile salts like ammonium formate and ammonium acetate are used. These salts' pH can accommodate both positive and negative ion modes in detection. It is important to pay attention to the solubility of these salts in organic solvents, especially acetonitrile.
In peptide and amino acid analyses, the use of perfluorocarboxylic acids (such as trifluoroacetic acid (TFA), heptafluorobutyric acid (HFBA), and pentafluoropropionic acid (PFPA)) has increased. These reagents can also suppress silanol activity and improve peak shape. TFA is widely used due to its good water solubility and low cutoff wavelength. HFBA, with its selectivity differing from TFA and PFPA, is also used in conjunction with MS, but it can somewhat contaminate the mass spectrometer.
Chaotropic Agents
Chaotropic agents are salts that can disrupt hydrogen bonding between water molecules and increase their disorder. In ion-pair chromatography, common examples include perchlorates, hexafluorophosphates, and tetrafluorobromates. Adding suitable chaotropic agents can enhance the hydrophobicity of ionic analytes, thereby improving their retention.
Precautions
1) In certain cases, it is necessary to strictly control the pH of the mobile phase and the column temperature during detection. Additionally, for mobile phases containing volatile ion-pair reagents, it is recommended to prepare them below the solvent surface to avoid reproducibility issues.
2) When using ion-pair reagents in the mobile phase, simultaneous appearance of positive and negative peaks may be observed, which can interfere with method development or routine analysis. Such issues are primarily due to the use of impure ion-pair reagents, buffering salts, or other mobile phase additives. Impurities in ion-pair reagents can also affect the retention time of analytes.
3) When using mobile phases containing ion-pair reagents, especially those with strong hydrophobicity, column equilibration may be slow. Insufficient equilibration time can result in retention time drift and unstable results. It is generally recommended to use isocratic elution conditions for mobile phases containing ion-pair reagents because gradient elution may cause baseline fluctuations and retention reproducibility issues. To ensure the chromatographic column reaches a good equilibrium state, it is best to equilibrate at a low flow rate overnight before use.
4) Weaker ion-pair reagents, such as TFA, PFPA, and elution agents do not slow down the column equilibration process. Typically, equilibrating the mobile phase for 10-20 column volumes is sufficient to achieve equilibrium.
5) When a sample contains both acidic and basic compounds, both types of IPC reagents may be effective, but both should not be added to the mobile phase simultaneously. These reagents may form ion pairs between them, which can counteract their net effects on separation, and using both reagents simultaneously complicates method development.
6) When selectively increasing the retention of basic solutes, the preferred IPC reagent should be alkyl sulfonates, while tetrabutylammonium salts are used when increasing the retention of acidic solutes.
