How Well Do You Know Ion-Pair Reagents?

Hello, I believe that those of you with laboratory experience are no strangers to the term “ion pair reagent“. It frequently appears in our daily tests and has indeed helped us solve many tricky problems in routine testing. In the following, I will briefly introduce this “great contributor” who has been of great help in our daily testing.

01 Principle

When using liquid chromatography to analyze samples with strong ionization ability, the retention time of the sample on the reverse phase column is very short or not retained at all. In this case, the corresponding ion pair reagent should be added to bind the ions in the analyte, forming a molecule that can be retained on the column.

02 Types of Ion-Pair Reagents

The type and concentration of ion-pair reagents used can have a significant impact on the separation efficiency. The selection of ion-pair reagents depends on the properties of the sample being analyzed.

● If the solute being separated is a strong acid or strong base, the ion-pair reagent can be a strong acid or strong base, or a weak acid or weak base.

● If the solute being separated is a weak acid or weak base, the ion-pair reagent used must be a strong base or strong acid.

● For mixed samples with significantly different solute structures, there are no special requirements for the selection of ion-pair reagents.

● The difference in the hydrophobicity of the ion-pair reagents can be adjusted by changing the concentration of the ion-pair reagent and the organic solvent concentration to achieve satisfactory separation efficiency.

1. For acidic compounds: tetrabutylammonium hydroxide (10% aqueous solution), tetrabutylammonium bromide, dodecyltrimethylammonium chloride.
2. For alkaline compounds: sodium pentanesulfonate, sodium hexanesulfonate, sodium heptanesulfonate, sodium octanesulfonate, sodium decanesulfonate.
3. Other ion-pair reagents: sodium perchlorate, trifluoroacetic acid, heptafluorobutyric acid, etc.

Commonly used ion-pair reagents and their CAS numbers:

1-sodium pentanesulfonate CAS: 22767-49-3

1-sodium hexanesulfonate CAS: 2832-45-3

1-sodium heptanesulfonate CAS: 22767-50-6

1-sodium octanesulfonate CAS: 5324-84-5

1-sodium decanesulfonate CAS: 13419-61-9

03 Ion-pair reagent selection steps:

1. Choose a suitable chromatographic column, often a C18 column.
2. Prepare the mobile phase using only HPLC grade water and chromatography grade reagents.
3. Select the mobile phase composition and concentration that provide the best separation.
4. If there are non-ionized substances in the sample, optimize separation before attempting ion-pair separation.
5. Choose a suitable series of ion-pair reagents to provide the corresponding ion pairs: use acidic ion-pair reagents for acidic analytes; use basic ion-pair reagents for basic analytes.
6. Select the ion-pair reagent that provides the best separation by comparison.
7. Once the ion-pair reagent has been determined, adjust the pH of the mobile phase to maximize separation; because small pH changes have a significant impact on retention and selectivity, adjust the pH carefully and slightly.
8. Ideally, the ion-pair reagent concentration in the mobile phase should be 0.005 M, but slightly increasing the ion-pair reagent concentration may slightly increase retention and optimize separation.

Advantages:

• Simple preparation of buffer solutions.
• Using reverse phase chromatography columns, there are many options for carbon chain length, which can increase retention time and improve separation properties.
• Even with larger ions, clear peak shapes can be obtained while maintaining high reproducibility of results.
• Shorter separation times compared to ion exchange.
• Suitable for separation of complex mixtures (different from ion suppression).
• Capable of separating both ionized and non-ionized analytes.
• Effectively improves peak shapes.

Drawbacks:

1. Ion-pair reagents cause significant damage to the chromatographic column. Ion-pair reagents can cause irreversible damage to the chromatographic column and irreversibly adsorb to the stationary phase, thereby affecting the active sites of the stationary phase. For example, in the case of an octadecylsilyl-bonded silica chromatographic column, this reaction has a significant impact on the column, and it is difficult to wash the ion-pair reagent off the column, which greatly shortens the service life of the chromatographic column.
2. Experimental conditions are not stable. The type and concentration of ion-pair reagents have a direct impact on the separation effect. In addition, ion-pair reagents are sensitive to pH, and high accuracy is required when preparing the mobile phase. Otherwise, it will directly affect the repeatability and reproducibility of the experiment.
3. The equilibration time for experimental chromatographic columns using ion-pair reagents is relatively long, and it is generally recommended to equilibrate with a small flow rate overnight.