Add Ion Pair Reagents, Don’t Forget These Questions

Ion pairing reagents are a common class of laboratory reagents that can be added to a reversed-phase chromatography mobile phase to enhance the retention of ionic samples. The use of ion-pairing reagents can improve the ion-exchange capacity of the surface of the hydrophobic bonded phase, allowing better control of the retention of acidic or basic samples under mild mobile phase pH conditions.

The ion-pairing reagent is adsorbed on the column bed through the interaction of the hydrophobic part (commonly heptyl and dodecyl) on the reagent with the stationary phase of the chromatographic column, so that the stationary phase has a certain ion exchange capacity, thereby enhancing the ion-pairing capacity. Retention of oppositely charged analytes.

For example, the common sodium octane sulfonate

The ion-pairing reagent is added to the mobile phase, but it is in the stationary phase of the column where it comes into play. Here is an example of sodium octane sulfonate. The ion-pairing reagent added to the mobile phase enters the column with the mobile phase, and the ion-pairing reagent then equilibrates between the stationary phase and the mobile phase of the column.

According to the principle of same-sex attraction, the carbon chain of 8 carbon atoms in sodium octane sulfonate will be adsorbed by the stationary phase of the weakly polar chromatographic column, while the sulfonic acid ions of greater polarity are exposed on the stationary phase of the chromatographic column. in the mobile phase outside. The column stationary phase that adsorbs the octane sulfonate ion is negatively charged, allowing it to adsorb and retain positively charged compounds.

In addition to improving the retention of target compounds, ion-pairing reagents also improve peak shape.

The matrix of the stationary phase of the reversed-phase column is mainly silica gel, and there are a lot of hydroxyl groups on the surface of silica gel. During the preparation of the column, the grafted stationary phase and the end-capping process will consume some hydroxyl groups, but there are still a large number of hydroxyl groups remaining on the surface of the silica matrix. The interaction of the silanol groups on the silica matrix surface with the compound can cause tailing peaks.

As mentioned earlier, the ion-pairing reagent pair will fill the gap of the stationary phase, and this part of the ion-pairing reagent can well shield the residual hydroxyl groups on the surface of the silica gel. Thus, the interaction between the target compound and the hydroxyl group on the silica surface is prevented, and the tailing peak is improved.

Commonly used ion pair reagents

Action on acidic compounds:

Tetrabutylammonium hydroxide, tetrabutylammonium chloride (tetrabutylammonium bromide), dodecyltrimethylammonium chloride.

Action on basic compounds:

Various alkyl sulfonates such as sodium octane sulfonate

Trifluoroacetic acid, pentafluoropropionic acid, heptafluorobutyric acid and other perfluoro-substituted linear organic acids. (Perfluoro-substituted linear organic acids are special, both ion-pairing reagents and pH-adjusting strong acids).

However, the use of ion-pairing reagents will inevitably cause some problems. Here is an introduction.

Possible problems and solutions

The column equilibration time is long

Compared with the reversed-phase chromatography method that does not add ion-pairing reagents, ion-pairing chromatography involves a variety of complex equilibration processes, so in general, the equilibration time of chromatographic columns using ion-pairing reagents is relatively long. Since the concentration of ion-pair reagents is 2-5mmol/L or even lower, they should be adsorbed on the surface of the packing materials of the reversed-phase chromatographic column, and the surface concentration is 0.5-2μmol/m2. A 4.6×250mm chromatographic column has about 3 g packing materials, 2 mmol of ion-pairing reagent is required for complete column equilibration. At a mobile phase concentration of 2 mmol, one liter of mobile phase is required for equilibration. These are of course extreme conditions, but it is normal to equilibrate a column with a few hundred milliliters of mobile phase.

Due to the long equilibration time, choosing a gradient elution method when using ion-pairing reagents can lead to a series of problems, such as poor retention reproducibility, unstable baselines, and other separation problems, so it is generally not recommended for ion-pairing methods. Use the gradient method. However, when using small molecule ion-pairing reagents such as trifluoroacetic acid (TFA) and triethylamine (TEA), the column equilibration is faster and gradient elution is generally not a problem.

If the column needs to be cleaned, it is recommended to use a 50% methanol-water solution for maintenance.

Peak shape problem

The addition of ion-pairing reagents usually improves peak shape problems caused by residual silanol groups on silica-based columns. If peak shape problems occur in ion-pairing methods, such as peak front extension, etc., it can be solved by changing the temperature. Therefore, when the peak shape is not good or the number of theoretical plates decreases, the effect of temperature change on the peak shape can be studied.

Blank Solvent Peak

In the ion pairing method, when a blank experiment is performed (ie, the sample solvent is injected), both positive and inverted peaks sometimes appear, and these abnormal peaks are also called blank solvent peaks. These blank solvent peaks interfere with the establishment or routine use of liquid phase methods, so blank experiments should be performed both before the establishment of an ion pair method and after a promising separation has been obtained.

Problems with blank solvent peaks are usually caused by differences between the mobile phase and the sample solvent, which can be magnified by insufficient buffer salt purity, the addition of ion-pair reagents, or other mobile phase additives.

Ion effects on variables in method development

Compared with the ordinary reversed-phase method, the ion-pair method can meet the requirements of separation degree and other requirements by adjusting parameters (including pH, type and concentration of ion-pair reagent, and temperature).

The pH of the mobile phase: In the ion-pairing method, pH not only affects the ionization degree of the analyte, but also affects the ionization progress of the added ion-pairing reagent and affects the ion-pairing effect with the analyte. Therefore, the change of pH affects the analyte. The effects of retention are more complex.
Temperature: Changes in temperature in an ion pair method can cause significant changes in relative retention. Temperature changes the amount of ion-pair reagent adsorbed on the column, so controlling temperature is especially important for separations.
Type and concentration of ion-pairing reagents: In most cases, the concentration of ion-pairing reagents in the stationary phase will affect the retention behavior of ion-exchange of solutes, while the type and concentration of ion-pairing reagents will directly affect its retention in the stationary phase. concentration.

When a certain retention is desired, a lower concentration of a more hydrophobic ion-pairing reagent or a higher concentration of a less hydrophobic ion-pairing reagent can be used.

When the ion-pairing reagent in the stationary phase is saturated, continuing to increase the concentration of the ion-pairing reagent will actually reduce the retention of the analyte, because the counterion concentration of the ion-pairing reagent increases, increasing the ion-exchange competition with the analyte.

By simultaneously changing the pH of the mobile phase and the concentration of the ion-pairing reagent, the retention range and relative retention of ionic samples can be controlled to a large extent.