What Happens to Ionic Compounds Inside a Chromatographic Column

In the process of reversed-phase chromatographic separation, it is essential for the analyte to maintain an appropriate and stable molecular/ion equilibrium state to ensure the effectiveness of chromatographic separation. However, do ionic compounds really behave so ‘honestly’ once they enter the chromatographic column?

The presence of silanol groups in the chromatographic column packing material can disrupt the original ion equilibrium state of ionic compounds, leading to secondary adsorption between silanol groups and the analyte, causing peak tailing. A typical example is the protonated alkaline compound BH+ undergoing ion-exchange interaction with the stationary phase’s silanol groups.

Taking the example of a potassium salt-containing buffer solution:

Ion-exchange interactions occurring in the chromatographic column can prolong the retention time of analytes, resulting in peak tailing. When using a Type B spherical silica gel chromatographic column for separation, the pH of the mobile phase can lead to different peak tailing phenomena if it is too high or too low. When pH ≥ 6, a tailing index peak similar to Figure (a) is often observed, while when the mobile phase pH is < 5, peak tailing generally leans towards the right side, as shown in Figure

At low pH:

Peak tailing occurs due to the charge repulsion between retained ionic molecules. For alkaline compounds, overloading on the column occurs faster compared to neutral compounds in the analysis. Therefore, when injecting alkaline compounds in amounts exceeding 0.5 μg (assuming a column inner diameter of 4-5 mm), it leads to significant peak tailing.

Improvement Methods:

1. Peak tailing observed at low pH can be mitigated by increasing the ionic strength of the mobile phase.
2. While keeping the molar concentration of the buffer solution constant, adjusting the pH of the mobile phase to enhance the buffer solution’s ionic strength can effectively reduce peak tailing for basic compounds. It is recommended to use volatile formate salts as buffer solutions for LC-MS detection and analysis.

Improvement Cases

Before Improvement

After Improvement

Adding a buffering salt to the mobile phase and adjusting the concentration of ion-pair reagents.

At pH ≥ 7:

Peak tailing phenomena still occur when analyzing alkaline samples using a Type B column, and there have been reports suggesting that this may be attributed to the slow adsorption-desorption process of BH+.

The degree of tailing depends on the type of organic solvent:
Acetonitrile (worst) > Methanol ≈ Tetrahydrofuran (best)

In general

Peak tailing can be improved by increasing the column temperature or increasing the content of the strong wash solvent. When the mobile phase pH is < 5, using columns with lower cation exchange capacity is advantageous for minimizing the degree of peak tailing. Mixed-particle matrix chromatography columns at pH < 8 can prevent the ionization of silica gel fillers, thus achieving ideal peak shapes for protonated alkaline compounds under these conditions.

In special cases

During the analysis with a lower pH mobile phase, when the sample quantity is <1 μg, un-ionized carboxylic acids can cause peak tailing. However, when the sample quantity is larger (1-50 μg), the peak shape improves. The exact reasons for this phenomenon are not clear, but it may be related to competition among high-sample-quantity analytes and can be considered independent of column factors.