Using the EZLC Modeler for Cannabinoid Separations–Part 2: Using EZLC Software to Monitor Effects of Ammonium Formate Concentrations on Cannabinoid Separations
24 Mar 2025Using the EZLC chromatogram modeler is a fast and easy way to see how different variables can affect separations. Part one of this series discussed how to use EZLC software as well as how to optimize an existing method. For cannabinoid analysis, Restek elected to use formic acid and ammonium formate as preferred mobile phase additives. This blog will demonstrate how ammonium formate buffer concentrations can impact cannabinoid separations.
Cannabinoid separations can be challenging. They can often be difficult due to overlapping hydrophobicity. However, the presence of acidic cannabinoids allows us to utilize an additional lever: pH. The pKa range of these acidic cannabinoids falls between 2.90–4.75. Depending on the pH of the mobile phase, the retention time, and sometimes the elution order, of cannabinoids can change. The neutral cannabinoids will remain the same, however the acidic cannabinoids can change retention time because they often fall on the pKa slope.
To dive a little deeper into this phenomenon, let’s discuss pKa. A low pKa indicates a strong acid, while a high pKa indicates a weak acid. When developing a chromatographic method, it is ideal to have a mobile phase with a pH 2 units away from the pKa value. This causes the analyte to become either fully protonated or deprotonated. When the pH is above the pKa value, the compound is deprotonated, and when the pH is lower than the pKa the compound will be fully protonated.
Figure 1: Retention behavior of acids and bases on a standard reversed-phase column
As the pH is lowered, the portion of the total molecules present in their protonated form increases and the number of deprotonated molecules decreases, so the overall polarity of the acid decreases, resulting in increased retention times. In relation to cannabinoids, most common cannabinoids are either neutral or acidic. By adding buffer to the mobile phase, the acidic cannabinoids can become deprotonated, depending on the pH of the buffer, resulting in higher affinity for the mobile phase and reduced retention.
Using EZLC software and the same method parameters from above, we can demonstrate the movement of these analytes by changing the concentration of the ammonium formate in the mobile phase. In the following examples, you will see the highlighted compounds show changes in retention times along with some changes in elution order as well. CBNA switches with Δ9-THC while THCA-A switches with CBC.
Figure 2: 0 mM ammonium formate + 0.1% formic acid
Figure 3: 4 mM ammonium formate + 0.1% formic acid
Figure 4: 8 mM ammonium formate + 0.1% formic acid
Figure 5: 12 mM ammonium formate + 0.1% formic acid
As you can see, adjusting the buffer concentration is another tool that can be utilized when developing methods for compounds that are ionizable. It is important that mobile phases are prepared fresh to ensure that the pH remains constant, so the method remains reproducible.
Tune in for part 3 of this series to learn why cannabinoids that are not in your panel still matter!
Resources and Further Reading
Dolan, J. (n.d.). Back to basics: The role of pH in retention and selectivity. Chromatography Online. https://www.chromatographyonline.com/view/back-basics-role-ph-retention-and-selectivity
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