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Exploring the Surprising Increase in Retention Time of Protonated Bases at Low pH in RPLC


Today we delve into a fascinating topic that challenges conventional wisdom in liquid chromatography: Can the retention of protonated bases increase as pH decreases? Typically, the retention time of a protonated base in a lower pH environment is shorter. However, there's an intriguing possibility that protonated bases may exhibit increased retention at lower pH levels. In this article, we'll explore this phenomenon and the theories that underpin it.

pH and Ionization:

The behavior of basic compounds in liquid chromatography is intricately linked to pH and pKa values. When pH exceeds pKa+2, the basic compound remains in its unionized form. This lack of ionization results in a less polar or even non-polar compound, which, in a reverse-phase liquid chromatography system, translates to longer retention times.

Conversely, if you adjust the mobile phase pH to be significantly lower than pKa-2, the basic compound undergoes complete ionization or protonation (BH+), an ionic and more polar species. In this scenario, reverse-phase liquid chromatography yields shorter retention times.

Contradictory Observations:

It's clear that the retention of protonated bases typically decreases at lower pH levels. However, there are instances where this trend is reversed. Here are some examples that challenge conventional thinking:

  1. The Influence of Acidic Modifiers: Studies have shown that increasing the concentration of acidic modifiers, such as perchlorate, trifluoroacetic acid, and phosphoric acid, in the mobile phase can lead to an increase in the retention time of protonated bases at lower pH values. For instance, as the pH was reduced from 3 to 1, the retention time of aminoindanol, a basic pharmaceutical compound, increased when acidic modifiers like perchloric, trifluoroacetic acid, nitric, and phosphoric acids were used. One theory suggests that at low pH when a basic compound is protonated (BH+), it can form a neutral complex with acidic modifiers like perchlorate. This complex is less polar and leads to increased retention time.

  2. Stationary Phase Modification: The modification of the hydrophobic character of the stationary phase is another intriguing factor. Using bonded stationary phases like C8 or C18 at a low pH where the basic compound is protonated, along with the presence of acidic modifiers, can lead to the deposition of anions on the stationary phase. This results in an anionic or ionic stationary phase. The interaction of the protonated base (BH+) with this charged stationary phase can lead to longer retention times.

  3. Desolvation of Protonated Analyte: This theory explores the role of solvent and solvation in retention. In the presence of water molecules, a protonated base is surrounded by a hydrophilic environment, which results in poor retention. However, increasing the concentration of acidic modifiers can disrupt the solvation, making the analyte slightly hydrophobic. This change in character results in higher retention times in reverse-phase liquid chromatography.


The unexpected increase in the retention time of protonated bases at low pH levels challenges our understanding of liquid chromatography. The presence of acidic modifiers and their interaction with


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