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pH, pKa, and Retention




1. Introduction:

In the world of chromatography, understanding the impact of pH on the retention of polarizable or polar compounds is essential. This knowledge plays a crucial role in the field of reverse-phase liquid chromatography (RPLC). We know that in RPLC, polar compounds tend to retain poorly while non-polar compounds exhibit strong retention. This phenomenon can be explained by the "like attracts like" rule. But what's the key to selecting the right pH during method development for optimal results? In this blog article, we will delve into how the Henderson-Hasselbalch equation can help determine the percent ionization of a compound and how this information can be used to predict retention in RPLC.


I'm Bhaskar Napte, founder of Pharma Growth, and today, we will explore the intricacies of pH and its role in reverse-phase liquid chromatography.


2. Understanding the Henderson-Hasselbalch Equation:

To determine the percent ionization of a weak acid, we need to consider two critical factors: the pH of the mobile phase and the pKa (the acid dissociation constant) of the compound.

10^(pH – pKa)

% Ionization (Weak Acid) = --------------------- x 100 (Eq. 1)

10^(pH – pKa) + 1



10^(pKa – pH)

% Ionization (Weak Base) = --------------------- x 100 (Eq. 2)

10^(pKa – pH) + 1



Once you have these values, you can easily calculate the percent ionization for the given compound.


3. Impact of pH and pKa on Retention of weak acid:


3a. Percent Ionization of weak acid vs. pH:

Let's take an example of a weak acid where the pKa of the compound is 5. At pH 0, the percent ionization is 0%, and it remains low at pH 1 and 2 (Use equation 1 to understand the above values). However, as the pH increases, the percent ionization gradually climbs. When the pH equals the pKa, you hit the 50% ionization mark. But the real revelation comes at higher pH values above 7 where percent ionization is almost 100%. You may refer to Fig. 1 for the %ionization of weak acid vs. pH.


Fig 1: % Ionization of weak acid vs. pH


3b. Retaining Non-Ionized weak acid:

Now, the impact of pH on weak acid retention becomes apparent. For pH ≤ pKa-2, the percent ionization is minimal. When the compound is negligibly ionized, it behaves like a less-polar compound. This is a significant insight. Below pH 3, the weak acid (with pKa= 5) remains non-ionic and less polar, resulting in stronger retention in RPLC. Refer to Fig 2 for details.


Fig 2: Retention of weak acid vs. pH


3c. Retaining Ionized weak acid:

Conversely, when the pH ≥ pKa+2, the weak acid undergoes complete ionization, becoming more polar. As a result, the compound retains poorly in reverse-phase liquid chromatography. Understanding this shift in ionization and its impact on polarity is crucial for method development. Refer to Fig 2 for details.


4. Impact of pH and pKa on Retention of weak base:


4a. Percent Ionization of weak base vs. pH:

Let's take an example of a weak base where the pKa of the compound is 5. At pH 9, the percent ionization is 0%, and it remains low at pH 7 and 8 (Use equation 2 to understand the above values). However, as the pH decreases, the percent ionization gradually climbs. When the pH equals the pKa, you hit the 50% ionization mark. But the real revelation comes at higher pH values above 3 where percent ionization is almost 100%. You may refer to Fig. 3 for the %ionization of a weak base vs. pH.


Fig 3: % Ionization of weak base vs. pH


4b. Retaining Non-Ionized weak base:

Now, the impact of pH on weak base retention becomes apparent. For pH ≥ pKa-2, the percent ionization is minimal. When the compound is negligibly ionized, it behaves like a less-polar compound. This is a significant insight. Above pH 7, the weak base (with pKa= 5) remains non-ionic and less polar, resulting in stronger retention in RPLC. Refer to Fig 4 for details.


Fig 4: Retention of weak base vs. pH


4c. Retaining Ionized weak base:

Conversely, when the pH ≤ pKa+2, the weak base undergoes complete ionization, becoming more polar. As a result, the compound retains poorly in reverse-phase liquid chromatography. Understanding this shift in ionization and its impact on polarity is crucial for method development. Refer to Fig 4 for details.


5. Choosing the Right pH: The key to achieving consistent retention time for a weak acid and weak base is selecting a pH value that either maximizes or minimizes ionization, depending on your compound's properties. For weak acids, a pH ≤ pKa-2 is ideal for strong retention. When working with weak bases, a pH ≥ pKa+2 ensures minimal ionization and better retention. Avoiding pH values near pKa ±2 is advisable to maintain consistent retention times.


6. Conclusion: Understanding the relationship between pH, percent ionization, and compound retention is vital for successful reverse-phase liquid chromatography. The Henderson-Hasselbalch equation and the pH values relative to pKa are valuable tools for method development. By leveraging this knowledge, chromatographers can optimize their separation processes, resulting in more accurate and reliable analyses.


Thank you for reading!

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Rated 5 out of 5 stars.

Very good information in a very simple manner, appreciate your efforts 👍

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Thank you for revising our concepts!

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