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pKa, Ionizable Drugs, And Pharmaceutical Discovery

Two out of three drugs have at least one ionization-capable group.

The pharmaceutical industry is a leading consumer of separation and purification products. A World Drug Index (WDI) survey of approximately 52,000 pharmaceutical compounds indicates that 62.5% (~32,500) had ionizable groups, with the majority also containing at least one basic center [1,2]. In other words, 2 out of 3 drugs on the market include at least one ionization-capable group. This propensity of the drugs to ionize has significant implications on the chromatography performance of reversed-phase (RP) stationary-phase materials - the most widely-used media for separation and purification [2]. A key term that is used when this issue is discussed is called pKa.


Knowing the drug’s pKa value is crucial to developing optimal chromatography methods.

So what is pKa? To understand pKa, a brief primer of the RP chromatography separation mechanism is necessary. In RP chromatography, “like dissolves like,” i.e., non-polar analytes (possessing neither positive nor negative charge) interact with non-polar stationary phases (hydrophobic surfaces such as C18, graphite, or carbon nanotube). Better separation are achieved through increased interactions of the analytes with the stationary phase. Neutral or ion-suppressed analytes, which are more non-polar than ionized analytes, have been shown to have improved retention on non-polar RP stationary phases. For ionizable analytes, its ionization state is significantly influenced by the pH levels of the mobile phase. Therefore, buffers are used to regulate the pH of the mobile phase in order to keep the analyte in the desired state.


This is a good stage to introduce pKa. The degree of ionization of a compound is indicated by the pKa value. At a pH = pKa, i.e., 50% of a compound is ionized. Thus, the analyte is present in both, ionized and neutral states. Thereby resulting in poor chromatograms (broad, tailing peaks). This is the pKa value presented for all practical uses. An acidic analyte (drug) present in an acidic environment (pH) is adjusted to at least two units below its pKa to remain in its ion-suppressed, neutral form, leading to improved retention of RP-HPLC. Conversely, a basic analyte (drug) present in an alkaline environment (pH) is adjusted to at least two units above its pKa to remain in its ion-suppressed, neutral form.

Fig. 1 Schematic plots of analyte retention k vs pH for analytes with acidic and basic

functional groups. Here carboxylic acid (-COOH) and amine (-NH2) are used as representative

examples of acidic and basic functional groups, respectively.


pKa has direct implications on chromatography method development.

During separation, the pH levels of most of the ~62.5% ionizable compounds with one basic center must be kept above 8. The effects of pH levels on a silica-based RP stationary phase material are well understood [3]. At very low pH levels (<2), the bonded stationary phase (long alkane chains such as C18) can be stripped off the silica support, whereas at high pH levels (>8), the silica itself can be damaged by dissolution. An increase in temperature further accelerates this degradation process. Advancements in silica bead technology have shown promise in slowing down this degradation.


It is plausible to assume that a significant proportion of future drug candidates will comprise of least one basic center. Additionally, since many of the new large molecule drugs being developed have multiple ionizable centers, particularly those based on proteins, oligonucleotides, DNA, and peptides. Thus, ionization suppression using excessive pH media (pH >10) could be necessary to discover and manufacture advance drug candidates with multiple ionizable centers.


At Millennial Scientific, we develop next-generation products for purification and pharmaceutical delivery. Get in touch with us to discuss how we can support your chromatography separation needs for small and large molecules with multiple ionizable centers.



[1] D.T. Manallack, The pK(a) Distribution of Drugs: Application to Drug Discovery, Perspectives in medicinal chemistry 1 (2007) 25-38.

[2] D. T Manallack, R. J. Prankerd, E. Yuriev, T. I. Oprea, and D. K. Chalmers, The Significance of Acid/Base Properties in Drug Discovery, Chem Soc Rev. 42(2) (2013) 485–496.

[3] R.E. Majors, Current Trends in HPLC Column Usage, LCGC Europe 25 (2012) 1-10. 

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