[Reader Insights] Acidic or Basic? Read Analyte Structure First When Developing a Method

Author: Sepuxianyun

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The Starting Point in Method Development

Introduction

“Please help! I cannot separate these peaks no matter what I try; can you suggest an optimization?”

This is what I often hear from fellow colleagues. Usually, it follows with a chromatogram, where two unresolved peaks are circled in red, plus the method conditions. When I ask what has been tried, the response is often something like “Slowed down the flow rate, changed to another C18 column — neither works.”

These attempts seemed logical at first glance, but what is missing is critical: the structure of the analyte. Ignoring the structure, or not analyzing the structure while knowing it, is nothing but setting ourselves up for trouble. Every choice in chromatography—column, mobile phase, etc. —should connect back to the analyte’s chemistry. A good method is not a product of endless trial-and-error, but designed with intent, with a philosophy of QbD (quality by design).

So, where do we start? For many analysts, organic chemistry may feel like a distant memory. Therefore, this guide is written to discuss about the structures of analytes, especially how to judge whether an analyte is acidic or basic, which matters in building an initial method.

Acidic or Basic?

Most drug molecules are built from carbon (C), hydrogen (H), oxygen (O), and nitrogen (N), with occasional appearances from sulfur (S), phosphorus (P), fluorine (F), chlorine (Cl), or bromine (Br). Basic groups are often nitrogen-based, while acidic groups include carboxyl groups (most common), phosphate groups, among others.

When judging whether a compound is acidic or basic, the key question is whether it tends to give up a proton (acidic) or accept one (basic). That’s where pKa comes in, defined as:

pKa = −log₁₀Ka

Ka describes how readily an acid (HA) dissociates in water:
HA + H₂O ⇌ H₃O⁺ + A⁻

For acids, lower pKa means stronger acidity. For bases, we look at the pKa of the conjugate acid—the higher it is, the stronger the base.

What Shapes Acid-Base Behavior?

Below is a table of some common compounds with their pKa values:

Acid/Base	Structure	Compound	pKa value
Bases	⌬-NH₂	Aniline	4.6
	⌬-NH-CH₃	N-Methylaniline	4.8
	⌬-CH₂-NH₂	Benzylamine	9.3
	⌬-CH₂-NH-CH₃	N-Methylbenzylamine	9.5
	⌬-CH₂-NH-CH₂CH₃	N-Ethylbenzylamine	9.7
Acids	HCOOH	Formic acid	3.75
	⌬-COOH	Benzoic acid	4.20
	CH₃-COOH	Acetic acid	4.76

The increase of pKa’s in the above basic compounds indicates an increase of alkalinity. This is particularly obvious between aniline and benzylamine where the addition of a methyl group increased pKa by 4.7. For acidic compounds, the functional groups connecting to the carboxyl group affect the acidity greatly.

In general, these structural factors play outsized roles in acid-base behavior:

1. Electronegativity of the central atom

More electronegative atoms pull harder on electrons, making it easier to release H⁺ (stronger acidity). Less electronegative atoms hold lone pairs less tightly, thus more willing to donate electrons and grab a proton (stronger basicity).
Example: fluorine, oxygen, nitrogen, and carbon have electronegativities of F>O>N>C, thus the hydrides HF, H₂O, NH₃, and CH₄ have acidities decrease in that order. NH₃ and CH₄ are practically not acidic at all.

2. Substituent effects

Electron-withdrawing groups (e.g., –NO₂, –CN, –COOH, halogens) pull electron density away, making protons easier to dissociate. This strengthens acids but weakens bases.
Example: Trifluoroacetic acid (CF₃COOH, pKa ≈ –0.3) is far stronger than acetic acid (CH₃COOH, pKa ≈ 4.76) due to having three highly electronegative fluorines.

Electron-donating groups (e.g., –CH₃, –OCH₃) push electron density back, refraining protons from dissociation, making acids weaker and bases stronger.
Example: N-methylbenzylamine (pKa ≈ 9.5) is slightly more basic than benzylamine (pKa ≈ 9.3) due to the methyl group giving electrons.

3. Conjugation and resonance

Whether a lone pair participates in conjugation matters. Nitrogen’s lone pair, for instance, can either stay localized (more basic) or be delocalized by resonance (less basic, e.g. p–π conjugation with benzene ring or carbonyl).
Example: Aniline (pKa ≈ 4.6) is much weaker as a base than benzylamine (pKa ≈ 9.3) because the nitrogen’s lone pair is delocalized into the benzene ring. On the acidic side, benzoic acid (pKa ≈ 4.20) is stronger than acetic acid (pKa ≈ 4.76) because the benzoate anion’s negative charge is stabilized by resonance with the ring. This is also why, as discussed in The Starting Point in Method Development, phosphoric acid works better than acetic acid in mobile phases.

4. Steric hindrance

Bulky substituents around a functional group can block access, changing its acid–base behavior. The effect depends on context and must be judged case by case.

A Real Example: Levofloxacin

Let’s apply this to a real molecule: the quinolone antibiotic levofloxacin.

N-1 is a tertiary amine. Its attached alkyl groups are electron-donating, so it’s reasonably basic. But steric hindrance from its three substituents makes it less basic than a primary or secondary amine. Its conjugate-acid pKa is around 8.
N-2 and N-3 are tied up in resonance with the benzene system, forming p-π conjugations. N-2, in particular, is adjacent to a strongly electron-withdrawing fluorine, making it the weakest in alkalinity. So both can largely be ignored when designing a method.
The molecule also carries a carboxyl group. Under acidic mobile-phase conditions, the tertiary amine at N-1 can cause peak tailing. Using an end-capped C18 or a C18 with polar-embedded groups helps improve peak shape.

Why Structure Matters

With these tools, analysts can make a quick assessment of most compounds. With drug molecules becoming increasingly complex and often with multiple heterocycles, a solid grounding in organic chemistry is more important than ever for chromatographers.

And circling back to the opening problem: if those two inseparable peaks represent one compound with an ionizable group and one without, sometimes all it takes is a simple adjustment of the mobile-phase pH to resolve them.

That’s why learning to read structure isn’t optional. It’s essential.