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Bob Bickler

How does an acidic pH affect reversed-phase chromatography separations?

November 1, 2022 at 2:19 PM / by Bob Bickler

Chromatography is as much an art as it is a science. Between synthetic reaction products and natural products, the range of compounds requiring separation, purification, and isolation is broad and diverse creating challenges from time to time. Because of this diversity, not all chromatographic separations can be performed with a “neutral” solvent system – one without added pH modifiers or buffers.

When separating ionizable/ionic compounds or inherently acidic/basic compounds mobile phase modifiers can be very beneficial. In this post, I show some examples where lowering mobile phase pH improves peak shape.

Low pKa compounds

When purifying compounds with low pKa, it is a good idea to acidify the mobile phase. An excellent rule of thumb is to lower the mobile phase pH 2 units below the target compound’s pKa. With reversed-phase, this can be accomplished by adding formic, acetic, or trifluoroacetic acid to each solvent or creating a third acidified solvent which can be blended isocratically. This is the approach I took in the following example – the separation of hippuric acid (pKa 3.6) and isatoic anhydride.

Under neutral conditions, we see major fronting of the hippuric acid peak and a distorted isatoic anhydride peak, Figure 1. These phenomena occur because the compounds, especially hippuric acid, are partially dissociated existing as both an intact compound and as an ionized compound. The ionized form is more polar and less retained than the parent compound.

Isatoic anhydride + hippuric acid neutral

Figure 1. Separation of hippuric acid (blue) and isatoic anhydride (red) using a water/acetonitrile gradient without pH modifiers. The hippuric acid peak is fronting indicating it is partially ionized.

By adding acid to the solvent system, both compounds are protonated into their native form which sharpens the elution bands and slightly increases retention, Figure 2.

Isatoic anhydride + hippuric acid + TFA

Figure 2. Separation of hippuric acid (yellow) and isatoic anhydride using the same water/acetonitrile gradient but with 0.1% TFA added isocratically as the pH modifier. The acid reduced ionization and improved peak shape.


High pKa compounds

Because acids protonate ionizable compounds, bases as well can have their elution profiles altered. Lowering mobile phase pH below the compound’s pKa will start the dissociation process changing the compound’s equilibrium during purification. This can cause peak splitting and even a complete separation of ionized and native species, Figure 3.

When purifying high pKa compounds, consider using either a buffer (ammonium formate, ammonium acetate, etc) to create an ion pair or operate at a pH > the compound’s pKa.

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Bob Bickler

Written by Bob Bickler