The document discusses the role of acid-base equilibria and pKa values in reversed-phase chromatography (RPC) method development. It explains how changes in mobile-phase pH can significantly affect the retention and separation of compounds, particularly when dealing with acids and bases. Additionally, it emphasizes the need to carefully control pH to achieve desired selectivity and resolution in sample analysis.

1. ACID–BASE EQUILIBRIAAND REVERSED-PHASE RETENTION
Basic understanding of pKa and its role in HPLC method development
C. P. SINGH
Research Scientist – ARD

2. ACID–BASE EQUILIBRIA AND REVERSED-PHASE RETENTION
Uncharged
molecule
Acid (HA)
Base (B)
Charged
molecule
Acid (A− )
Base (BH+)
(Acids) HA ⇔ A− + H+
(Bases) B + H+ ⇔ BH+
Ionization
Hydrophobic
Less Polar
More retained in RPC
Hydrophilic
More polar
Less retained in RPC
As a result its retention factor k in RPC can be reduced 10-fold or more

3. Acids
HA ⇔ A− + H+
Bases
B + H+ ⇔ BH+
When the mobile-phase
pH is increased
When mobile-phase pH
decreases
Bases gain a proton
and become ionized
Acids lose a proton and
become ionized
Acid Bases
(Acids) Ka = [A−][H+]
[HA]
(Bases) Ka = [B][H+]
[BH+]
Acidity constant Ka
𝑨𝑪𝑰𝑫 𝑷 𝑲𝒂 = 𝒑𝑯 − 𝐥𝐨𝐠
𝑨−
𝑯𝑨
𝑩𝑨𝑺𝑬𝑺 𝑷 𝑲𝒂 = 𝒑𝑯 − 𝐥𝐨𝐠
𝑩
𝑩𝑯 +
Henderson–Hasselbalch equation
pKa value = −logKa
ACID–BASE EQUILIBRIA AND REVERSED-PHASE RETENTION

4. Hypothetical illustration of the RPC separation of an acidic compound HA from a basic compound B as a function of pH.
Ionization of HA and B as a function of
mobile-phase pH and effect on k.
Sample separation as a function of mobile-
phase pH.

5. Section - I
Reproducible
Section -II
Less reproducible
Section - III
Reproducible
The mobile-phase pH - In order to control selectivity and resolution.
Section -II
A change in pH will provide a maximum change in
retention and separation.
Chosen if we want to change selectivity and
resolution by varying pH.
When an acid or base is half-ionized
A change in pH of 0.1 unit.
Will result in a change of k by about 10%
Can result in a change in resolution of
as much as ±2.5Rs units.
A possible change in separation from baseline
resolution (Rs >1.5) to complete overlap (Rs = 0).

6. Effect of mobile-phase pH on RPC retention as a function of solute type.
Sample: 1, salicylic acid; 2, Phenobartitone; 3, Phenacetin; 4, Nicotine; 5, Methylamphetamine
Compounds 2 and 4 are
seen to have pKa
values of about 8 and
6.5, respectively.
Compounds whose retention
increases significantly as pH
increases are bases (4 and 5).
The shape of a plot of retention versus pH for a peak allows a determination of its sample type (acid, base, or neutral), and a
rough estimate of its pKa value.
Compounds whose
retention decreases with an
increase in pH are acids
(1 and 2).
Compounds that show little
change in retention with pH
(3) are either neutral or are
fully ionized over the pH
range studied.
While the pKa values of
compounds 1 and 5
cannot be estimated
accurately (a complete
retention vs. pH curve is
required), it is safe to say
that pKa ≥ 9 for
compound 5, and pKa ≤ 3
for compound 1.

7.  A change in mobile-phase pH can be a powerful means of controlling relative retention (selectivity) and
separation for samples that contain acids and/or bases.
 This sample contains acids and bases with a wide range in pKa values and therefore exhibits sizable changes
in retention for small changes in pH throughout the range 3 < pH < 9. Consequently, either a careful control
of mobile pH will be required for the separation of this sample or conditions must be selected that provide
excess resolution (Rs 2).
 In order to avoid pH-related variations in retention, the mobile-phase pH can be selected to be different from
the pKa values of all sample components, by at least ±1.5 pH-units (regions I and III of Fig).
 As the majority of compounds have pKa values >4, low-pH separations (2 ≤ pH ≤ 3) are more likely to be less
sensitive to small changes in pH—which is one reason for beginning method development with a low-pH
mobile phase.
ACID–BASE EQUILIBRIA AND REVERSED-PHASE RETENTION

8.  if a solute is half-ionized, a change in mobile-phase pH by 0.1 unit can cause a complete loss of resolution. This
suggests that mobile-phase pH may need to be controlled within about 0.02 units for such a separation, which
could prove difficult for many laboratories.
 Values of pKa in the literature for different acids or bases usually refer to solutions in buffered-water at near-
ambient temperatures. If the mobile phase contains organic solute, or if the temperature is much different from
ambient, values of both pH and pKa can change significantly.
Thanks
ACID–BASE EQUILIBRIA AND REVERSED-PHASE RETENTION