|As factors of a mobile phase affecting the elution profile of a solute
(sample), polarity, pH, etc. are given. Continued from the previous Q&A,
let us examine the effect of pH associated with a sample with readily dissociating
Assuming that an aqueous solvent with a high polarity is used as the mobile
phase for separation of a sample component, particularly a weak acidic
or weak basic component, with a readily dissociating functional group,
hydrogen ion concentration and ion strength have greater effects on separation
than the polarity of the solvent. This is because the degree of ionization
of these components differ and k' values vary considerably depending on
the pH of the mobile phase.
In a case of reverse phase partition chromatography, for example, a decrease
in pH of the mobile phase accelerates dissociation of components with a
basic functional group, weakens affinity to the stationary phase and k'
becomes smaller. Dissociation of components with an acidic functional group
is suppressed, affinity to the stationary phase is strengthened and k'
becomes larger. When the pH of the mobile phase is raised, on the contrary,
dissociation of the components with a basic functional group is suppressed,
affinity to the stationary phase is strengthened and k' becomes larger.
Dissociation of components with an acidic functional group is accelerated,
affinity to the stationary phase is weakened and k' becomes smaller (Table
In other words, it is advised to add a stronger acid to lower the pH and
suppress dissociation of ions if a substance with an acidic functional
group is analyzed by the reverse phase chromatography. For analysis of
a substance with a basic functional group, a stronger alkali is added to
raise the pH and suppress dissociation to retain the substance longer (ion
Now, assuming that dissociation equilibrium is reached for an organic acid
AH as shown by
AH ↔ A− + H+
Ka = [A−][H+] / [AH],
k' is given by the following equation.
k' = a/(1 + Ka/[H+]) + b/(1+[H+]/Ka)
where a and b are constants, and it corresponds to k' value when the concentration
of a non-dissociating or dissociating component becomes 0.
The relationship between pH and k' taking pKa of an organic acid as 5.0
is shown in Fig. 1.
It can be seen from this relation that k' changes sharply at a point where
pH equals pKa.
Apart from the ion retardation method, ion pair chromatography (ion pair
method) is used to regulate retention of a sample with a readily dissociating
Fig. 2 shows a schematic diagram of separation by the reverse phase ion
pair method. When a suitable counterion is added to an ionic or ionizable
solute in a mobile phase of an aqueous solvent, ionic sample A−H+ produces ion pair reagent B+OH− and ion pair B+A−. As this ion pair BA is not charged, it is retained in the chemically
bound stationary phase of the reverse phase system and separated in the
same manner as ordinary organic compounds. Retention of the ion pair is
regulated by the type and concentration of the counterion and pH of the
As ion pair reagents, quaternary alkylammonium salts, tertiary amines,
etc. are used for components with an acidic functional group while alkylsulfonates,
perchloric acid, alkyl sulfuric acid, etc. are used for components with
a basic functional group. The concentration range of counterion is usually
between 0.001 and 0.1 M and retention is increased with an increase in the counterion concentration.
The above may be summarized as follows:
(1) Determine the best suited pH from the relationship between the retention
time of individual components and pH.
(2) Separate by the ion pair method by the addition of a counterion, if
necessary, for separation of acidic or basic ionic components when dissociation
of a sample component cannot be suppressed by changing the pH alone.
These are general methods. Pay attention to the pH range applicable to
the carrier in both ion retardation method and ion pair method.
|Table 1 Relationship between pH and retention of readily dissociating compounds
||Component with a basic functional group
||Component with an acidic functional group