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Cromatography Handbook of HPLC - Rizzi A.

Rizzi A. Cromatography Handbook of HPLC - John Wiley & Sons, 2005. - 14 p.
Download (direct link): chromatographyhandbook2005.pdf
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A. Acid-Base Equilibria
Acid-base equilibria are extremely important in chromatography when aqueous mobile phases are used. They determine the degree of dissociation of weakly acidic groups and the degree of protonation of weakly basic groups, whether these groups are constituents of the analyte, constituents of the stationary phase (e.g., weak ion-exchanging groups or the silanol groups of the support material), or constituents of the buffer, or whether they belong to the matrix components of the sample. In all these instances, the pH of the aqueous mobile phase deter-mines the position of the equilibrium and thus the retention and separation of the analytes.
1. pKa Values and the Degree of Dissociation and Protonation
For weak acids or weakly acidic moieties within a molecule HA, which undergo dissociation in water according to the equilibrium
HA + H20 ~ H30+ + A" (43)
with the equilibrium constant Ka,
_ [1
[HA] v
the degree of dissociation ct1 is defined as
Table 11 Secondary Chemical Equilibria Frequently Employed in HPLC
Analytes Mobile phase Stationary phase Type of chromatography
Dissociation and protonation equilibria Anion-exchanger IEC
Acidic compounds Aqueous Alkyl-silica RPLC
Basic compounds Aqueous Cation-exchanger IEC
Ion-pairing equilibria Alkyl-silica RPLC
Charged analytes Aqueous + ion-pairing Alkyl-silica RPLC
Charged ligands Aqueous + metal ion Alkyl-silica RPLC
Metal ions Aqueous + charged Ion-exchanger IEC
lipand Alkvl---silica RPLC
Host-guest complexation Alkyl-silica RPLC
Organic compounds Aqueous + host
=--------E^J------- (45)
{[A] + [HA]} ^ ]
where square brackets symbolize the molar concentrations of the species. Combining the two equations yields the fundamental Eq. (46).
1 1 (46)
ryd ---
{1 + [H30+]/Ka} 1 + l(PA*-pH
This equation shows that the degree of dissociation is determined by the difference between
the pH value of the mobile phase and the pKa value of the compound.
An analogous formalism can be given for the degree of protonation ap of weakly basic moieties B, according to the equilibrium (written here as deprotonation)
HB+ + H20 H30+ + (47)
with the equilibrium constant Ka,
K-~ [HB-]--------------------------------------------------------------------------^
In this case, ap is defined as
.. -- -----------------------------------------------------------m
------ + [B]>---------------------------------------------------------_
being the unprotonated basic species and HB+ the protonated (acidic) species. The degree of protonation is given by Eq. (50).
ap =------------1---------=----------1------- (50)
{i + /s:a/[H3o+]} + (-*> v ;
The graphs showing the degree of dissociation and protonation, respectively, according to the Eqs. (46) and (50) are given in Fig. 10. When the pH equals the pKa, the degree of dissociation or protonation is V2. ad and ap change significantly only in a range of about 2 pH units on either sides of the pKa value. Outside this range any changes in pH have a marginal influence on the dissociation or protonation and thus on the retention factor.
The pKa values of many compounds of interest (analytes and buffer compounds) can be found in tables in the literature [76]. Given these data and with a certain chemical intuition one can very approximately predict pKa values of homologous, analogous, and similar
compounds not found in the tables. This might be quite useful during the development of a chromatographic method. Table 12 collects pKa ranges typically found for some acidic and basic groups and Table 13 gives a collection of pKa values for various selected molecules taken from the literature [76]. The table illustrates the influence of the molecular structural environment on the pKa values of acidic and basic groups.
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