Wako Pure Chemical Industries, Ltd.Wako Pure Chemical Industries, Ltd. - Laboratory Chemicals
Home > Products > Analysis and Environment > Wako FAQ -Chromatography- Q9

Wako FAQ -Chromatography- Q9


FAQ
Q9: We are examining the conditions of a mobile phase in the HPLC. We would like to know how to regulate retention of a sample that has a readily dissociating functional group.



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 functional group.

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 1).

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 retardation method).

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 functional group.

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 AH+ 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 mobile phase.

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
pH Component with a basic functional group Component with an acidic functional group
Decreased Low retention High retention
Increased High retention Low retention

Fig.1_Relationship between pH and k'

Fig.2_Schematic Diagram of Separation by the Reverse Phase Ion Pair Method








INDEX
Q1Which reference standard do you recommend for use to check performance of a column?
Q2Is there any simple method to change the solvent concentrations in the mobile phase once prepared?
Q3How are ODS columns, Wakosil-II HG, AR and RS, used in different situations?
Q4Ion pair reagents for low wavelength are on the market. What effects and differences do they show with actual samples?
Q5We wish to fractionate by HPLC. How can we determine the optimal separating conditions?
Q6We wish to perform fractionation by the reverse phase flash chromatography. How can we determine the optimal separating conditions?
Q7Is column performance improved by narrowing the inside diameter of a column?
Q8We are examining mobile phase conditions of HPLC.We intend to examine by changing solvents.Is there any method to estimate the elution profile in advance?
Q9We are examining the conditions of a mobile phase in the HPLC. We would like to know how to regulate retention of a sample that has a readily dissociating functional group.
Q10I have used a semi-microcolumn (2φ mm) but cannot obtain improvement of sensitivity generally offered.
I wonder what is the cause as I am sure to have used the system applicable to microcolumns.
Home > Products > Analysis and Environment > Wako FAQ -Chromatography- Q9