Eluent Concentration and pH Value

In addition to the type of eluent, its concentration is one of the most important parameters affecting retention. The pH value of the mobile phase is closely connected to the eluent concentration. When a carbonate/bicarbonate mixture is selected as an eluent, parameters such as ionic strength and pH value cannot be considered separately, because the selectivity changes as the concentration ratio of the two components is varied. 

In general, the retention of mono- and divalent ions shifts forward as the eluent concentration increases. On the other hand, a change in pH primarily affects the retention behavior of multivalent ions, because the valency of such ions depends on the pH value of the mobile phase. This effect is illustrated using orthophosphoric acid as an example. Dissociation occurs in three steps [122]  

An ion chromatographic separation of the three orthophosphate species is impossible because of these pK equilibria. When using the relatively weak sodium hydroxide eluent, the pH value increases as the concentration increases. With a 1 mmol/L NaOH (pH 11) eluent, orthophosphate and sulfate do not elute at all, primarily because the concentration of the monovalent eluent ions is too low for divalent analyte ions to elute. Moreover, at pH 11 about 10% of the orthophosphate ions exist as ions, which have a much longer retention 

A more complex situation is the carbonate/bicarbonate buffer mixture. Due to the dissociation equilibrium. 

Tabie 3.21 In k values of inorganic anions at different ionic strengths but constant pH value. 

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While the parameters listed in Section 3.3.4.1 that determine the retention are fundamental in character, the parameters concerning the eluent discussed below depend on the detection system being used. This particularly applies to conductivity detection, which is possible directly or in combination with a suppressor system. These two modes of conductivity detection are fundamentally different and require eluents that not only differ with regard to their type but also to their concentrations and pH values, respectively. Therefore, the influence of these parameters on both modes is discussed separately for the most important detection system. 

Global LSER calculations have also been applied to the study of the retention of ioniz-able analyses in RP-HPLC. While the retention of neutral analyses does not depend on the pH of the mobile phase the retention of analyses with one or more ionizable substructures considerably depends on the pH even at the same concentration of organic modifier in the eluent. The relationship between the retention and pH of the mobile phase and pK value of the analyte can be described by... 

The maximum retention factor (kQ) is related to the log P value and k and k are the retention factors of the cationic and anionic forms, respectively. The pKa values are known, and the retention factor in a given eluent can therefore be predicted in reversed-phase liquid chromatography using an alkyl-bonded silica gel or polystyrene gel column. The separation conditions can be adjusted according to their logP and pKa values by the selection of a suitable organic modifier concentration and the pH of the eluent.3,4... 

Measurements for pH determinations in a mixture of aqueous and organic solvents should be described as apparent pH. The true pH value can only be measured in aqueous solutions. In general, the apparent pH of a buffer solution increases as the proportion of organic solvent in the aqueous mixture increases. When preparing an eluent, it is usually best to dissolve the required buffer salts in distilled water at the appropriate concentration, adjust the pH, then mix this solution with the required organic solvents. 

Acetonitrile shows in mixtnres with water, a better solnbility for salts. It is therefore recommended in ion-pair chromatography [52], Basic analytes also show better peak shapes in acetonitrile-buffer mixtures than with methanol. The proper selection, whether acetonitrile or methanol, should be used as the organic component in the mixtnre with a bnffer, however, the type of RP column used classical RP or a shielded RP column is also important. For demonstration with basic analytes, a standard mixture of anti depressives is used. Iso-eluotropic mixtures of methanol and acetonitrile are used. For standardization, the concentration of buffer components are also be kept constant. The analyte structures and the eluent mixtures are summarized in Table 2.2. As selectivity is worse in acidic eluents, a pH value of 7 has been used. Two phases... 

The respective data are summarized in Table 3-14. Clearly, the retention times of all anions investigated decrease with increasing addition of carbonate at a constant bicarbonate content of the solution (pH 8.35 to 10.23). However, when bicarbonate is added at constant carbonate concentrations (pH range 10.98 to 10.28), the retention times hardly decrease. This effect is understandable inasmuch as bicarbonate exhibits only a small elution power. The decisive factor for the retention behavior of multivalent ions is the pH value resulting from the concentration ratio of both eluent components. An inspection of Fig. 3-58 reveals that bromide and nitrate are superimposed by orthophosphate within a very narrow pH range between 9.6 and 10.0. Interferences also occur at pH > 10.8. In both cases, this may be attributed to the dissociation equiblibria of orthophosphoric acid. 

If this equilibrium is disturbed by a sample injection, a new equilibrium is established via relaxation i.e., the kind of relaxation process depends on the pH value of the sample injected. If the sample pH is lower than the pH value of the mobile phase, benzoate ions in the mobile phase are protonated due to the sample injection. Thus, the concentration of molecular benzoic acid in the mobile phase increases as does the concentration of the amount adsorbed to the stationary phase. The amount not adsorbed travels through the column and appears as a chromatographic signal the system peak. A qualitatively similar chromatogram is obtained when a sample containing the solute ions and the corresponding eluent component is injected into the system. However, only the position of the system peak is comparable, not its area and direction. 

Straight lines are obtained for the various inorganic anions when the eluent concentration is varied at constant pH value and when the logarithm of the net retention volume is plotted versus the logarithm of the eluent concentration. The slope of the straight line depends on the type of eluent and analyte ion, Sy, respectively. Fig. 3-69 illustrates this effect on the eluents and solute ions investigated by Small [81]. The coelution of some anions and the reversal of the elution order under specific chromatographic conditions is remarkable. The retention behavior is described mathematically as ... 

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