Unified pH Values of Liquid Chromatography Mobile Phases

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Unified pH values of liquid chromatography mobile phases Agnes Suu, Lauri Jalukse, Jaanus Liigand, Anneli Kruve, Daniel Himmel, Ingo Krossing, Marti Roses, and Ivo Leito Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac504692m • Publication Date (Web): 09 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Analytical Chemistry

Unified pH values of liquid chromatography mobile phases Agnes Suua, Lauri Jaluksea, Jaanus Liiganda, Anneli Kruvea, Daniel Himmelb, Ingo Krossingb, Martí Rosésc, Ivo Leitoa*

a

Institute of Chemistry, University of Tartu, 14a Ravila Street, 50411 Tartu, Estonia, e-mail: [email protected]

b

Institute for Inorganic and Analytical Chemistry and Freiburger Materialforschungszentrum (FMF), Albert-Ludwigs-Universität Freiburg, Albertstrasse 21, 79104 Freiburg, Germany

c

Departament de Química Analítica, Universitat de Barcelona, Martí í Franquès, 1-11, E-08028 Barcelona, Spain

ABSTRACT This work introduces a conceptually new approach of measuring pH of mixed-solvent liquid chromatography (LC) mobile phases. Mobile phase pH is very important in LC, but its correct measurement is not straightforward and all commonly used approaches have deficiencies. The new approach is based on the recently introduced unified pH (pHabs) scale, which enables direct comparison of acidities of solutions made in different solvents, based on chemical potential of the proton in the solutions. This work represents the first experimental realization of the pHabs concept using differential potentiometric measurement for comparison of the chemical potentials of the proton in different solutions (connected by a salt bridge), together with earlier published reference points for obtaining the pHabs values (referenced to the gas phase) or pH values

(referenced to the aqueous solution). The liquid junction potentials were estimated in the framework of Izutsu’s three-component method.

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pHabs values for a number of common LC and LC-MS mobile phases have been determined. The pHabs scale enables for the first time direct comparison of acidities of any LC mobile phases: with different organic additives, different buffer components etc. A possible experimental protocol of putting this new approach into chromatographic practice has been envisaged and its applicability tested. It has been demonstrated that the ionization behavior of bases (cationic acids) in the mobile phases can be better predicted by using the pH values and aqueous pKa

values than by using the alternative means of expressing mobile phase acidity. Description of the values is possible if the change of their pKa ionization behavior of acids on the basis of pH

values with solvent composition change is taken into account.

INTRODUCTION The pH of the mobile phase affects the retention of compounds with acid-base properties in liquid chromatography (LC) and its measurement is needed to understand and control the separation process.1 LC mobile phases are almost always mixtures of water and organic solvents and in principle every aqueous, non-aqueous and mixed solvent medium has its own pH scale.2 As a complication, the pH values of these solutions made in different solvents are incomparable due to the different zero points (linked to the activity of the solvated proton in the particular solvent) of the scales. Until recently an “intersolvental” pH scale, which would provide comparable pH data for different media has been considered non-attainable.4 True, there are acidity measures that can be compared across solutions in different solvents, such as the H0 function, but these are neither direct measures of the hydrogen ion activity5 nor of its chemical potential and all of them have limitations.6


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There are three common ways to measure the pH of a mixed-solvent LC mobile phase:1 (1) measurement of pH in the aqueous buffer before addition of the organic solvent (the so-called pH);

(2) measurement of pH in the mobile phase after pH calibration with aqueous standard

solutions (the so-called

pH)

and (3) measurement of pH in the mobile phase after pH

calibration with standard solutions prepared in the same medium as the mobile phase (the socalled pH). Only pH has a fully rigorous physical meaning for the mixed-solvent mobile phase1, but it is also the least accessible in practice, because of limited availability of standard buffer solutions7,8 for the different mobile phases, as well as the pKa values of compounds in such mixtures. Most importantly, the pH values of mobile phases made in different solvents (or solvent mixtures) obtained according to any of the three ways are not rigorously comparable. The pH values measured in pH scale can in principle be converted into aqueous pH scale by using standard Gibbs energy of transfer of hydrogen ion ∆tG°(H+) and these converted values are comparable. However, the limited availability of ∆tG°(H+) values limits the applicability of this approach. In addition, in the case of e.g. acetonitrile-water mixtures the available data are inconsistent.9 In this work a fourth approach is proposed, which enables direct comparison of acidities of LC mobile phases, even if prepared using different solvents or their mixtures. It is based on the concept of unified pH scale – pHabs scale – proposed by Himmel et al.10 In the pHabs scale the solvated proton activity is based on its absolute chemical potential and uses the (hypothetical) ideal proton gas at 1 bar as the standard state, thereby setting the chemical potential of the gaseous proton at 1 bar to 0 kJ/mol.10 In solution, the H+ activity is lowered by solvation and the absolute chemical potential of the proton is given as H , solv = ∆ ° H − [pH × ln 10]


(1)

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where µabs(H+,solv) is the proton’s absolute chemical potential in the given solvent, ∆solvG°(H+) is the Gibbs standard free energy of proton solvation, pH is the conventional pH (in chromatography it is useful to use pH defined via the molar scale although the latest IUPAC recommendations do not directly address this definition)3, R is molar gas constant, T is the absolute temperature. The absolute pHabs value is defined as follows: pH = −

%&'( ) , *+,-.

(2)

For practical realization of the scale differential potentiometry11 is a suitable technique. It enables direct comparison of solvated proton activity (related to the solvated proton’s chemical potential) difference, measured by potential difference between two H+-sensitive indicator electrodes immersed in the compared solutions S1 and S2 without a reference electrode and thus offers the possibility of direct comparison of the activities of the H+ ion in the solutions. Although elegant in its design, this scale has not yet been experimentally realized, because of experimental difficulties. The main difficulty that arises is that if two electrolyte solutions in different solvents are in contact then at their junction the so-called liquid junction potential (LJP) emerges.12,13 It is caused by transfer of ions with different mobilities and different solvation energies in the solvents of S1 and S2 across the junction, as well as by the contact of the two solvents themselves.12 The experimentally measured potential difference inevitably includes contribution from the LJPs, which can by no means be considered as “small corrections”: at the junctions of solvents of different nature can be in the range of 50-250 mV, corresponding to 1 - 4 pH units (!). In this work the LJP is estimated using the most advanced approach available, developed by Izutsu and his co-workers.14 This work presents the first experimental realization of the unified pH scale concept and envisages an experimental protocol how the unified pH could be introduced in the LC practice.


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The most common LC and LC-MS mobile phases, in respect to both pH regulating additives (ammonia, acetic buffers, formic acid) and organic modifiers (acetonitrile, methanol), are values. The notion pH means that all characterized with pHabs values, as well as the pH

the acidities are expressed on the common pHabs scale and are directly comparable, but the point of origin is shifted by a constant value (equal to ∆ ° H , H/ O⁄ 1210), as expressed in the following equation, which is obtained by combining equations 1 and 2: pH = pH +

∆(567 8°9) , : *+;? ] @AB


(5)

8

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where [A−] is the equilibrium concentration of anion and CHA is analytical concentration of acid. For bases (cationic acids) the principle is similar BH+

H+ + B α=

[C]

@DA)

(6)

where [B] is the equilibrium concentration of base and CBH+ is analytical concentration of the conjugate acid of the base. Procedure of comparing acidities of solutions. The H+-sensitive electrodes were immersed in solutions connected by the salt bridge (Figure 1) and after stabilisation (approximately 15 min) the reading (∆Emeasured) was recorded. Then the positions of the electrodes were switched and the measurement was repeated. The sign of the reading changes, when electrodes are switched between the solutions. Two measurements were made with the same solutions and then all solutions were renewed. Absolute values of four measurements, two with one polarity and two with the other polarity, were averaged for one potential difference. Nine mobile phases in Table 3 were measured a year later with improved salt bridge design and two values instead of four were averaged. In the cell (4) two liquid junctions are formed, so that the ∆Emeasured does not correspond to the potential difference ∆E of the H+ sensitive electrodes Ind2 and Ind1 (eq 8 below), but also includes contributions from the LJPs across the two junctions Ej(B,S1) and Ej(B,S2). Therefore, ∆E is found as follows: ∆E = ∆Emeasured + Ej(B,S1) – Ej(B,S2)

(7)

The LJPs were calculated as described in SI. ∆E can be converted into the difference of pHabs values (∆pHabs values) of the solutions,11 i.e. their relative acidity: ∆E = E Ind/ − E Ind- = −

*+ , -. H

9pH S/ − pH S- :


(8)

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The theoretical slope RTln10/F in eq 8 was replaced by the experimentally determined slope (see SI). The obtained ∆pHabs values can be combined into a continuous scale (so-called “ladder”). Measurement of

L LJK

values. Evikon pH Meter E6115 was used with several combination

electrodes to measure the pH of diluted standard buffers used as anchor points. The pH of the aqueous component of the mobile phase was only measured with Hydromet electrode. The calibration was done with standard aqueous buffers with pH values of 4 and 7. Anchoring of the scale. For assigning the pH values to the solutions the ladder was

anchored to the aqueous solutions with pH values 4.15 and pH 7.16 (two independent anchor points lead to more accurate anchoring). The pHabs values (with 1 bar proton gas as point of origin) were found from the pH values by eq 3.

Anchoring was done by keeping the ladder (Table 3) intact, i.e. without altering any of the pH differences between the different solutions on the ladder. This was achieved by two consecutive least squares procedures. During the first minimization all assigned pHabs values except that of the anchor point (which was arbitrary at this stage) were allowed to change so that the sum of squared differences between the assigned value differences and the actual ∆pH measurement results was minimized. As the second step, a constant offset was found in such a way that by adding it to all pHabs values on the ladder the sum of squared differences between the pH values of the standard solutions (4.15 and 7.16) and the pHabs values of the respective solutions on the ladder was minimized. The least squares algorithm is described in detail in ref 18.


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RESULTS AND DISCUSSION Validation. The differential potentiometry method was validated with aqueous standard buffer solutions with reference pH values 2.00, 4.00, 7.00 and 10.01 at (25 ± 1) °C. Saturated aqueous solution of KCl was used as the salt bridge electrolyte and in this case – because the compared solutions have low ionic strength and are made in the same solvent – the LJP at the two junctions can be assumed to cancel. The buffer solution with pH 7.00 ± 0.02 was used as the reference point. The results are given in Table 2. The consistency of the validation data (expressing the mutual agreement between the different relative acidity measurements) is evaluated with the consistency standard deviation of the scale sconsistency defined as follows: MN,OPQ,NR

=S

∑Y9∆UVWX,Y Z∆U&((Y[\V],Y :

Unified pH Values of Liquid Chromatography Mobile Phases

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