Improved pH measurement of mobile phases in reversed-phase liquid chromatography.

Mobile phase pH is a critically important parameter in reversed-phase liquid chromatographic (RPLC) separations involving analytes that display acidic or basic properties in the pH range used for the mobile phase. The main problem in measuring mobile phase pH lies in the fact that RPLC mobile phases are typically aqueous-organic mixtures. In addition to experimental difficulties, the pH values refer to different aqueous-organic compositions that cannot be correctly compared. Given this situation, the unified pH (wabspH, also termed as ) based on the absolute chemical potential of the solvated proton has been proposed as a rigorous way of characterising mobile phase acidity that is fully inter-comparable between mobile phases of any composition. Here we report the wabspH values of 78 reversed-phase liquid chromatography-mass spectrometry mobile phases that were carefully measured by potential differences in a symmetric cell with two glass electrode half-cells and almost ideal ionic liquid triethylamylammonium bis((trifluoromethyl)sulfonyl)imide [N2225][NTf2] salt bridge with multiple overlapping measurements. The system of altogether 300 ΔwabspH values was anchored to the pH value of standard pH 7.00 aqueous buffer solution. The consistency standard deviation of the whole set of measurements was 0.09 pH units. In addition to the differential potentiometric reference method, simpler measurement methods that use double junction reference or double junction combined electrodes were tested and were found suitable for routine laboratories if high accuracy is not required.

Ionization and lipophilicity in nonpolar media mimicking the cell membrane interior.

Ionization and lipophilicity may vary with the environment. Therefore, in this study we provide some insight in the performances of different experimental techniques (potentiometry, UV-vis, shake-flask and chromatography) to determine ionization and lipophilicity in more nonpolar systems than those commonly used in drug discovery. To this purpose a pool of 11 compounds of pharmaceutical interest was firstly submitted to a few experimental techniques to measure pKa in water, water/acetonitrile mixtures and pure acetonitrile. Then we measured logP/logD with shake-flask and potentiometry in octanol/water and toluene/water and also determined a chromatographic lipophilicity index (log k'80 PLRP-S) in a nonpolar system. Results show that ionization decreases for both acids and bases in a coherent, significant but not dramatical extent when water is present in the system, but the picture is completely different in pure acetonitrile. Lipophilicity may vary or not with the environment according to the chemical structure of the investigated compounds as also revealed by electrostatic potential maps. Since the internal core of cell membranes is largely nonpolar, our results support the need of extending the pool of physicochemical descriptors to be determined in the various stages of drug discovery programs and indicate some experimental strategies for their determination.

Modular Design of Chiral Conjugate-Base-Stabilized Carboxylic Acids: Catalytic Enantioselective [4 + 2] Cycloadditions of Acetals.

Readily available 1,2-amino alcohols provide the framework for a new generation of chiral carboxylic acid catalysts that rival the acidity of the widely used chiral phosphoric acid catalyst (S)-TRIP. Covalently linked thiourea sites stabilize the carboxylate conjugate bases of these catalysts via anion-binding, an interaction that is largely responsible for the low pKa values. The utility of the new catalysts is illustrated in the context of challenging [4 + 2] cycloadditions of salicylaldehyde-derived acetals with homoallylic and bishomoallylic alcohols, providing polycyclic chromanes in a highly enantioselective fashion.

Highly Acidic Conjugate-Base-Stabilized Carboxylic Acids Catalyze Enantioselective oxa-Pictet-Spengler Reactions with Ketals.

Acyclic ketone-derived oxocarbenium ions are involved as intermediates in numerous reactions that provide valuable products, however, they have thus far eluded efforts aimed at asymmetric catalysis. We report that a readily accessible chiral carboxylic acid catalyst exerts control over asymmetric cyclizations of acyclic ketone-derived trisubstituted oxocarbenium ions, thereby providing access to highly enantioenriched dihydropyran products containing a tetrasubstituted stereogenic center. The high acidity of the carboxylic acid catalyst, which exceeds that of the well-known chiral phosphoric acid catalyst TRIP, is largely derived from stabilization of the carboxylate conjugate base through intramolecular anion-binding to a thiourea site.

Experimental Basicities of Superbasic Phosphonium Ylides and Phosphazenes.

Experimental basicities of some of the strongest superbases ever measured (phosphonium ylides) are reported, and by employing these compounds, the experimental self-consistent basicity scale of superbases in THF, reaching a pKα (estimate of pKa) of 35 and spanning more than 30 pKa units, has been compiled. Basicities of 47 compounds (around half of which are newly synthesized) are included. The solution basicity of the well-known t-Bu-N═P4(dma)9 phosphazene superbase is now rigorously linked to the scale. The compiled scale is a useful tool for further basicity studies in THF as well as in other solvents, in particular, in acetonitrile. A good correlation between basicities in THF and acetonitrile spanning 25 orders of magnitude gives access to experimentally supported very high (pKa > 40) basicities in acetonitrile, which cannot be directly measured. Analysis of structure-basicity trends is presented.

(15)N NMR Spectroscopy, X-ray and Neutron Diffraction, Quantum-Chemical Calculations, and UV/vis-Spectrophotometric Titrations as Complementary Techniques for the Analysis of Pyridine-Supported Bicyclic Guanidine Superbases.

Pyridine substituted with one and two bicyclic guanidine groups has been studied as a potential source of superbases. 2-{hpp}C5H4N (I) and 2,6-{hpp}2C5H3N (II) (hppH = 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine) were protonated using [HNEt3][BPh4] to afford [I-H][BPh4] (1a), [II-H][BPh4] (2), and [II-H2][BPh4]2 (3). Solution-state (1)H and (15)N NMR spectroscopy shows a symmetrical cation in 2, indicating a facile proton-exchange process in solution. Solid-state (15)N NMR data differentiates between the two groups, indicating a mixed guanidine/guanidinium. X-ray diffraction data are consistent with protonation at the imine nitrogen, confirmed for 1a by single-crystal neutron diffraction. The crystal structure of 1a shows association of two [I-H](+) cations within a cage of [BPh4](-) anions. Computational analysis performed in the gas phase and in MeCN solution shows that the free energy barrier to transfer a proton between imino centers in [II-H](+) is 1 order of magnitude lower in MeCN than in the gas phase. The results provide evidence that linking hpp groups with the pyridyl group stabilizes the protonation center, thereby increasing the intrinsic basicity in the gas phase, while the bulk prevents efficient cation solvation, resulting in diminished pKa(MeCN) values. Spectrophotometrically measured pKa values are in excellent agreement with calculated values and confirm that I and II are superbases in solution.

Experimental Basicities of Phosphazene, Guanidinophosphazene, and Proton Sponge Superbases in the Gas Phase and Solution.

Experimental gas-phase superbasicity scale spanning 20 orders of magnitude and ranging from bicyclic guanidine 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene to triguanidinophosphazenes and P3 phosphazenes is presented together with solution basicity data in acetonitrile and tetrahydrofuran. The most basic compound in the scale-triguanidinophosphazene Et-N═P[N═C(NMe2)2]3-has the highest experimental gas-phase basicity of an organic base ever reported: 273.9 kcal mol(-1). The scale includes besides the higher homologues of classical superbasic phosphazenes and several guanidino-substituted phosphazenes also a number of recently introduced bisphosphazene and bis-guanidino proton sponges. This advancement was made possible by a newly designed Fourier transform ion cyclotron resonance (ICR) mass spectrometry setup with the unique ability to generate and control in the ICR cell sufficient vapor pressures of two delicate compounds having low volatility, which enables determining their basicity difference. The obtained experimental gas-phase and solution basicity data are analyzed in terms of structural and solvent effects and compared with data from theoretical calculations.

Solution and gas-phase acidities of all-trans (all-E) retinoic acid: an experimental and computational study.

Retinoic acid is of fundamental biological importance. Its acidity was determined in the gas phase and in acetonitrile solution by means of mass spectrometry and UV/Vis spectrophotometry, respectively. The intrinsic acidity is slightly higher than that of benzoic acid. In solution, the situation is opposite. The experimental systems were described theoretically applying quantum chemical methods (wave function theory and density functional theory). This allowed the determination of the molecular structure of the acid and its conjugate base, both in vacuo and in solution, and for computational estimates of its acidity in both phases.

Fluoro- and perfluoralkylsulfonylpentafluoroanilides: synthesis and characterization of NH acids for weakly coordinating anions and their gas-phase and solution acidities.

Fluoro- and perfluoralkylsulfonyl pentafluoroanilides [HN(C6F5)(SO2X); X = F, CF3, C4F9, C8F17] are a class of imides with two different strongly electron-withdrawing substituents attached to a nitrogen atom. They are NH acids, the unsymmetrical hybrids of the well-known symmetrical bissulfonylimides and bispentafluorophenylamine. The syntheses, the structures of these perfluoroanilides, their solvates, and some selected lithium salts give rise to a structural variety beyond the symmetrical parent compounds. The acidities of representative subsets of these novel NH acids have been investigated experimentally and quantum-chemically and their gas-phase acidities (GAs) are reported, as well as the pKa values of these compounds in acetonitrile (MeCN) and DMSO solution. In quantum chemical investigations with the vertical and relaxed COSMO cluster-continuum models (vCCC/rCCC), the unusual situation is encountered that the DMSO-solvated acid Me2SO-H-N(SO2CF3)2, optimized in the gas phase (vCCC model), dissociates to Me2SO-H(+)-N(SO2CF3)2(-) during structural relaxation and full optimization with the solvation model turned on (rCCC model). This proton transfer underlines the extremely high acidity of HN(SO2CF3)2. The importance of this effect is studied computationally in DMSO and MeCN solution. Usually this effect is less pronounced in MeCN and is of higher importance in the more basic solvent DMSO. Nevertheless, the neglect of the structural relaxation upon solvation causes typical changes in the computational pKa values of 1 to 4 orders of magnitude (4-20 kJ mol(-1)). The results provide evidence that the published experimental DMSO pKa value of HN(SO2CF3)2 should rather be interpreted as the pKa of a Me2SO-H(+)-N(SO2CF3)2(-) contact ion pair.

Basicity Limits of Neutral Organic Superbases.

The potential limits of superbasicity achievable with different families of neutral bases by expanding the molecular framework are explored using DFT computations. A number of different core structures of non-ionic organosuperbases are considered (such as phosphazenes, guanidinophosphazenes, guanidino phosphorus ylides). A simple model for describing the dependence of basicity on the extent of the molecular framework is proposed, validated, and used for quantitatively predicting the ultimate basicities of different compound families and the rates of substituent effect saturation. Some of the considered bases (guanidino phosphorus carbenes) are expected to reach gas-phase basicity around 370 kcal mol(-1), thus being the most basic neutral bases ever reported. Also, the classical substituted alkylphosphazenes were predicted to reach pK(a) values of around 50 in acetonitrile, which is significantly higher than previously expected.

Very strong organosuperbases formed by combining imidazole and guanidine bases: synthesis, structure, and basicity.

New structural motives for organosuperbases, that are easy to prepare and highly basic are urgently required in many areas of chemistry. The synthesis of N,N'-bis(imidazolyl)guanidine bases (BIG bases) is reported. Their pKα  values are determined as 26.1-29.3 in THF. They are thus probably the strongest known phosphorous-free organic bases both in solution and in the gas phase. Calculations help to determine the structural and electronic factors giving rise to the high basicity.

A new class of organosuperbases, N-alkyl- and N-aryl-1,3-dialkyl-4,5-dimethylimidazol-2-ylidene amines: synthesis, structure, pK(BH+) measurements, and properties.

A series of stable organosuperbases, N-alkyl- and N-aryl-1,3-dialkyl-4,5-dimethylimidazol-2-ylidene amines, were efficiently synthesized from N,N'-dialkylthioureas and 3-hydroxy-2-butanone and their basicities were measured in acetonitrile. The derivatives with tert-alkyl groups on the imino nitrogen were found to be more basic than the tBuP(1) (pyrr) phosphazene base in acetonitrile. The origin of the high basicity of these compounds is discussed. In acetonitrile and in the gas phase, the basicity of the alkylimino derivatives depends on the size of the substituent at the imino group, which influences the degree of aromatization of the imidazole ring, as measured by (13)C NMR chemical shifts or by the calculated ΔNICS(1) aromaticity parameters, as well as on solvation effects. If a wider range of imino-substituents, including electron-acceptor substituents, is treated in the analysis then the influence of aromatization is less predominant and the gas-phase basicity becomes more dependent on the field-inductive effect, polarizability, and resonance effects of the substituent.

Retention mechanisms of acidic and basic analytes on the Pentafluorophenyl stationary phase using fluorinated eluent additives.

This work explores the effects of three selected fluoroalcohols - 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), 1,1,1,3,3,3-hexafluorotert­butyl alcohol (HFTB) and hexafluoro-2,3-(trifluoromethyl)-2,3-butanediol (PP) as novel eluent additives and their effect on the retention of basic and acidic analytes, using a reversed phase (RP) column with a fluorophenyl (PFP) stationary phase. In order to observe the changes in the model analytes' retention, chromatograms were obtained at multiple (5.0; 6.0; 7.0; 8.5; 9.0 and 9.5) pH values depending on the eluent. The retention observed with fluoroalcohols was compared with that of a conventional eluent additive - ammonium acetate. When fluoroalcohols were used as eluent additives, a decrease in the retention factors (compared with ammonium acetate) was generally observed for strong acids. The retention factors of strong bases were generally higher when using HFIP and HFTB as eluent additives. The behaviour of weak bases and weak acids was more nuanced, potentially enabling interesting selectivity. The extent of the effect regarding different fluoroalcohols also varied, with HFIP and HFTB having a more significant effect on the retention of analytes than PP. The retention data were interpreted in terms of the hypothesis that four interactions are at play: (a) hydrophobic retention typical to RP; (b) π-π interactions between the analytes containing an aromatic ring and the aromatic rings on the stationary phase; (c) charge-charge or hydrogen bond interactions between the analytes and partially deprotonated fluoroalcohols adsorbed on the stationary phase and (d) a hydrogen bond or charge-charge interaction between the free silanol groups or their deprotonated forms on the stationary phase and the analytes (either neutral or ionic). Alternative selectivity obtained through fluoroalcohols on the PFP stationary phase was compared with the C18 and biphenyl stationary phases. It was demonstrated that at the same eluent pH but with a different buffer system and/or different RP stationary phases, very different selectivity and retention order can be obtained.

Equilibrium acidities of superacids.

In this paper, we report the most comprehensive equilibrium superacidity scale that is available to date. Contrary to most of the past works, this scale is set up in a medium of constant composition and the obtained acidity values characterize the acidities of molecules rather than acidities of media. The current scale is thus complementary to the well-known H(0) scale in the information that it provides. The solvent used is 1,2-dichloroethane (DCE). DCE has very weak basic properties (but sufficiently high polarity) and is an appropriate solvent for measuring acidities of very strong acids of diverse chemical nature. DCE acidities of well-known superacids (CF(3)SO(2)OH, (CF(3)SO(2))(2)NH, cyanocarbon acids, etc.) as well as common mineral acids (H(2)SO(4), HI, HBr, etc.) are reported. Acidities of altogether 62 acids have been determined from 176 interlinked relative acidity measurements. The scale spans 15 orders of magnitude (from picric acid to 1,1,2,3,3-pentacyanopropene) and is expected to be a useful tool in design, use, and further acidity measurements of superacidic molecules.

The perfluorinated alcohols (F5C6)(F3C)2COH and (F5C6)(F10C5)COH: synthesis, theoretical and acidity studies, spectroscopy and structures in the solid state and the gas phase.

The syntheses of the perfluorinated alcohols (F(5)C(6))(F(3)C)(2)COH (1) and (F(5)C(6))(C(5)F(10))COH (2) are described. Both compounds were prepared in reasonable yields (1: 65%, 2: 85%) by reacting the corresponding ketone with C(6)F(5)MgBr, followed by acidic work-up. The alcohols were characterized by NMR, vibrational spectroscopy, single-crystal X-ray diffraction, acidity measurements and gas-phase electron diffraction. A combination of appropriate 2D NMR experiments allowed the unambiguous assignment of all signals in the (19)F spin systems, of which that of 2 was especially complex. High acidity of the alcohols is indicated by acidity measurements as well as the calculated gas phase acidities. It is also supported by the crystal structure of 2, which exhibits only a single weak intermolecular hydrogen bridge with an O...O distance of 301 pm. This shows the low donor strength of the oxygen atom in the compound, which is partly compensated through formation of two intramolecular CF...H contacts of 220 and 232 pm length to the proton not involved in the hydrogen bridge. The pK(a) values in acetonitrile are 22.2 for 1 and 22.0 for 2; their calculated gas phase acidities are 1367 and 1343 kJ mol(-1) (MP2/TZVPP level).

Rifampicin as an example of beyond-rule-of-5 compound: Ionization beyond water and lipophilicity beyond octanol/water.

Ionization and lipophilicity in early drug discovery are commonly characterized in water and octanol/water, respectively and thus do not consider the non-polar features of the biomembrane core. This is particularly limiting for bRo5 compounds which may adapt their properties (e.g. ionization and lipophilicity) to the environment. In this paper we used experimental methods to characterize rifampicin for its ionization properties in various water/cosolvent mixtures and in pure MeCN and its lipophilicity in octanol/water and toluene/water systems. Moreover, we also measured log k'80 PLRP-S, a chromatographic index of lipophilicity in non-polar media. Results show that the existence domain of neutral rifampicin is limited compared to the zwitterion, but the lipophilic cationic species is extremely relevant in non-polar environments.

Evaluation and validation of detailed and simplified models of the uncertainty of unified [Formula: see text] measurements in aqueous solutions.

The use of the unified pH concept, [Formula: see text] , applicable to aqueous and non-aqueous solutions, which allows interpreting and comparison of the acidity of different types of solutions, requires reliable and objective determination. The [Formula: see text] can be determined by a single differential potentiometry measurement referenced to an aqueous reference buffer or by a ladder of differential potentiometric measurements that allows minimisation of inconsistencies of various determinations. This work describes and assesses bottom-up evaluations of the uncertainty of these measurements, where uncertainty components are combined by the Monte Carlo Method (MCM) or Taylor Series Approximation (TSM). The MCM allows a detailed simulation of the measurements, including an iterative process involving in minimising ladder deviations. On the other hand, the TSM requires the approximate determination of minimisation uncertainty. The uncertainty evaluation was successfully applied to measuring aqueous buffers with pH of 2.00, 4.00, 7.00, and 10.00, with a standard uncertainty of 0.01. The reference and estimated values from both approaches are metrologically compatible for a 95% confidence level even when a negligible contribution of liquid junction potential uncertainty is assumed. The MCM estimated pH values with an expanded uncertainty, for the 95% confidence level, between 0.26 and 0.51, depending on the pH value and ladder inconsistencies. The minimisation uncertainty is negligible or responsible for up to 87% of the measurement uncertainty. The TSM quantified measurement uncertainties on average only 0.05 units larger than the MCM estimated ones. Additional experimental tests should be performed to test these uncertainty models for analysis performed in other laboratories and on non-aqueous solutions.

Revision of the gas-phase acidity scale below 300 kcal mol(-1).

The gas-phase acidity (GA) scale from (CF(3)CO)(2)NH to (C(2)F(5)SO(2))(2)NH--about a 24 kcal mol(-1) range of gas-phase acidities--was reexamined using the Fourier transform ion cyclotron resonance equilibrium measurement approach. Some additions and modifications to the standard methodology of GA measurements were introduced (estimation of partial pressures from mass spectra of the compounds, instead of the pressure gauge readings and use of long reaction times) to achieve higher reliability. Gas-phase acidities of 18 compounds were determined for the first time. The results reveal a contraction of the previously published values in this part of the scale. In particular, the GA values of (CF(3)SO(2))(2)NH and (C(2)F(5)SO(2))(2)NH (important components of lithium ion battery electrolytes and ionic liquids) were revised toward stronger acidities from 291.8 kcal mol(-1) to 286.5 kcal mol(-1) and from 289.4 kcal mol(-1) to 283.7 kcal mol(-1) (i.e., by 5.3 and 5.7 kcal mol(-1)), respectively. Experimental and computational evidence is presented in support of the current results.

Alternative eluent composition for LC-MS analysis of perfluoroalkyl acids in raw fish samples.

A wide range of anthropogenic pollutants that possess serious environmental and health risks are known. One type of these harmful substances that have become a focus of interest during the past decade are perfluoroalkyl acids (PFAAs), which are extensively used in industry for different purposes. Due to the harmful effects that these compounds might cause in living organisms, EFSA and EU CONTAM panel have issued a monitoring program for PFAAs in foodstuffs. This has given rise to intense research dedicated to the analysis of PFAAs over the past few years. This work focuses on chromatographic analysis of three PFAAs in fish. The analytes, perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorooctanesulfonic acid (PFOS), are commonly associated with the production of fluoropolymers. Fluorinated alcohols are used as eluent components, and their possible advantages as eluent modifiers in LC-MS analysis of PFAAs, alternative retention mechanism and enhanced ionization efficiency, are examined. The analyzed fish samples originating from Estonian fresh and marine waters had low contents of PFAAs.