The relative influences of acidity and polarity on responsiveness of small organic molecules to analysis with negative ion electrospray ionization mass spectrometry (ESI-MS)

The purpose of the work presented here was to evaluate the influence of solution composition and analyte characteristics on responsiveness to analysis with negative ion electrospray ionization mass spectrometry. The responses of a series of structurally diverse acidic molecules were compared in various solvents. Response was generally observed to be higher in methanol than acetonitrile and response for all analytes was poorer when water was mixed with the organic solvent. A positive correlation between negative ion ESI-MS response and log P was observed when either acetonitrile or methanol was used as the electrospray solvent. This result was expected because analytes with significant nonpolar character should be particularly responsive to ESI-MS analysis due to their higher affinity for electrospray droplet surfaces. It was also predicted that highly acidic analytes would be most responsive to analysis with negative ion ESI-MS due to their tendency to form negative ions. However, for the analytes studied here, acidity was found not to have a consistent influence on ESI-MS response. Many of the highly acidic molecules were quite polar and, consequently, were poorly responsive. Furthermore, the deprotonated molecular ion was detected for a number of molecules with very high pKa values, which would not be expected to form negative ions in the bulk solution. Ultimately, these results indicate that acidity is not a conclusive parameter for prediction of the relative magnitudes of negative ion ESI-MS response among a diverse series of analytes. Analyte polarity does; however, appear to be useful for this purpose.     

Processes that affect electrospray ionization-mass spectrometry of nucleobases and nucleosides

Due to the complexity of electrospray ionization processes and the many factors that affect the ion signal, optimization of electrospray ionization methods to gain ultimate sensitivity for analysis of nucleobases and nucleosides may not be straightforward. In this work, we investigated the effect of the pK _a and the gas-phase basicity of analyte and other electrolytes on the [M+H]⁺ ion signal for 11 select nucleobases and nucleosides in 50% methanol:water solution. Solution chemistry plays a role in the electrospray signal for all analytes, but gas-phase chemistry may be important for compounds with pK _a <3 depending on the solution composition. For compounds with pK _a <3, gas-phase proton transfer reactions can be promoted to increase analyte electrospray response by the addition of ammonium acetate to the solution.     

Capillary electrophoresis in N,N-dimethylformamide

N,N-Dimethylformamide (DMF) is a dipolar protophilic solvent with physicochemical properties that makes it suitable as solvent for capillary electrophoresis (CE). It is prerequisite for the proper application of CE to adjust and to change the pH of the background electrolyte (BGE) in a defined manner. This was done in the present work using benzoic acid-benzoate by selecting different concentration ratios of acid and salt, and calculating the theoretical pH from the activity-corrected Henderson-Hasselbalch equation. The mobilities of the analytes (chloro- and nitro-substituted phenolates) were found to follow reasonably well the typical sigmoid mobility versus pH curve as predicted by theory. The actual mobilities and pK(a) values (at 25 degrees C) of the analytes were derived from these curves. pK(a) values were in the range of 11.1-11.7, being thus 3-4.4 units higher than in water. This pK(a) shift is caused by the destabilization of the analyte anion and the better stability (solubility) of the molecular analyte acid in DMF, which overcome the higher basicity of DMF compared to water. Absolute mobilities were calculated from the actual mobilities; they were between 32x10(-9) and 42x10(-9) m(2)/Vxs. Slight deviations of the measured mobilities from the theoretical mobility versus pH curve were discussed on the bases of ion pairing and heteroconjugation and homoconjugation of either buffer components or buffer components and analytes. Heteroconjugation was used as a mechanism for the electrically driven separation of neutral analyte molecules in a BGE where salicylate acted as complex forming ion. Rough estimation of the complexation constants for the phenolic analytes gave values in the range of 100-200 L/mol. Addition of water to the solvent decreased the effect of heteroconjugation, but it was still present up to the surprisingly high concentration of 20% water. Electrophoretically relevant parameters like ionic mobilities and pK(a) values, and conjugation and ion pairing are dependent on the water content of the solvent. The water uptake of DMF was measured when exposed to humidity of ambient air. The resulted behavior of the water uptake was found rather similar to that for acetonitrile and methanol.     

Importance of gas-phase proton affinities in determining the electrospray ionization response for analytes and solvents

The effect of gas-phase proton transfer reactions on the mass spectral response of solvents and analytes with known gas-phase proton affinities was evaluated. Methanol, ethanol, propanol and water mixtures were employed to probe the effect of gas-phase proton transfer reactions on the abundance of protonated solvent ions. Ion-molecule reactions were carried out either in an atmospheric pressure electrospray ionization source or in the central quadrupole of a triple-quadrupole mass spectrometer. The introduction of solvent vapor with higher gas-phase proton affinity than the solvent being electrosprayed caused protons to transfer to the gas-phase solvent molecules. In mixed solvents, protonated solvent clusters of the solvent with higher gas-phase proton affinity dominated the resulting mass spectra. The effect of solvent gas-phase proton affinity on analyte response was also investigated, and the analyte response was suppressed or eliminated in solvents with gas-phase proton affinities higher than that of the analyte.     

Formation and decompositions of chloride adduct ions, [M + Cl]−, in negative ion electrospray ionization mass spectrometry

The ability to promote chloride-attachment ions of the form [M + Cl]- in negative ion electrospray ionization mass spectrometry (ESI-MS) has been developed using chlorinated solvents such as chloroform and carbon tetrachloride. This approach expands the current capabilities of negative ion ESI-MS by enabling detection of analytes that lack acidic sites and thus exhibit weak [M - H]- signals. In contrast to the remote-site collision-induced dissociation (CID) often observed in positive ion ESI-MS/MS for alkali metal cation adducts, the decomposition of chloride adducts usually proceeds via competitive dissociations to form Cl-, which is not structurally informative, or [M - H]-. The latter can provide structural information via consecutive decompositions. For compounds having higher gas-phase acidities than HCl, a low CID collision energy can promote the formation of [M - H]-, whereas for the majority of compounds with lower gas phase acidities than HCl, higher collision energies generally improve the relative yield of [M- H] . Because chloride attachment occurs primarily at electrophilic hydrogens, the daughter ion ratio, Cl-/[M - H]-, depends primarily upon the difference in gas phase acidity between the analyte molecule and HCl. At higher collision energies, entropic factors take on increased importance in determining the product ratio. The difference between the deltaS(0) terms for formation of Cl and formation of [M - H]- has been estimated for a series of substituted phenols and a series of acetic acid analogs. Finally, a novel neutral loss of CH3Cl from glycerophosphocholine and from ganglioside GM3 methyl ester is reported.     

Quantitative aspects of and ionization mechanisms in positive-ion atmospheric pressure chemical ionization mass spectrometry

The behavior in atmospheric pressure chemical ionization of selected model polycyclic aromatic compounds, pyrene, dibenzothiophene, carbazole, and fluorenone, was studied in the solvents acetonitrile, methanol, and toluene. Relative ionization efficiency and sensitivity were highest in toluene and lowest in methanol, a mixture of molecular ions and protonated molecules was observed in most instances, and interferences between analytes were detected at higher concentrations. Such interferences were assumed to be caused by a competition among analyte molecules for a limited number of reagent ions in the plasma. The presence of both molecular ions and protonated analyte molecules can be attributed to charge-transfer from solvent radical cations and proton transfer from protonated solvent molecules, respectively. The order of ionization efficiency could be explained by incorporating the effect of solvation in the ionization reactions. Thermodynamic data, both experimental and calculated theoretically, are presented to support the proposed ionization mechanisms. The analytical implications of the results are that using acetonitrile (compared with methanol) as solvent will provide better sensitivity with fewer interferences (at low concentrations), except for analytes having high gas-phase basicities.     

Using a nanoelectrospray-differential mobility spectrometer-mass spectrometer system for the analysis of oligosaccharides with solvent selected control over ESI aggregate ion formation

Differential mobility spectrometry (DMS), also commonly referred to as high field asymmetric waveform ion mobility spectrometry (FAIMS) is a rapidly advancing technology for gas-phase ion separation. The interfacing of DMS with mass spectrometry (MS) offers potential advantages over the use of mass spectrometry alone. Such advantages include improvements to mass spectral signal/noise, orthogonal/complementary ion separation to mass spectrometry, enhanced ion and complexation structural analysis, and the potential for rapid analyte quantitation. In this report, we demonstrate the successful use of our nanoESI-DMS-MS system, with a methanol drift gas modifier, for the separation of oligosaccharides. The tendency for ESI to form oligosaccharide aggregate ions and the negative impact this has on nanoESI-DMS-MS oligosaccharide analysis is described. In addition, we demonstrate the importance of sample solvent selection for controlling nanoESI oligosaccharide aggregate ion formation and its effect on glycan ionization and DMS separation. The successful use of a tetrachloroethane/methanol solvent solution to reduce ESI oligosaccharide aggregate ion formation while efficiently forming a dominant MH(+) molecular ion is presented. By reducing aggregate ion formation in favor of a dominant MH(+) ion, DMS selectivity and specificity is improved. In addition to DMS, we would expect the reduction in aggregate ion complexity to be beneficial to the analysis of oligosaccharides for other post-ESI separation techniques such as mass spectrometry and ion mobility. The solvent selected control over MH(+) molecular ion formation, offered by the use of the tetrachloroethane/methanol solvent, also holds promise for enhancing MS/MS structural characterization analysis of glycans.     

Solvent effect on analyte charge state,signal intensity,and stability in negative ion electrospray mass spectrometry; implications for the mechanism of negative ion formation

Department of Chemistry, University of New Orleans, New Orleans, Louisiana, USA The effect of solvent composition on negative ion electrospray ionization (ESI) mass spectrometry was examined. The onset potentials for ES1 of a series of chlorinated solvents and methanol were found to be within the range predicted by D. P. H. Smith, based on differences in the surface tension of the solvents used. The tendency toward electric discharge decreased with increasing percent weight of chlorine in the solvent. This effect has been attributed to an increasing propensity for electron capture for more highly chlorinated solvents. Addition of the electron scavenger gas SF, was even more effective at suppressing corona discharge phenomena. In a comparison of ultimate signal intensity obtainable for a test analyte in 10% methanol, the highest signal, which was stable over the widest range of temperatures, was exhibited by chloroform compared to dichloromethane, 1,2-dichloroethane, carbon tetrachloride, and methanol (100%). Chloroform, thus, is a recommended solvent for negative ion electrospray mass spectrometry (ES/MS) when solubility is not a limiting issue. Solvent polarity was shown to exhibit a profound influence on the distribution of charge states in negative ion ES/MS. For both chlorinated and nonchlorinated organic solvents, the higher the solution dielectric constant, the more the charge-state distribution is shifted toward higher charge states. These observations build on the “electrophoretic” mechanism of droplet charging. Solvents with high solution dielectric constants are considered to be most effective at stabilizing multiply charged ions (where charge separation is greatest), and they are likely to increase the level of droplet charging. Solvents with high basicities (gas phase and solution phase) and high proton affinities, yet low dielectric constants, favor lower charge states in ES mass spectra of lipid A and cardiolipin from Escherichia coli. This indicates that gas-phase processes and solvent basicity contribute much less toward ion formation than solution-phase solvation via preferred orientation of the solvent dipole.     

Gas sensing mechanism in chemiresistive cobalt and metal-free phthalocyanine thin films

The gas sensing behaviors of cobalt phthalocyanine (CoPc) and metal-free phthalocyanine (H2Pc) thin films were investigated with respect to analyte basicity. Chemiresistive sensors were fabricated by deposition of 50 nm thick films on interdigitated gold electrodes via organic molecular beam epitaxy (OMBE). Time-dependent current responses of the films were measured at constant voltage during exposure to analyte vapor doses. The analytes spanned a range of electron donor and hydrogen-bonding strengths. It was found that, when the analyte exceeded a critical base strength, the device responses for CoPc correlated with Lewis basicity, and device responses for H2Pc correlated with hydrogen-bond basicity. This suggests that the analyte-phthalocyanine interaction is dominated by binding to the central cavity of the phthalocyanine with analyte coordination strength governing CoPc sensor responses and analyte hydrogen-bonding ability governing H2Pc sensor responses. The interactions between the phthalocyanine films and analytes were found to follow first-order kinetics. The influence of O2 on the film response was found to significantly affect sensor response and recovery. The increase of resistance generally observed for analyte binding can be attributed to hole destruction in the semiconductor film by oxygen displacement, as well as hole trapping by electron donor ligands.     

Evaluation of doped and undoped poly (o‐anisidine) as sensing materials for a sensor array for volatile organic compounds

Poly (o‐anisidine) (PoANI) and PoANI doped with nickel oxide and zinc oxide were evaluated as sensing materials for four gas analytes (methanol, ethanol, acetone, and benzene). The sensing materials had high sensitivity (showing an affinity towards the target analytes even at low concentrations, in the range of 1‐5 ppm), but rather poor selectivity, especially when the gas analytes were in a mixture. To exploit the poor selectivity, the three sensing materials were combined into a sensor array using principal component analysis (PCA) as a sensing algorithm. It was found that using a sensor array, the four individual gases could be separated. However, when all four gases were present (in analyte mixtures), there was too much overlap in the responses to distinguish between individual gas analytes and their related mixtures.     

Identification and counting of carbonyl and hydroxyl functionalities in protonated bifunctional analytes by using solution derivatization prior to mass spectrometric analysis via ion-molecule reactions

A mass spectrometric method has been developed for the identification of carbonyl and hydroxyl functional groups, as well as for counting the functional groups, in previously unknown protonated bifunctional oxygen-containing analytes. This method utilizes solution reduction before mass spectrometric analysis to convert the carbonyl groups to hydroxyl groups. Gas-phase ion-molecule reactions of the protonated reduced analytes with neutral trimethylborate (TMB) in a FT-ICR mass spectrometer give diagnostic product ions. The reaction sequence likely involves three consecutive steps, proton abstraction from the protonated analyte by TMB, addition of the neutral analyte to the boron reagent, and elimination of a neutral methanol molecule. The number of methanol molecules eliminated upon reactions with TMB reveals the number of hydroxyl groups in the analyte. Comparison of the reactions of the original and reduced analytes reveals the presence and number of carbonyl and hydroxyl groups in the analyte.     

Minimizing analyte electrolysis in an electrospray emitter

The electrospray (ES) ion source is a controlled-current electrolytic flow cell. Electrolytic reactions in the ES emitter capillary are continually ongoing to sustain the production of charged droplets and ultimately gas-phase ions from this device. Under certain circumstances, the analytes under study may be directly involved in these electrolytic processes. It is demonstrated that a simple means to minimize analyte electrolysis is to exchange the normal metal emitter capillary of commercial ES sources with one made of fused silica. This change is shown to provide an ES mass spectrometric system of similar performance in terms of gas-phase ion signal generated for non-electroactive analytes and also assures minimal oxidation of electroactive analytes even at low (2.0 microl x min(-1)) solution flow-rates and high (millimolar) solution electrolyte concentrations.     

Suppression and enhancement of secondary ion formation due to the chemical environment in static-secondary ion mass spectrometry

Through analyzing mixtures of compounds of known gas-phase basicities, the importance of this property on the secondary ions emitted from a surface under primary ion bombardment is investigated. The aim is to obtain a greater understanding of the ionization mechanisms that occur in secondary ion mass spectrometry (SIMS). The commonly used matrix assisted laser desorption/ionization (MALDI) matrix 2,4,6-trihydroxyacetophenone (THAP) and a range of low molecular weight biomolecules were used to investigate whether analyte/matrix suppression effects that have been observed in analogous MALDI experiments were also present in static-SIMS. The outcome of the experiments demonstrates that strong suppression of the quasi-molecular signal of one molecule in a mixture can occur due to the presence of the other, with the gas-phase basicity of the compounds being a good indicator of the secondary ions detected. It is also demonstrated that the suppression of the quasi-molecular ion signal of a compound in a two-component mixture can be minimized by the inclusion of a third compound of suitable gas-phase basicity.     

Taylor dispersion monitored by electrospray mass spectrometry: a novel approach for studying diffusion in solution

This work describes a novel approach for monitoring analyte diffusion in solution that is based on electrospray ionization mass spectrometry (ESI-MS). A mass spectrometer at the end of a laminar flow tube is used to measure the Taylor dispersion of an initially sharp boundary between two solutions of different analyte concentration. This boundary is dispersed by the laminar flow profile in the tube. However, this effect is diminished by analyte diffusion that continuously changes the radial position, and hence the flow velocity of individual analyte molecules. The steepness of the resulting dispersion profile therefore increases with increasing diffusion coefficient of the analyte. A theoretical framework is developed to adapt the equations governing the dispersion process to the case of mass spectrometric detection. This novel technique is applied to determine the diffusion coefficients of choline and cytochrome c. The measured diffusion coefficients, (11.9 +/- 1.0) x 10(-10) m(2) s(-1) and (1.35 +/- 0.08) x 10(-10) m(2) s(-1), respectively, are in agreement with the results of control experiments where the Taylor dispersion of these two analytes was monitored optically. Due to the inherent selectivity and sensitivity of ESI-MS, it appears that the approach described in this work could become a valuable alternative to existing methods for studying diffusion processes, especially for experiments on multicomponent systems.     

Negative ion-atmospheric pressure photoionization-mass spectrometry

The ionization mechanism in the novel atmospheric pressure photoionization mass spectrometry (APPI-MS) in negative ion mode was studied thoroughly by the analysis of seven compounds in 17 solvent systems. The compounds possessed either gas-phase acidity or positive electron affinity, whereas the solvent systems had different polarities and gas-phase acidities and some of them positive electron affinities. The analytes that possessed gas-phase acidity formed deprotonated ions in proton transfer; in addition, fragments and solvent adducts were observed. The compounds of positive electron affinity formed negative molecular ions by electron capture or charge exchange and substitution products of form [M - X + O](-) by substitution reactions. The efficiency of deprotonation was decreased if the solvent used possessed higher gas-phase acidity than the analyte. Solvents of positive electron affinity captured thermal electrons and deteriorated the ionization of all the analytes. Also, the proportion of substitution products was affected by the solvent. Finally, the performances of negative ion APPI and negative ion APCI were compared. The sensitivity for the studied compounds was better in APPI, but the formation of substitution products was lower in APCI.     

A glass capillary ultramicroelectrode with an electrokinetic sampling ability.

A glass capillary ultramicroelectrode (tip diameter approximately 1.2 microm) having an electrokinetic sampling ability is described. It is composed of a pulled glass capillary filled with an inner solution and three internal electrodes (Pt working and counter electrodes and an Ag/AgCl reference electrode). The voltammetric response of the capillary electrode is based on electrokinetic transport of analyte ions from the sample solution into the inner solution across the conical tip. It was found that the electrophoretic migration of analytes at the conical tip is faster than electroosmotic flow, enabling electrokinetic transport of analyte ions into the inner solution of the electrode. By using [Fe(CN)6]4- and (ferrocenylmethyl)trimethylammonium (FcTMA+) ions as model analytes, differential pulse voltammetric responses of the capillary electrode were investigated in terms of tip diameter of the capillary, sampling voltage, sampling time, detection limit and selectivity. The magnitude of the response depends on the size and charge of analyte ions. With a capillary electrode having a approximately 1.2-microm tip diameter, which minimizes non-selective diffusional entry of analytes, the response after 1 h sampling at +1.7 V is linearly related to [Fe(CN)6]4- concentration in the range of 0.50-5.0 mM with the detection limit of 30 microM. Application of a potential of the same sign as that of the analyte ion forces the analyte to move out from the electrode to the solution, enabling reuse of the same capillary electrode. The charge-selective detection of analytes with the capillary electrode is demonstrated for [Fe(CN)6]4- in the presence of FcTMA+.     

Identification of Epoxide Functionalities in Protonated Monofunctional Analytes by Using Ion/Molecule Reactions and Collision-Activated Dissociation in Different Ion Trap Tandem Mass Spectrometers

A mass spectrometric method has been delineated for the identification of the epoxide functionalities in unknown monofunctional analytes. This method utilizes gas-phase ion/molecule reactions of protonated analytes with neutral trimethyl borate (TMB) followed by collision-activated dissociation (CAD) in an ion trapping mass spectrometer (tested for a Fourier-transform ion cyclotron resonance and a linear quadrupole ion trap). The ion/molecule reaction involves proton transfer from the protonated analyte to TMB, followed by addition of the analyte to TMB and elimination of methanol. Based on literature, this reaction allows the general identification of oxygen-containing analytes. Vinyl and phenyl epoxides can be differentiated from other oxygen-containing analytes, including other epoxides, based on the loss of a second methanol molecule upon CAD of the addition/methanol elimination product. The only other analytes found to undergo this elimination are some amides but they also lose O = B-R (R = group bound to carbonyl), which allows their identification. On the other hand, other epoxides can be differentiated from vinyl and phenyl epoxides and from other monofunctional analytes based on the loss of (CH₃O)₂BOH or formation of protonated (CH₃O)₂BOH upon CAD of the addition/methanol elimination product. For propylene oxide and 2,3-dimethyloxirane, the (CH₃O)₂BOH fragment is more basic than the hydrocarbon fragment, and the diagnostic ion (CH₃O)₂BOH₂⁺ is formed. These reactions involve opening of the epoxide ring. The only other analytes found to undergo (CH₃O)₂BOH elimination are carboxylic acids, but they can be differentiated from the rest based on several published ion/molecule reaction methods. Similar results were obtained in the Fourier-transform ion cyclotron resonance and linear quadrupole ion trap mass spectrometer.     

Separation mechanism of chiral impurities, ephedrine and pseudoephedrine, found in amphetamine-type substances using achiral modifiers in the gas phase

A new mechanism is proposed that describes the gas-phase separation of chiral molecules found in amphetamine-type substances (ATS) by the use of high-resolution ion mobility spectrometry (IMS). Straight-chain achiral alcohols of increasing carbon chain length, from methanol to n-octanol, are used as drift gas modifiers in IMS to highlight the mechanism proposed for gas-phase separations of these chiral molecules. The results suggest the possibility of using these achiral modifiers to separate the chiral molecules (R,S) and (S,R)-ephedrine and (S,S) and (R,R)-pseudoephedrine which contain an internal hydroxyl group at the first chiral center and an amino group at the other chiral center. Ionization was achieved with an electrospray source, the ions were introduced into an IMS with a resolving power of 80, and the resulting ion clusters were characterized with a coupled quadrupole mass spectrometer detector. A complementary computational study conducted at the density functional B3LYP/6-31g level of theory for the electronic structure of the analyte–modifier clusters was also performed, and showed either “bridged” or “independent” binding. The combined experimental and simulation data support the proposed mechanism for gas-phase chiral separations using achiral modifiers in the gas phase, thus enhancing the potential to conduct fast chiral separations with relative ease and efficiency.     

Binding energies of protonated betaine complexes: a probe of zwitterion structure in the gas phase

The dissociation kinetics of proton-bound dimers of betaine with molecules of comparable gas-phase basicity were investigated using blackbody infrared radiative dissociation (BIRD). Threshold dissociation energies were obtained from these data using master equation modeling. For bases that have comparable or higher gas-phase basicity, the binding energy of the protonated base.betaine complex is ~1.4 eV. For molecules that are ~2 kcal/mol or more less basic, the dissociation energy of the complexes is ~1.2 eV. The higher binding energy of the former is attributed to an ion-zwitterion structure which has a much larger ion-dipole interaction. The lower binding energy for molecules that are ~2 kcal/mol or more less basic indicates that an ion-molecule structure is more favored. Semiempirical calculations at both the AM1 and PM3 levels indicate the most stable ion-molecule structure is one in which the base interacts with the charged quaternary ammonium end of betaine. These results indicate that the measurement of binding energies of neutral molecules to biological ions could provide a useful probe for the presence of zwitterions and salt bridges in the gas phase. From the BIRD data, the gas-phase basicity of betaine obtained from the kinetic method is found to be 239.2 +/- 1.0 kcal/mol. This value is in excellent agreement with the value of 239.3 kcal/mol (298 K) from ab initio calculations at the MP2/6-31+g** level. The measured value is slightly higher than those reported previously. This difference is attributed to entropy effects. The lower ion internal energy and longer time frame of BIRD experiments should provide values closer to those at standard temperature.     

Capillary zone electrophoresis of cationic and anionic drugs in methanol

The actual mobilities and dissociation constants of acidic and basic pharmaceuticals were determined in methanol. Actual mobilities were derived from the dependence of the effective mobilities of the analytes on the pH of the methanolic background electrolyte solution (pH(MeOH)). The pKa values of the pharmaceuticals in methanol (pK(a,MeOH)) were calculated by non-linear curve fitting to the measured mobility values. It was found that the shift in pKa value (when compounds were transferred from water to methanol) increased with the acidity of the analyte. The average pKa shift for compounds exhibiting acidic properties in water was ca. 5.5 units, and the shift for basic compounds about 2 units. As was shown for a mixture of beta-blockers, the calculated actual mobilities and pKa values can be utilised in the optimisation of pH conditions for separation. The practical value of the method was illustrated by the analysis of urine samples.