Physicochemical characterization of phosphinic pseudopeptides by capillary zone electrophoresis in highly acidic background electrolytes

Phosphinic pseudopeptides (i.e., peptide isosteres with one peptide bond replaced by a phosphinic acid moiety) were analyzed and physicochemically characterized by capillary zone electrophoresis in the pH range of 1.1-3.2, employing phosphoric, phosphinic, oxalic and dichloroacetic acids as background electrolyte (BGE) constituents. The acid dissociation constant (pK(a)) of phosphinate group in phosphinic pseudopeptides and ionic mobilities of these analytes were determined from the pH dependence of their effective electrophoretic mobilities corrected to standard temperature and constant ionic strength of the BGEs. It was shown that these corrections are necessary whenever precise mobility data at very low pH are to be determined. Additionally, it was found that the ionic mobilities of the phosphinic pseudopeptides and pK(a) of their phosphinate group are affected by the BGE constituent used. The variability of migration behavior of the pseudopeptides can be attributed to their ion-pairing formation with the BGE components.     

Analysis and characterization of phosphinic pseudopeptides by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) was applied to analysis and characterization of phosphinic pseudopeptides with the general structure N-Ac-Val-Ala(psi)(PO2(-)-CH(2)) Leu-Xaa-NH(2), where Xaa represents one of 20 proteinogenic amino acid residues. Pseudopeptides containing neutral or acidic amino acid residues in position Xaa were analyzed as anions in weakly alkaline (pH 8.1) Tris-Tricine background electrolyte (BGE), pseudopeptides with basic amino acid residues in position Xaa were analyzed as cations in acid BGEs (Tris-phosphate buffers). Acidity of phosphinic acid moiety in peptides with basic amino acid residues was determined from the dependence of effective mobility of these peptides on pH in the acid pH region (pH 1.4-2.8). Additionally, separation of diastereomers of some peptides was achieved.     

Evaluation of carrier ampholyte-based capillary electrophoresis for separation of peptides and peptide mimetics

Carrier ampholyte-based capillary electrophoresis (CABCE) has recently been introduced as an alternative to CE (CZE) in the classical buffers. In this study, isoelectric BGEs were obtained by fractionation of Servalyt pH 4-9 carrier ampholytes to cuts of typical width of 0.2 pH unit. CABCE feasibility was examined on a series of insect oostatic peptides, i.e. proline-rich di- to decapeptides, and phosphinic pseudopeptides--tetrapeptide mimetics synthesized as a mixture of four diastereomers having the -P(O)(OH)-CH(2)- moiety embedded into the peptide backbone. With identical selectivity, the separation efficiency of CABCE proved to be as good as classical CE for the insect oostatic peptides and better for diastereomers of the phosphinic pseudopeptides. In addition, despite the numerous species present in the narrow pH cuts of carrier ampholytes, CABCE seems to be free of system zones that could hamper the analysis. Peak symmetry was good for moderately to low mobile peptides, whereas some peak distortion due to electromigration dispersion, was observed for short peptides of rather high mobility.     

Separation of diastereomers of phosphinic pseudopeptides by capillary zone electrophoresis and reverse phase high‐performance liquid chromatography

Capillary zone electrophoresis (CZE) and reverse phase high‐performance liquid chromatography (RP‐HPLC) were used for separation of diastereomers of phosphinic pseudopeptides in achiral separation media. A set of phosphinic pseudopeptides, i. e. peptides with one peptide bond substituted by phosphinic acid moiety ‐PO₂^–‐CH₂‐ derived from the structure N‐Ac‐Val‐AlaB(‐CH₂)Leu‐His‐NH₂ synthesized as a mixture of four diastereomers was used. Separations of diastereomers by CZE were carried out in Tris‐phosphate background electrolytes in the pH range 1.1–3.2 and at least partial separation of the four diastereomers of each pseudopeptide was achieved. A routinely used RP‐HPLC method (C₁₈‐silica column and water/acetonitrile/trifluoroacetic acid mobile phase) was also capable of resolving the diastereomers. In addition, since individual diastereomers of majority of the pseudopeptides were isolated by RP‐HPLC it was possible to check the purity of these RP‐HPLC separated diastereomers and to compare the migration order of the diastereomers in CZE with their elution order in RP‐HPLC. The results obtained by CZE and RP‐HPLC demonstrate a complementarity of both methods in analysis and separation of phosphinic pseudopeptides including their diastereomers.     

Investigation of the effect of ionic strength of Tris-acetate background electrolyte on electrophoretic mobilities of mono-, di-, and trivalent organic anions by capillary electrophoresis

The effect of ionic strength of the background electrolyte (BGE) composed of tris(hydroxymethyl)aminomethane (Tris) and acetic acid on the electrophoretic mobility of mono-, di- and trivalent anions of aliphatic and aromatic carboxylic and sulfonic acids was investigated by capillary zone electrophoresis (CZE). Actual ionic mobilities of the above anions were determined from their CZE separations in Tris-acetate BGEs of pH 8.1 to 8.2 in the 3 to 100 mM ionic strength interval at constant temperature (25 degrees C). It was found that the ionic strength dependence of experimentally determined actual ionic mobilities does not follow the course supposed by the classical Onsager theory. A steeper decrease of actual ionic mobilities with the increasing ionic strength of BGE and a higher estimated limiting mobility of the anions than that found in the literature could be attributed to the specific behavior of the Tris-acetate BGEs. Presumably, not only a single type of interaction of anionic analytes with BGE constituents but rather the combination of effects, such as ion association or complexation equilibria, seems to be responsible for the observed deviation of the concentration dependence of the actual ionic mobilities from the Onsager theory. Additionally, several methods for the determination of limiting ionic mobilities from CZE measured actual ionic mobilities were evaluated. It turned out that the determined limiting ionic mobilities significantly depend on the calculation procedure used.     

Synthesis of mono-6-deoxy-6-N,N,N',N',N'-pentamethylethylenediammonio-cyclomaltoheptaose, a single-isomer, monosubstituted, permanently dicationic beta-CD and its use for enantiomer separations by CE

The dichloride salt of mono-6-deoxy-6-N,N,N',N',N'-pentamethylethylenediammonio-cyclomaltoheptaose (PEMEDA-BCD), the first single-isomer, monosubstituted, permanently dicationic beta-CD has been synthesized, analytically characterized, and used for the capillary electrophoretic separation of the enantiomers of a group of analytes in acidic and basic BGEs. When the concentration of PEMEDA-BCD was changed in the BGEs, the resulting effective mobilities of the analytes and the respective separation selectivities followed the predictions of the ionic strength-corrected charged resolving agent migration model. Good separation selectivities and favorable normalized EOF mobilities allowed for the rapid, efficient separation of the enantiomers of anionic, weak acid and nonionic analytes in the low- and/or high-pH BGEs.     

Isoelectric buffers, part 3: determination of pKa and pI values of diamino sulfate carrier ampholytes by indirect UV-detection capillary electrophoresis

Ampholytes with close pK(a) values (i.e., good carrier ampholytes (CAs)) are needed as buffers in pH-biased isoelectric trapping (IET) separations. The syntheses of two families of such good CAs were reported recently. Members of the family of diamino sulfate ampholytes (first series) had pI values in the 5.7 < pI < 9.0 range. Members of the family of quaternary ammonium dicarboxylic acid ampholytes (second series) had pI values in the pI < 4.3 range. To further characterize the diamino sulfate ampholytes, their effective mobilities were measured by indirect UV-absorbance detection capillary electrophoresis in a series of background electrolytes (BGEs) with different pH values. The pK(a) and limiting ionic mobility values of the CAs were obtained by fitting these mobility values, as a function of the pH and the ionic strength of the BGEs, to the theoretical mobility expression. These diamino sulfates complete the list of CAs suitable for IET separations.     

Use of 1-alkyl-3-methylimidazolium-based ionic liquids as background electrolytes in capillary electrophoresis for the analysis of inorganic anions

Separation of inorganic anions in CE is often a challenging task because the electrophoretic mobilities of inorganic anions are comparable to or even greater than the EOF mobility. In this study, we present the use of ionic liquids (ILs) as background electrolytes (BGEs) in CE of inorganic anions. The 1-alkyl-3-methylimidazolium-based ILs as BGEs dynamically coated the capillary wall and induced a reversed EOF. This allowed the anions to comigrate with the EOF and yielded a rapid separation. Increasing the alkyl chain length of the ILs and BGE concentration can significantly improve the separation resolution. With 40 mM 1-butyl-3-methylimidazolium tetrafluoroborate as BGE, good separations of five model anions (Br-, I-, NO2(-), NO3(-), and SCN-) were achieved in a range of buffer pH values. The separation efficiency was as high as 34 600-155 000, and the RSDs of the migration times were less than 0.8% (n = 5).     

Capillary zone electrophoresis in methanol: migration behavior and background electrolytes

Nonaqueous (NA) solutions are often used as background electrolytes (BGEs) and NA solvents are added to aqueous BGEs as organic modifiers in capillary zone electrophoresis (CZE), in order to optimize the separations. This can be tricky, however, because the pH* and pK* concepts may be totally different in NA solvents, whereas often less knowledge is available concerning phenomena, such as system zones, applying NA solvents. In this paper, the concepts of pH* and pK* are considered for methanol as a solvent and pK* values are determined for several components in mixtures of water and methanol. With a mathematical model, adapted for calculations in methanol, parameters are calculated describing the fronting or tailing character of peaks and the question of peaks or dips, and the existence of system zones is discussed for pure methanol as a solvent. These aspects are experimentally verified, applying BGEs useful for the separation of cationic species in the indirect UV mode. It can be concluded that the mathematical model developed for aqueous BGEs is applicable to BGEs in methanol, too, and that the behavior of BGEs in methanol is comparable with that in water concerning the fronting or tailing character of peaks and the question of peaks and dips, although the mobilities and pK values can change significantly.     

Determination of thermodynamic acidity constants and limiting ionic mobilities of weak electrolytes by capillary electrophoresis using a new free software AnglerFish

Thermodynamic acidity constants (acid or acid-base dissociation constants, sometimes called also as ionization constants) and limiting ionic mobilities (both of them at defined temperature, usually 25°C) are the fundamental physicochemical characteristics of a weak electrolyte, that is, weak acid or weak base or ampholyte. We introduce a novel method for determining the data of a weak electrolyte by the nonlinear regression of effective electrophoretic mobility versus buffer composition dependence when measured in a set of BGEs with various pH. To correct the experimental data for zero ionic strength we use the extended Debye-Hückel model and Onsager-Fuoss law with no simplifications. Contrary to contemporary approaches, the nonlinear regression is performed on limiting mobility data calculated by PeakMaster's correction engine, not on the raw experimental mobility data. Therefore, there is no requirement to perform all measurements at a constant ionic strength of the set of BGEs. We devised the computer program AnglerFish that performs the necessary calculations in a user-friendly fashion. All thermodynamic pKa values and limiting electrophoretic mobilities for arbitrarily charged substances having any number of ionic forms are calculated by one fit. The user input consists of the buffer composition of the set of BGEs and experimentally measured effective mobilities of the inspected weak electrolyte.     

Determination of acid-base dissociation constants of amino- and guanidinopurine nucleotide analogs and related compounds by capillary zone electrophoresis

CZE has been applied for determination of acid-base dissociation constants (pKa) of ionogenic groups of newly synthesized amino- and (amino)guanidinopurine nucleotide analogs, such as acyclic nucleoside phosphonate, acyclic nucleoside phosphonate diesters and other related compounds. These compounds bear characteristic pharmacophores contained in various important biologically active substances, such as cytostatics and antivirals. The pKa values of ionogenic groups of the above compounds were determined by nonlinear regression analysis of the experimentally measured pH dependence of their effective electrophoretic mobilities. The effective mobilities were measured by CZE performed in series of BGEs in a broad pH range (3.50-11.25), at constant ionic strength (25 mM) and temperature (25 degrees C). pKa values were determined for the protonated guanidinyl group in (amino)guanidino 9-alkylpurines and in (amino)guanidinopurine nucleotide analogs, such as acyclic nucleoside phosphonates and acyclic nucleoside phosphonate diesters, for phosphonic acid to the second dissociation degree (-2) in acyclic nucleoside phosphonates of amino and (amino)guanidino 9-alkylpurines, and for protonated nitrogen in position 1 (N1) of purine moiety in acyclic nucleoside phosphonates of amino 9-alkylpurines. Thermodynamic pKa of protonated guanidinyl group was estimated to be in the range of 7.75-10.32, pKa of phosphonic acid to the second dissociation degree achieved values of 6.64-7.46, and pKa of protonated nitrogen in position 1 of purine was in the range of 4.13-4.89, depending on the structure of the analyzed compounds.     

Synthesis of a single-isomer sulfated beta-cyclodextrin carrying nonidentical substituents at all of the C2, C3, and C6 positions and its use for the electrophoretic separation of enantiomers in acidic aqueous and methanolic background electrolytes. Part 2: Heptakis(2-O-methyl-6-O-sulfo) cyclomaltoheptaose

The sodium salt of heptakis(2-O-methyl-6-O-sulfo)cyclomaltoheptaose (HMS), the second single-isomer, sulfated beta-CD carrying nonidentical substituents at all of the C2, C3, and C6 positions, has been synthesized, analytically characterized, and used for the capillary electrophoretic separation of the enantiomers of a group of 23 weak base analytes in acidic aqueous and methanolic BGEs. HMS interacted strongly with only about half of the analytes studied. The good separation selectivities and favorable normalized EOF mobilities allowed for rapid, efficient separation of the enantiomers of 19 of the 23 weak base analytes in the aqueous BGEs, often with separation selectivity values complimentary to those obtained with other single-isomer sulfated CDs. HMS did not prove to be as good a resolving agent in acidic methanolic BGEs as its counterpart, heptakis(2-O-methyl-3-O-acetyl-6-O-sulfo)cyclomaltoheptaose.     

Ampholytes as background electrolytes in capillary zone electrophoresis: sense or nonsense? Histidine as a model ampholyte

A lot of phenomena, occuring in capillary zone electrophoresis (CZE), are linked with the ionic concentration of the background electrolyte (BGE). If weak bases and acids are used as BGEs in CZE, at a pH where they are scarcely ionized, the ionic concentration of the BGE is very low and this brings a strong peak broadening, limited sample stacking and low sample load. Because the electromigration dispersion increases extremely, moreover, the existence of low-conductivity BGEs in CZE is a contradiction in terms. The behavior of ampholytes as BGE in CZE is examined, by means of histidine as a model ampholyte. For BGEs consisting of histidine, important parameters, including the ionic concentrations, buffer capacity, transfer ratio, and the indicator for electromigration dispersion E(1)m(1)/E(2)m(2), are calculated at various pH. Although the transfer ratio is fairly constant over the whole pH traject, the ionic concentration and buffer capacity decrease whereas the electromigration dispersion strongly increases near the pI of histidine. I.e., that ampholytes can be applied as BGEs in CZE, however, just not at pH near their pI value, except as the difference between the pK values of the basic and acidic group, the deltapK value, is very small. For ampholytes with a low deltapK value or at high concentrations, all the before-mentioned effects are less fatal, but in that case we can not speak of a real low-conductivity BGE. If ampholytes are used at pH near their pK values, the use of ampholytes as BGE is not advantageously compared with simple weak bases and acids. This has been confirmed by calculations and experiments.     

Temperature dependence of acidity constants, a tool to affect separation selectivity in capillary electrophoresis

The mathematical models of migration and dispersion in capillary zone electrophoresis of small molecules form a sound basis for separation strategies of complex mixtures. It turned out that the key property is the effective mobility of the sample ions. To tune resolution parameters such as pH, complexation constants and ionic strength are widely used; temperature however is not although mobilities and pK(a) values depend in a more or less degree on temperature. From the temperature dependences of pK(a) values of a number of compounds listed in the literature a general rule can be derived: for carboxylic and inorganic acids dpK(a)/dT values are very small and the pK(a) values change less than +/-0.05 units/10K. Thermodynamically speaking, these compounds exhibit dissociation enthalpies close to zero. Phenols and amines, on the other hand, have systematically larger dpK(a)/dT values of about -0.1 to -0.2 units per 10K (the results of dissociation enthalpies of 20-70 kJ/mole). Based on this classification, a distinction can be made between different situations in capillary electrophoresis: (i) selectivity changes with temperature are largely due to the temperature dependence of the pK(a) of the buffering compound in the background electrolyte, (ii) selectivity changes mainly result from the temperature dependence of the pK(a) of the sample ions, and (iii) temperature effects on the pK(a) values of both, sample and buffer play a role. This work demonstrates such effects on selectivity in capillary electrophoresis highlighting the fact that in some instances temperature can be used to fine-tune separations.     

CE determination of the thermodynamic pKa values and limiting ionic mobilities of 14 low molecular mass UV absorbing ampholytes for accurate characterization of the pH gradient in carrier ampholytes-based IEF and its numeric simulation

Fourteen low molecular mass UV absorbing ampholytes containing 1 or 2 weakly acidic and 1 or 2 weakly basic functional groups that best satisfy Rilbe's requirement for being good carrier ampholytes (ΔpK_a = pKa_monoanion ‒ pKa_monocation < 2) were selected from a large group of commercially readily available ampholytes in a computational study using two software packages (ChemSketch and SPARC). Their electrophoretic mobilities were measured in 10 mM ionic strength BGEs covering the 2 < pH < 12 range. Using our Debye-Hückel and Onsager-Fuoss laws-based new software, AnglerFish (freeware, https://echmet.natur.cuni.cz/software/download ), the effective mobilities were recalculated to zero ionic strength from which the thermodynamic pK_a values and limiting ionic mobilities of the ampholytes were directly calculated by Henderson-Hasselbalch equation-type nonlinear regression. The tabulated thermodynamic pK_a values and limiting ionic mobilities of these ampholytes (pI markers) facilitate both the overall and the narrow-segment characterization of the pH gradients obtained in IEF in order to mitigate the errors of analyte ampholyte pI assignments caused by the usual (but rarely proven) assumption of pH gradient linearity. These thermodynamic pK_a and limiting mobility values also enable the reality-based numeric simulation of the IEF process using, for example, Simul (freeware, https://echmet.natur.cuni.cz/software/download ).     

Determination of acid-base dissociation constants of azahelicenes by capillary zone electrophoresis

CZE was employed to determine acid-base dissociation constants (pK(a)) of ionogenic groups of azahelicenes in methanol (MeOH). Azahelicenes are unique 3-D aromatic systems, which consist of ortho-fused benzene/pyridine units and exhibit helical chirality. The pK(a) values of pyridinium groups of the studied azahelicenes were determined from the dependence of their effective electrophoretic mobility on pH by a nonlinear regression analysis. The effective mobilities of azahelicenes were determined by CZE at pH range between 2.1 and 10.5. Thermodynamic pK(a) values of monobasic 1-aza[6]helicene and 2-aza[6]helicene in MeOH were determined to be 4.94 +/- 0.05 and 5.68 +/- 0.05, respectively, and pK(a) values of dibasic 1,14-diaza[5]helicene were found to be equal to 7.56 +/- 0.38 and 8.85 +/- 0.26. From these values, the aqueous pK(a) of these compounds was estimated.     

Separation and investigation of structure-mobility relationship of gonadotropin-releasing hormones by capillary zone electrophoresis in conventional and isoelectric acidic background electrolytes

Capillary zone electrophoresis (CZE) has been applied to qualitative and quantitative analysis, separation and physicochemical characterization of synthetic gonadotropin-releasing hormones (GnRHs) and their analogs and fragments. Structurally related peptides were separated in conventional and isoelectric acidic background electrolytes (BGEs), pH 2.18-2.50. Best separation was achieved in isoelectric BGE composed of 200 mM iminodiacetic acid, pH 2.32. The effective electrophoretic mobilities, m(ep), of GnRHs in five BGEs were determined and four semiempirical models correlating effective mobility with charge, q, and relative molecular mass, M(r), (m(ep) versus q/M(r)(k), where k is related to the molecular shape) were tested to describe the migration behavior of GnRHs in CZE. None of the models was found to be quite definitively applicable for the whole set of 10 GnRHs differing in size (tetrapeptide-decapeptide) and positive charge (0.91-3.00 elementary charges). Nevertheless, for the dependence of m(ep) on q/M(r)(k), the highest coefficient of correlation, R=0.995-0.999, was obtained for k close to the value 0.5 in all five acidic BGEs. This indicates that the most probable structure of GnRHs in these BGEs can be predicted as a random coil.     

Peptide separations and dissociation constants in nonaqueous capillary electrophoresis: comparison of methanol and aqueous buffers

Nonaqueous capillary electrophoresis was evaluated for its potential to separate peptides in methanolic background electrolytes in comparison to aqueous-methanol (50% v/v) and water. Isomeric aspartyl dipeptides and Leu- and Met-enkephalin served as model compounds. pK(a) values were determined in the three solvent systems based on the apparent pH scale and in the case of methanol additionally based on the conventional pH scale. Changing from water to methanol led to an increase of the ionization constants describing the dissociation equilibria of the carboxyl group and the amino group, respectively. The pK(a) shift was more pronounced for the carboxylic acid function leading to a compression of the mobility-pH curve. As reported for aqueous buffers, efficient separations of the peptides were achieved in methanolic background electrolytes including the resolution of the diastereomers of the isomeric alpha- and beta-aspartyl dipeptides. In contrast to aqueous buffers, the separation of Leu- and Met-enkephalin could also be obtained in buffers in methanol at high pH.     

Determination of acid-base dissociation constants of very weak zwitterionic heterocyclic bases by capillary zone electrophoresis

Thermodynamic acid-base dissociation (ionization) constants (pK(a)) of seven zwitterionic heterocyclic bases, first representatives of new heterocyclic family (2,3,5,7,8,9-hexahydro-1H-diimidazo[1,2-c:2',1'-f][1,3,2]diazaphosphinin-4-ium-5-olate 5-oxides), originally designed as chiral Lewis base catalysts for enantioselective reactions, were determined by capillary zone electrophoresis (CZE). The pK(a) values of the above very weak zwitterionic bases were determined from the dependence of their effective electrophoretic mobility on pH in strongly acidic background electrolytes (pH 0.85-2.80). Prior to pK(a) calculation by non-linear regression analysis, the CZE measured effective mobilities were corrected to reference temperature, 25°C, and constant ionic strength, 25 mM. Thermodynamic pK(a) values of the analyzed zwitterionic heterocyclic bases were found to be particularly low, in the range 0.04-0.32. Moreover, from the pH dependence of effective mobility of the bases, some other relevant characteristics, such as actual and absolute ionic mobilities and hydrodynamic radii of the acidic cationic forms of the bases were determined.     

Determination of the microenvironment-pH and charge and size characteristics of amino acids through their electrophoretic mobilities determined by CZE

Effective electrophoretic mobility data of 20 amino acids reported in the literature are analyzed and interpreted through simple physicochemical models, which are able to provide estimates of coupled quantities like hydrodynamic shape factor, equivalent hydrodynamic radius (size), net charge, actual pK values of ionizing groups, partial charges of ionizing groups, hydration number, and pH near molecule (microenvironment-pH of the BGE). It is concluded that the modeling of the electrophoretic mobility of these analytes requires a careful consideration of hydrodynamic shape coupled to hydration. In the low range of pH studied here, distinctive hydrodynamic behaviors of amino acids are found. For instance, amino acids with basic polar and ionizing side chain remain with prolate shape for pH values varying from 1.99 to 3.2. It is evident that as the pH increases from low values, amino acids get higher hydrations as a consequence each analyte total charge also increases. This result is consistent with the monotonic increase of the hydrodynamic radius, which accounts for both the analyte and the quite immobilized water molecules defining the electrophoretic kinematical unit. It is also found that the actual or effective pK value of the alpha-carboxylic ionizing group of amino acids increases when the pH is changed from 1.99 to 3.2. Several limitations concerning the simple modeling of the electrophoretic mobility of amino acids are presented for further research.