Aleksander Jaworski

Comparison of orthogonality estimation methods for the two-dimensional separations of peptides.

In two-dimensional chromatography, the orthogonality of separation is important for achieving high peak capacity. In this paper, a number of different metrics are compared as measures of orthogonality. Six peptide elution data sets acquired on different stationary phases are plotted against reversed phase retention data and examined as two-dimensional chromatographic pairs. The data, including six in silico prepared data pairs, are utilized to challenge and compare selected orthogonality metrics. The metrics include correlation coefficients, mutual information, box-counting dimensionality, and surface fractional coverage with different hulls. Although correlation coefficients were found to be less suited for the intended purpose, other methods can provide a suitable measure of orthogonality. The presented results are discussed in terms of method utility, simplicity, and applicability for statistically small sets of chromatographic data. Two of the methods, box counting dimensionality and fractional coverage, were found to be mathematically related.

Utility of retention prediction model for investigation of peptide separation selectivity in reversed-phase liquid chromatography: impact of concentration of trifluoroacetic acid, column temperature, gradient slope and type of stationary phase.

Peptide separation selectivity in reversed-phase liquid chromatography was investigated using a training set of 165 peptides with a total of 1698 amino acid residues. Gradient separation was performed at selected chromatographic conditions, varying column temperature, gradient slope, ion-pairing reagent concentration, and type of stationary phase. The retention times for each set of experiments were utilized to calculate the amino acid retention coefficients using a published prediction model (Rapid Commun. Mass Spectrom. 2007, 21, 2813-2821). The calculated retention coefficients reflect the contribution of each type of amino acid residue to peptide retention at a given chromatographic condition. For example, the concentration of ion-pairing reagent (trifluoroacetic acid) had the strongest impact on the retention of peptides containing an increasing number of basic (charged) amino acids, such as arginine, lysine, and histidine, while the retention coefficients of other amino acids are minimally affected. Increasing the separation temperature resulted in a moderate decrease of amino acid retention coefficients with the exception of isoleucine, leucine, valine, and proline. This finding suggests that these residues enhance peptide retention at elevated temperature. Amino acid residue retention coefficients were also helpful to understand the impact of sorbent pore size (130 vs 300 A) on peptide retention selectivity. In addition we investigated the selectivity differences of various reversed-phase chromatographic sorbents. The trends suggested by calculated retention coefficients were confirmed using a set of synthetic peptides with specifically designed sequences.

Retention behavior of peptides in hydrophilic-interaction chromatography.

Selected hydrophilic interaction chromatography (HILIC) columns packed with bare silica, bridge-ethyl hybrid silica, or an amide sorbent chemistry were utilized for an investigation of chromatographic behavior and separation selectivity of tryptic peptides. Retention model was proposed allowing for retention prediction of peptides with correlation coefficient R(2)~0.92-0.97 for various columns. The values of optimized amino acid retention coefficients were compared to those obtained for reversed-phase liquid chromatography (Gilar et al., Anal. Chem. 2010, 82, 265-275) and used to elucidate the impact of different amino acid on peptide HILIC retention. In contrast to reversed-phase chromatography, where presence of Phe, Trp, Ile, and Leu amino acid residues in sequence strongly promoted, and presence of hydrophilic His, Lys and Arg residues strongly reduced peptide retention, the effects of these amino acid residues in HILIC were opposite (His, Lys and Arg promote, Phe, Trp, Ile and Leu demote peptide retention in HILIC). Retention coefficient optimized for pH experiments illustrated the impact of silanols on HILIC retention.

Voltammetric reduction of hydrogen ion in solutions of polyprotic strong acids with and without supporting electrolyte

Abstract Voltammetric reduction of strong polyprotic acids in solutions with excess and without supporting electrolyte was studied both theoretically and experimentally. A theoretical model based on the transport (diffusion and migration) equations and the electroneutrality principle was used to compute voltammograms by finite difference simulation under both steady state and transient conditions. Simulated voltammograms are compared with experimental reduction curves obtained at platinum microelectrodes without and with excess electrolyte for two strong polyprotic acids, sulfuric (H2SO4) and tungstosilicic (H4W12SiO40) acids. Perchloric acid (HClO4) was used as the reference for comparison. The experimental results agree well with the calculated voltammetric curves both without and with excess supporting electrolyte. The dependence of voltammetric response on the concentration of acids is also discussed.

Conditions of Strict Voltammetric Reversibility of the H+/H2 Couple at Platinum Electrodes

Cyclic voltammetric curves obtained at Pt electrodes for the hydrogen couple, H(+)/H(2), fit very well the Shuman theory, as corrected, for reversible electrode processes of other than 1:1 stoichiometry. Good agreement was obtained for acid concentrations in the millimolar range and for normal scan rates, which minimize the effect of the adsorption peaks. An error in Shumans equation for potential is corrected. Voltammograms obtained at Pt microelectrodes fit well the theoretical simulated data.

Migration and diffusion coupled with a fast preceding reaction. Voltammetry at a microelectrode.

A mathematical model implemented by simulation is presented for voltammetry of a reversible couple that involves a fast preceding chemical reaction and mixed diffusional and migrational transport. The hydrogen couple, H(+)/H(2), fulfills the above criteria. For strong acids there is no preceding reaction, whereas for weak acids the preceding reaction is HA = H(+) + A(-). The computed voltammograms are compared with experimental voltammograms for the reduction of strong and weak acids at Pt microelectrodes with excess of and without supporting electrolyte. The key assumption in the calculations is that the flux of hydrogen ion is independent of the anion. This assumption is supported by the experimental fact that the wave heights in the absence of supporting electrolyte of several strong acids of equal concentration and with anions of various size are identical.

Chronoamperometry of strong acids without supporting electrolyte

The steady-state reduction current of the hydrogen ion in a 1:1 strong acid without adding supporting electrolyte at a microelectrode is known to be independent of the diffusion coefficient of the anion, although the anion diffuses to the electrode together with the hydrogen ion in order to maintain electroneutrality. This paper aims at resolving this inconsistency of the diffusion of the anion by investigating transient reduction currents of the hydrogen ion without supporting electrolyte. The time-dependent diffusion equation associated with migration was solved under the condition of the potential step toward the limiting current-domain at a hemi-spherical electrode. The theoretical transient current has a linear relation with the inverse square root of the time. The anion behaves as if it were electroactive. The slope of the line is expressed by an average of diffusion coefficients of the hydrogen ion and the interacted anion. Chronoamperometric measurements were made in hydrochloric acid including various concentrations of supporting electrolyte.

The Strength of Acids in Alcohols As Determined by Steady-State Voltammetry

Steady-state voltammograms for reduction of acids of various strengths in alcohols with excess supporting electrolyte and without any supporting electrolyte can be used to infer charge type and strength of the acid on the basis of the phenomenon of migration. For strong and moderately weak acids (K(a)/[Formula: see text] > 10(-)(3)) in alcohols, the ratio of steady-state transport-limited current to diffusion-limited current, corrected appropriately for ion-ion interactions, the presence of ionic impurities, and changes in viscosity, for hydrogen ion reduction without supporting electrolyte and with excess supporting electrolyte equals 2. For acetic acid, which is very weak (K(a)/[Formula: see text] < 10(-)(6)), the value of the steady-state transport-limited current is, under the experimental conditions applied here, independent of supporting electrolyte concentration. In the case of a homogeneous acid-base equilibrium, a novel analytical procedure yields diffusion coefficients of both hydrogen ion and undissociated weak acid molecules from the diffusional and migrational currents. Limiting currents obtained in alcohols with excess supporting electrolyte and without supporting electrolyte are compared by means of an extended formula that incorporates the ionic strength dependence of diffusion coefficients.

Solvent selectivity and strength in reversed-phase liquid chromatography separation of peptides.

A set of tryptic peptides was analyzed in reversed-phase liquid chromatography using gradient elution with acetonitrile, methanol, or isopropanol. We used these retention data as training sets to develop retention prediction models of peptides for the three organic eluents used. The coefficients of determination, R(2), between predicted and observed data were approximately 0.95 for all systems. Retention coefficient values of twenty amino acids calculated from a model were utilized to investigate differences in separation selectivity between acetonitrile, methanol, or isopropanol eluents. The experimentally observed difference in separation selectivity appears to be a complex interplay of multiple amino acids, each contributing to a different degree to overall peptide retention. While retention contribution of hydrophilic amino acids was higher in methanol than acetonitrile, peptides containing aromatic amino acids (tyrosine, phenylalanine, tryptophan) exhibit relatively lower retention in methanol compared to acetonitrile. The differences between acetonitrile and isopropanol eluents were less pronounced. We also compared the relative elution strength of the three organic eluents for peptides. The relationship between the elution strength of two solvents is not linear, rather it was best fitted by a cubic polynomial function. Three solvents can be arranged in the order of increasing elution power as methanol

Oxidation of mercury microelectrodes in complexing media in the presence and absence of supporting electrolyte: Formation of thiocyanate complexes

Abstract Normal pulse- and staircase-voltammetric oxidation of a mercury microelectrode in thiocyanate media both with and without supporting electrolyte was investigated. An equation describing the normal pulse voltammetric limiting currents flowing through a nearly spherical segment microelectrode was developed theoretically and verified experimentally. The diffusion coefficients of mercury(II) complexes containing different numbers of the thiocyanate anions were determined. A general time-dependent digital simulation scheme was developed for calculating the current for the oxidation of mercury(0) in the presence of ligands and excess supporting electrolyte and for any pulse voltammetric technique. The time-dependent mass balance equation recently developed for a system, in which diffusion coefficients of reactant and products are different, was applied. A brief review was given on the standard potentials of various mercury systems available in the literature. A general procedure for the evaluation of the stability constants of metal complexes based on the shape of the voltammograms was described and applied to the case of thiocyanato complexes of Hg(II). The stability constant results agreed well with those of Nyman and Alberts [Anal. Chem. 32 (1960) 207]. Also discussed was the determination of the formation constant β1 for Hg(SCN)+.