Mobile Phases Of Different Compositions Research Articles

Analysis of peptides and protein digests by reversed phase high performance liquid chromatography–electrospray ionisation mass spectrometry using neutral pH elution conditions

In this study, the advantages of carrying out the analysis of peptides and tryptic digests of proteins under gradient elution conditions at pH 6.5 by reversed-phase liquid chromatography (RP-HPLC) and in-line electrospray ionisation mass spectrometry (ESI-MS) are documented. For these RP separations, a double endcapped, bidentate anchored n-octadecyl wide pore silica adsorbent was employed in a capillary column format. Compared to the corresponding analysis of the same peptides and protein tryptic digests using low pH elution conditions for their RP-HPLC separation, this alternative approach provides improved selectivity and more efficient separation of these analytes, thus allowing a more sensitive identification of proteins at different abundance levels, i.e. more tryptic peptides from the same protein could be confidently identified, enabling higher sequence coverage of the protein to be obtained. This approach was further evaluated with very complex tryptic digests derived from a human plasma protein sample using an online two-dimensional (2D) strong cation-exchange (SCX)-RP-HPLC-ESI-MS/MS system. Again, at pH 6.5, with mobile phases of different compositions, improved chromatographic selectivities were obtained, concomitant with more sensitive on-line electrospray ionisation tandem mass spectrometric (ESI-MS/MS) analysis. As a consequence, more plasma proteins could be confidently identified, highlighting the potential of these RP-HPLC methods with elution at pH 6.5 to extend further the scope of proteomic investigations.

Adsorption interaction parameter of polyethers in ternary mobile phases: The critical adsorption line

It is shown that in LC of polymers, the interaction parameter in ternary mobile phases can be described by a plane, which is determined by the dependencies in binary mobile phases. Instead of a critical adsorption point, critical conditions are observed along a straight line of composition between the two critical points in binary mobile phases. Consequently, a separation of block copolymers under critical conditions for one block by an adsorption mechanism for the other block can be achieved in ternary mobile phases of different compositions, which allows an adjustment of the retention of the adsorbing block.

Evaluation of the influence of residual silanol groups on the total retention in reversed-phase chromatography

It was demonstrated that the characteristics of stationary phases for reversed-phase chromatography can be compared by analyzing the retention of several compounds belonging to a homologous series during elution with one and the same composition of mobile phase or the retention of a nonpolar compound during elution with several mobile phases of different compositions. The slope of the corresponding linear correlations can be used to characterize the hydrophobicity of the columns (stationary phases) under study. To analyze the polar interactions between the sorbate and the stationary phase it is advantageous to study the retention of a polar substance in several (at least in two) mobile phases of different compositions. The degree of displacement of the linear correlation relative to that for nonpolar substances can be considered a measure of the influence of residual silanol groups on the total retention characteristics.

Separation of free sterols by high temperature liquid chromatography

Increasing the column temperature accelerates markedly elution in HPLC. The separation of five free sterols was studied on three packing materials that can withstand high temperatures. These stationary phases included graphitic carbon, a polymeric C18 silica, and a zirconia-based adsorbent. Measurements of retention data were made at up to 150 °C with mobile phases of different compositions. Since the columns tested afford different retention mechanisms, a variety of elution patterns were observed, with some being more advantageous than others for certain sterol separations. Effects observed include some selectivity improvements and some elution order reversals. The separation of free sterols in selected fruit juices is also presented. Albeit at the expense of a longer analysis time, the graphitic carbon column produced the best separation of the sterols in this study.

High-performance liquid chromatography of chalcones: Quantitative structure-retention relationships using partial least-squares (PLS) modeling

In this study, the multivariate partial least squares projections to latent structures (PLS) technique was used for modeling the RP-HPLC retention data of 17 chalcones, which were determined with methanol-water mobile phases of different compositions. The PLS model was based on molecular descriptors which can be calculated for any compound utilizing only the knowledge of its molecular structure. The PLS analysis resulted in a model with the following statistics: r=0.976, Q=0.933, s=0.076, and F=43.63. The adequacy of the developed model was assessed by means of crossvalidation and also, by PLS modeling of the retention data of several chalcones reported by Walczak et al. [J. Chromatogr. 353, 123, (1986)], which were obtained using stationary phases of different polarity (-NH2, DIOL,-CN, ODS, C8). The structural interpretation of the developed PLS model was accomplished by means of comparative correlations between the nonempirical descriptors used in the model and the solvation parameters developed by Abraham. The results obtained in this work provides evidence for the great potential of the topological approach for the development of quantitative structure-retention relationship (QSRR) models.

Restricted diffusion model for surface diffusion in reversed-phase liquid chromatography

The analysis of experimental results in reversed-phase liquid chromatography (RPLC) allows further discussion of the restricted diffusion model of surface diffusion formulated on the basis of the absolute rate theory. Chromatographic data were acquired on different RPLC systems with two series of homologous compounds, several stationary phases having different alkyl ligand densities and ligands of various lengths, and methanol/water mobile phases of different compositions. The enthalpy-entropy compensation observed and the linear free energy relationships found for surface diffusion suggest that the surface diffusion mechanism remains probably the same in all RPLC conditions studied. Whereas the isosteric heat of adsorption approaches zero with decreasing retention, the activation energy of surface diffusion tends toward a finite limit and the surface diffusion coefficient tends toward a value near the corresponding molecular diffusivity. These results support the validity of the restricted diffusion model. The influence of different factors on the validity of this model (i.e., the activation energy and the frequency factor of surface diffusion, and the surface tortuosity) was also considered.

Molecular Topology and Quantum Chemical Descriptors in the Study of Reversed-Phase Liquid Chromatography. Hydrogen-Bonding Behavior of Chalcones and Flavanones

The reversed-phase liquid chromatographic hydrophobicity parameters (logK′wandS) of several chalcones and flavanones were determined with methanol:water mobile phases of different compositions and with trace quantities ofn-decylamine and 1-octanol added to the eluent to minimize the silanophilic interactions present in alkylsilane-bonded phases. It has been reported that qualitative hydrogen bonding information can be obtained from the relationship betweenSand logK′wdetermined with these chromatographic conditions. To quantitatively describe the hydrogen-bonding discrimination effects observed for the compounds under study, the parameter Δ was defined as follows: Δ(s–logKw). With topological and molecular orbital calculations, several molecular descriptors were computed for test compounds, and multivariate statistical techniques were used to examine the informative value of the parameter proposed. The results obtained indicate that Δ(s–logKw) encodes structural information mainly related to the molecular polarity and the ability of compounds to participate in hydrogen-bonding interactions. Similar structural information was also obtained for Δ(s–logKw) values of 31 structurally dissimilar compounds.

Hydrophobicity parameters determined by reversed-phase liquid chromatography: VIII. Hydrogen-bond effects of ester and amide groups in heteroaromatic compounds on the relationship between the capacity factor and the octanol—water partition coefficient

The logarithms of the capacity factors, log k′, for several heteroaromatic systems (furan, pyrrole, benzofuran, indole, benzene and their alkyl, ester and amide derivatives) were determined on a Capcell Pak C 18 column using methanol—buffer (pH 7.4) mobile phases of different compositions. These results and the log k w values, derived by a linear extrapolation of the plot of log k′ against the volume fraction of methanol to 0% methanol, were correlated with experimental log P values by taking into account the hydrogen-acceptor and hydrogen-donor effects. Whereas the hydrogen-donor effect was found to be minimized by using the log k w parameter, the hydrogen-acceptor effect was found to become insignificant in an eluent containing 50% methanol. Isocratic data determined at this eluent composition gave the simplest and best correlation.

Evaluation of stationary phases for the separation of buckminsterfullerenes by high-performance liquid chromatography

The high-performance liquid chromatographic separation of the buckminsterfullerenes C 60 and C 70 was studied using different chromatographic systems. Normal-phase (silica), non-aqueous reversed-phase (C 18-bonded silica and graphitized carbon black) and charge-transfer complexation with dinitrobenzoylphenylglycine (DNBPG) and 2-(2,4,5,7-tetranitro-9-fluorenylideneaminooxy)propionic acid (TAPA) were considered as retention mechanisms. Mobile phases of different compositions, but all based on hydrocarbons (toluene, hexane, heptane) as the main solvent, were used. Although it was found that the adsorption of the fullerenes on all the other phase systems is exothermic, their interactions with the charge-transfer phases exhibit an unusual endothermic behavior. The retention times of C 60 and C 70 on these phases increase with increasing temperature. The adsorption enthalpy and entropy derived from the Van 't Hoff plots confirm this rare temperature dependence. The adsorption enthalpy is positive whereas the adsorption entropy is strongly positive.

Hydrophobicity parameters determined by reversed-phase liquid chromatography : I. Relationship between capacity factors and octanol-water partition coefficients for monosubstituted pyrazines and the related pyridines

The capacity factors ( k′) of monosubstituted pyrazines and 2-substituted pyridines were measured on a Capcellpack C 18 column using methanol-buffer (pH 9.2) mobile phases of different compositions, and the relationship between log P ( n-octanol-water partition coefficient) and log k′ was analysed. In all instances the amphiprotic substituents were eluted faster than expected on the assumption of log P-log k′ linearity, reflecting that these groups act as hydrogen donors. For other substituents, a good linear relationship between log P and log k′ was obtained with eluents containing 50–70% (v/v) of methanol. However, as the methanol content decreased, linearity no longer held and correction terms for the electronic effects and specific effects attributed to ester and amide groups were required. The relationships between log k′(pyridine) was studied by applying a modified bidirectional Hammett-type correction for the component in π-value attributable to electronic effects of substituents and aza functions on the hydrogen-bonding solvation effect. The retention behaviour changed systematically with changes in mobile phase composition.

Effects of solvent composition in the normal-phase liquid chromatography of alkylphenols and naphthols

The normal-phase liquid chromatographic models of Scott, Snyder, and Soczewinski were considered for a ..mu..-Bondapak NH/sub 2/ stationary phase. n-Heptane:2-propanol and n-heptane:ethyl acetate mobile phases of different compositions were used. Linear relationships were obtained from graphs of log K' vs. log mole fraction of the strong solvent for both n-heptane:2-propanol and n-heptane:ethyl acetate mobile phases. A linear relationship was obtained between the reciprocal of corrected retention volume and % wt/v of 2-propanol but not between the reciprocal of corrected retention volume and % wt/v of ethyl acetate. The slopes and intercept terms from the Snyder and Soczewinski models were found to approximately describe interactions with ..mu..-Bondapak NH/sub 2/. Capacity factors can be predicted for the compounds by using the equations obtained from mobile phase composition variation experiments.