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Abstract: In this study, we describe the application of a new analytical procedure based on capillary electrochromatographic (CEC) techniques for the characterization of different basic and acidic peptides using isocratic eluent conditions containing acetonitrile and ammonium acetate buffers of different molarities between pH 3.8 and 5.2. In particular, 10 immunogenic peptide analogs with isoelectric points ranging from 3.7 to 10.1 were investigated; nine of these peptides, 1–9, were truncated analogs of the parent peptide, 10, which is a peptidomimetic related to a HIV-1 gp120 epitope. Several of these peptides have the propensity to form α-helical secondary structures in solution. Electrochromatographic separations of these peptides were achieved with packed fused silica capillaries (25 cm packed length, 100 µm i.d.) containing 3 µm n-octadecylsilica particles. The influence of temperature on the CEC elution behavior of these peptides, as well as the impact of changes in the eluent composition, e.g. pH, buffer concentration and acetonitrile content, were examined. The results confirm that improvements in the resolution and analysis of synthetic peptides by CEC procedures result from the increase in electro-osmotic flow (EOF) as the temperature is increased. These findings emphasize the dominant influence of the temperature-dependent viscosity parameter, η, on the EOF and thus on peptide resolution in CEC. Moreover, these investigations have shown that eluent properties can be specifically chosen to favor either electrophoretic mobility or chromatographic retention, with the overall CEC selectivity peptides of different sequence or composition reflecting the summated contributions from both separation mechanisms. Over the pH range 4.0–5.0, and using eluents with ionic strengths ranging from 6.2 to 15 mm ammonium acetate but containing a fixed volume fraction, ψ, of acetonitrile above ψ=0.40, the CEC retention behavior of peptides 1–10 correlated with a linear relationship linking the retention coefficient, κ[sub cec], and the differential frictional size-to-mass ratio parameter, ξ[sub fric], of these peptides. However, using eluents with a low acetonitrile content and low pH values, linear correlations were also observed between the incremental retention coefficient, Δκ[sub cec], and the product term [- 0.66(Δ∑χ[sub n]) log(M[sub i]/M[sub j])], which links the difference in intrinsic hydrophobicities and molecular masses of two peptides, P[sub i] and P[sub j]. This study thus demonstrates the power of CEC procedures in the analysis of synthetic bioactive peptides and provides a general experimental framework to evaluate, using CEC procedures, the influence of the key molecular attributes of peptides on their structure-retention dependencies. Finally, these studies provide additional, practical insights into the use of CEC procedures for the analysis, resolution and biophysical characterization of closely related peptide analogs derived from solid-state peptide synthesis under conditions of different eluent composition or temperature. [ABSTRACT FROM AUTHOR]

Abstract: Native and n-alkyl-bonded (n-octadecyl) monolithic silica rods with mesopores in the range between 10 and 25nm and macropores in the range between 1.8 and 6.0μm were examined by mercury intrusion/extrusion, inverse size exclusion chromatography (ISEC) and nitrogen sorption. Our results reveal very good agreement for the mesopore size distribution obtained from nitrogen adsorption (in combination with an advanced NLDFT analysis) and ISEC. Our studies highlight the importance of mercury porosimetry for the assessment of the macropore size distribution and show that mercury porosimetry is the only method which allows obtaining a combined and comprehensive structural characterization of macroporous/mesoporous silica monoliths. Our data clearly confirm that mercury porosimetry hysteresis and entrapment have different origin, and indicate the intrinsic nature of mercury porosimetry hysteresis in these silica monoliths. Within this context some silica monoliths show the remarkable result of no entrapment of mercury after extrusion from the mesopore system (i.e. for the first intrusion/extrusion cycle). The results of a systematic study of the mercury intrusion/extrusion behavior into native silica monoliths and monoliths with bonded n-alkyl groups reveals that the macro (through) pore structure, which controls the mass transfer to and from the mesopores, here mainly controls the entrapment behavior. Our data suggest that mercury intrusion/extrusion porosimetry does not only allow to obtain a comprehensive pore structure analysis, but can also serve as a tool to estimate the mass transport properties of silica monoliths to be employed in liquid-phase separation processes. [Copyright &y& Elsevier]

The development of standard operation procedures for the manufacture of a n-octadecyl bonded spherical silica packing from partially condensed tetraethoxysilane as silica source is described. The synthesis comprises five intermediate products and six synthesis steps which were examined according to their reproducibility and robustness. The results led to the optimisation of the manufacturing process for a n-octadecyl bonded silica. Correlations were drawn between the dynamic viscosity of the poly(ethoxy)siloxane (PES), the synthesis parameters, the resulting pore structural properties and particle size distribution of the silicas. Validated procedures were developed to manufacture spherical porous ultra-pure silicas with a specific surface area of 350 m2 g−1±5% R.S.D., a specific pore volume of 1.0 ml g−1±3.7% R.S.D., an average pore diameter of 12.0 nm±0.5% R.S.D. and an average particle diameter of 5 μm. Results are presented on trial batches and the final master batch which were both used as packing materials in reversed-phase liquid chromatography (RP-LC) columns. The latter columns were certified and accepted as an HPLC column as reference material (BCR-722) by the European Commission, Institute for Reference Materials and Measurements (IRMM), Geel, Belgium. [Copyright &y& Elsevier]

Novel general expressions are constructed and presented that describe the behavior of the height equivalent of a theoretical plate (plate height), H, as a function of the linear velocity, Vx, along the axis, x, of the column and the kinetic parameters that characterize the mass transfer and adsorption mechanisms in chromatographic columns. Open tube capillaries as well as columns packed with either non-porous or porous particles are studied. The porous particles could have unimodal or bimodal pore-size distributions and intraparticle convective fluid flow and pore diffusion are considered. The expressions for the plate height, H, presented in this work could be applicable to high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC) systems, and could be used together with experimental plate height, H, versus linear velocity, Vx, data to determine the values of the parameters that characterize intraparticle convective fluid flow and pore diffusion. Furthermore, chromatographic systems under unretained as well as under retained conditions are examined. The experimental values of the plate height, H, versus the linear velocity, Vx, for a CEC system involving charged porous silica C8 particles and an uncharged analyte are compared with the theoretical results for the plate height, H, obtained from the expressions presented in this work. The agreement between theory and experiment is good, and the results indicate that the magnitude of the intraparticle electroosmotic flow (EOF) in the pores of the particles is substantial while the pore diffusion coefficient was of small magnitude. But the overall intraparticle mass transfer resistance in these particles was low because of the significant contribution of the intraparticle EOF. Simulation results are also presented (i) for a hybrid HPLC–CEC system, and (ii) for different CEC systems involving open capillaries as well as packed columns having non-porous or porous particles. The analysis of the results indicates (a) the reasons for the superior performance exhibited by the hybrid HPLC–CEC system over the performance obtained when the system is operated only in the HPLC mode, and (b) the operational configuration and the properties that the structure of the porous particles would have to have in CEC systems involving uncharged or charged analytes under unretained or retained conditions in order to obtain high CEC efficiency (low values of the plate height, H). [Copyright &y& Elsevier]

Abstract: In order to elucidate the role of the flow-through characteristics with regard to the column performance in high-performance liquid chromatography (HPLC) native and n-octadecyl bonded monolithic silica rods and columns, respectively of 100mm length and 4.6mm ID with mesopores in the range between 10 and 25nm and macropores in the range between 0.7 and 6.0μm were examined by mercury intrusion/extrusion, scanning electron microscopy, image analysis and permeability. The obtained data of the flow-through pore sizes and porosity values as well as surface-to-volume ratio of the stationary phase skeleton enabled to predict their influence to the chromatographic separation efficiency. Our data demonstrate that mercury porosimetry is a reliable technique to obtain all the characteristic parameters of the flow-through pores of silica monoliths. An important result of our examination was that the surface-to-volume ratio of monolithic silica skeletons had more significant impact to the separation process, rather than the average flow-through pore sizes. We could also show the essential differences between the particulate and monolithic stationary phases based on theoretical computation. The results, obtained from other characterization methods also indicated the structural complexity of monolithic silica samples. Permeability of columns is a generally applicable parameter to characterize all chromatographic phases no matter the chemistry or format. The correlation coefficient obtained for mercury intrusion and permeability of water was 0.998, though our investigation revealed that the surface modification is more likely influencing the obtained results. Further, the assumption of the cylindrical morphology of flow-through pores is not relevant to the investigated monolithic silica columns. These results on the morphology of the flow-through pores and of the skeletons were confirmed by the image analysis as well. Our main finding is that the flow-through pore sizes are not relevant for the estimation of the chromatographic separation efficiency of monolithic silica columns. [Copyright &y& Elsevier]

Abstract: This paper describes an initial investigation of the surface properties of three lunar soil samples from the Apollo 11, 12 and 16 missions, respectively. We report on density measurements using a helium pycnometer, adsorption isotherms of krypton applied for the determination of specific surface area of the samples and gravimetric measurement of the isotherms of water, heptane and octane. Electron-microscopic photographs are described and discussed. [Copyright &y& Elsevier]

Abstract: In this work, the pore structural parameters and size exclusion properties of LiChrospher strong cation-exchange and reverse phase restricted access materials (RAM) are analysed. The molecular weight size exclusion limit for polystyrenes was found to be about 17.7kDa, while for standard proteins, the molecular weight size exclusion limit was higher, at approximately 25kDa. The average pore diameter on a volume basis calculated from the pore network model changes from 8.5nm (native LiChrospher) to 8.6nm (diol derivative) to 8.2nm (sulphonic acid derivative) to 6.9nm (n-octadecyl derivative). Additional characterisations were performed on restricted access materials with nitrogen sorption at 77K, water adsorption at 25°C, intrusion–extrusion of water (in order to evaluate the hydrophobic properties of the pores of the hydrophobic RAM), and zeta potential measurements by microelectrophoresis. For peptide analysis out of the biofluids, the strong cation-exchange functionality seems to be particularly suitable mainly because of the high loadability of the strong cation-exchange restricted access material (SCX-RAM) and the fact that one can work under non-denaturing conditions to perform effective chromatographic separations. For bacitracin, the dynamic capacity of the SCX-RAM columns does not reach its maximum value in the analysed range. For lysozyme, the dynamic capacity reaches a value of 0.08mg/ml of column volume before column is overloaded. Additionally, the proper column operating conditions that lead to the total effective working time of the RAM column to be equal to approximately 500 injections (depending on the type of sample), is comprehensively described. The SCX-RAM column was used in the same system analysing urine samples for the period of 1 month (∼150 injections) with run-to-run reproducibility below 5% RSD and below 10% RSD for the relative fractions. [Copyright &y& Elsevier]

Abstract: This paper describes the on-line sample pretreatment and analysis of proteins and peptides with a poly(methylmethacrylate) (PMMA) microfluidic device (IonChip™). This chip consists of two hyphenated electrophoresis channels with integrated conductivity detectors. The first channel can be used for sample preconcentration and sample clean-up, while in the second channel the selected compounds are separated. Isotachophoresis (ITP) combined with zone electrophoresis (CZE) was used to preconcentrate a myoglobin sample by a factor of about 65 before injection into the second dimension and to desalt a mixture of six proteins with 100mM NaCl. However, ITP–CZE could not be used for the removal of two proteins from a protein/peptide sample since the protein zone in the ITP step was too small to remove certain compounds. Therefore, we used CZE–CZE for the removal of proteins from a protein/peptide mixture, thereby injecting only the peptides into the second CZE separation channel. [Copyright &y& Elsevier]

Abstract: Complex biological samples require very high resolution separation strategies. The platform introduced here capitalises on the hyphenation of liquid chromatographic (LC) and electric potential gradient electrochromatographic multi-dimensional separation genres. First-dimension selectivity is provided by simultaneous size exclusion (SEC) and strong cation exchange (SCX) chromatography modes, while the second dimension comprises reversed phase (RP) characteristics in a dynamic (time-variant) electric field. The time-variant potential gradient with reversal of polarity is applied across the second dimension monolithic capillary throughout the duration of the solvent strength gradient elution. Hence, the platform offers comprehensive on-line sample clean-up (matrix depletion, analyte enrichment), fractionation (first dimention LC), and separation (second dimension LC) with the prospect of altering selectivity via polarity reversal dynamic electric field tuning. [Copyright &y& Elsevier]

Small cyclic peptides have been employed to elucidate the performance of novel sorbents as stationary phases in capillary electrochromatography (CEC). In this paper chain length dependencies for ordinary liquid chromatographic sorbents are reported together with findings acquired on beads specifically designed to suit CEC. The latter, tailor-made, spherical, porous silica exhibits a distinguished surface modification to meet the criteria anticipated to enhance performance profiles in CEC. With well-characterised peptides resembling the analytes, probing of the CEC system in a systematic manner (predominantly via the organic modifier content of the background electrolyte (BE)) reveals insight into the complex interplay occurring in such analytical systems at the molecular and sub-molecular level in particular upon various modes of interaction. [Copyright &y& Elsevier]

The resolution of several structurally related synthetic peptides, derived from the loop 3 region of the activin β[sub A]-β[sub D] subunits, has been studied using capillary electrochromatography (CEC) with Hypersil n-octadecylsilica as the sorbent. The results confirm that the CEC migration of these peptides can be varied in a charge-state-specific manner as the properties of the background electrolyte, such as pH, salt concentration and content of organic modifier, or temperature are systematically changed. Acidic peptides followed similar trends in retention behaviour, which was distinctly different to that shown by more basic peptides. The CEC separation of these peptides with the Hypersil n-octadecyl-silica involved distinguishable contributions from both electrophoretic mobility and chromatographic retention. Temperature effects were reflected as variations in both the electro-osmotic flow and the electrophoretic mobility of the peptides. When the separation forces acting on the peptides were synergistic with the electro-osmotic flow, as, for example, with the positively charged peptides at a particular pH and buffer electrolyte composition, their retention coefficient, κ[sub cec], decreased with increasing capillary temperature, whereas when the separation forces worked in opposite directions, as for example with negatively charged peptides, their κ[sub cec] values increased slightly with increasing temperature. Moreover, when the content of organic modifier, acetonitrile, was sufficiently high, e.g. >40% (v/v) and nonpolar interactions with the Hypersil n-octadecyl-silica sorbent were suppressed, mixtures of both the basic and acidic synthetic peptides could be baseline resolved under isocratic conditions by exploiting the mutual processes of electrophoretic mobility and electrostatic interaction. A linear relationship between the In κ[sub cec] values and the volume fractions, ψ, of the organic modifier over a limited... [ABSTRACT FROM AUTHOR]

In 1999, we published a comprehensive survey of standards and certified reference materials for dispersed and porous solids. The present paper summarises and reports on new standards and proposals. [Copyright &y& Elsevier]