Flow-through pore characteristics of monolithic silicas and their impact on column performance in high-performance liquid chromatography.

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 100 mm length and 4.6 mm ID with mesopores in the range between 10 and 25 nm and macropores in the range between 0.7 and 6.0 microm 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.

Textural characterization of native and n-alky-bonded silica monoliths by mercury intrusion/extrusion, inverse size exclusion chromatography and nitrogen adsorption.

Native and n-alkyl-bonded (n-octadecyl) monolithic silica rods with mesopores in the range between 10 and 25 nm and macropores in the range between 1.8 and 6.0 microm 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.

Sulphonic acid strong cation-exchange restricted access columns in sample cleanup for profiling of endogenous peptides in multidimensional liquid chromatography. Structure and function of strong cation-exchange restricted access materials.

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 17.7 kDa, while for standard proteins, the molecular weight size exclusion limit was higher, at approximately 25 kDa. The average pore diameter on a volume basis calculated from the pore network model changes from 8.5 nm (native LiChrospher) to 8.6 nm (diol derivative) to 8.2 nm (sulphonic acid derivative) to 6.9 nm (n-octadecyl derivative). Additional characterisations were performed on restricted access materials with nitrogen sorption at 77 K, water adsorption at 25 degrees 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 0.08 mg/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 (approximately 150 injections) with run-to-run reproducibility below 5% RSD and below 10% RSD for the relative fractions.

Comparative study of morphometric properties characterizing the complexity of silicate pore networks probed by adsorption of nitrogen and methanol.

In this work, we compare the surface and morphometric properties of the pore networks in four silicas (code names Fr1428, Fr474, Fr1386, and MM1164) with different random porosities using the adsorption isotherms of two different probe adsorbents, nitrogen and methanol. The parent material Fr1428 was a pure silica 25 microm sample. The Fr474 sample was the same one with bonded electroneutral diol groups on its outer surface. Fr1386 was the parent material with bonded electroneutral diol groups on its outer surface and sulfonic groups on its inner surface, and the MM1164 sample was the original sample with external electroneutral diol groups and internal n-octadecyl groups. The properties examined were the specific surface area S(p) and the specific pore volume V(p), the pore connectivity c, the pore anisotropy b, the tortuosity tau, and the lacunarity lambda of the pore network as well as the percentage microporosity. These properties provide a complete characterization of complexity of the porous network. The surface areas of the solids were estimated via the traditional BET plots (S(BET)) and the I-point method (S(I)). The two sets of values S(BET) and S(I) were practically identical and they decrease as the size of the functional group increases. The values of percentage microporosity were also determined by the same I-point method using the variation of the C parameter of the BET equation. The total pore volume V(p) was found to be higher in the case of methanol adsorption, compared to nitrogen, which might be related to increase condensation. The networks of the pores were simulated using a dual site bond model (DSBM) and Monte Carlo (MC) techniques for achieving their proper arrangement into the solids. From the resulting simulating networks, the pore connectivity distributions (PCD) and their mean values c(mean) were estimated and favorably compared to the values of connectivity c(Seaton) determined according to the method of Seaton. Both values decrease with the size of the functional groups and are weakly affected by the adsorbent employed. From the simulation pore network, the mean values of tortuosity tau(mean) were also estimated and found to be lower when N2 was used as adsorbate compared to MeOH. The values of lacunarity lambda, estimated according to the method by Allain and Cloitre using the moving box technique in the DSBM/MC simulation matrix of the pore network, indicate that the distribution of the poreless mass into the matrix increases with the size of the functional group. Finally, the internal relationships observed between the pore anisotropy b and the percentage microporosity as well as between the tortuosity tau and the pore connectivity c are discussed.

Selective protein removal and desalting using microchip CE.

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 100 mM 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.

Dynamic electric field assisted multi-dimensional liquid chromatography of biological samples.

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.

Evaluation and comparison of tailor-made stationary phases based on spherical silica-based beads for capillary electrochromatography via peptide separation analysis.

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.

Development of standard operation procedures for the manufacture of n-octadecyl bonded silicas as packing material in certified reference columns for reversed-phase liquid chromatography.

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 (-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 microm. 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.

The CEC behaviour of several synthetic peptides related to the activin betaA-betaD subunits.

The resolution of several structurally related synthetic peptides, derived from the loop 3 region of the activin betaA-betaD 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, kappacec, decreased with increasing capillary temperature, whereas when the separation forces worked in opposite directions, as for example with negatively charged peptides, their kappacec 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 ln kappacec values and the volume fractions, psi, of the organic modifier over a limited range of psi-values, was established for the negatively charged peptides under these isocratic conditions. These findings thus provide useful guidelines in a more general context for the resolution and analysis of structurally related synthetic peptides using CEC methods.

Novel general expressions that describe the behavior of the height equivalent of a theoretical plate in chromatographic systems involving electrically-driven and pressure-driven flows.

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).

Application of CEC procedures for the analysis of synthetic peptides: characterization of linear immunogenic peptides that mimic a HIV-1 gp120 epitope.

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 alpha-helical secondary structures in solution. Electrochromatographic separations of these peptides were achieved with packed fused silica capillaries(25 cm packed length, 100 microm i.d.) containing 3 microm 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 inelectroosmotic flow (EOF) as the temperature is increased. These findings emphasize the dominant influence of the temperature-dependent viscosity parameter, eta, 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, psi, of acetonitrile above psi = 0.40, the CEC retention behavior of peptides 1-10 correlated with a linear relationship linking the retention coefficient, kappta(cec), and the differential frictional size-to-mass ratio parameter, Xi(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, Delta(Kappa)cec, and the product term [-0.66(Delta(Sigma[Xn]) log(Mi/Mj)], which links the difference in intrinsic hydrophobicities and molecular masses of two peptides, Pi and Pj. 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.

Capillary electrochromatography analysis of hormonal cyclic and linear peptides.

The retention behavior of linear and cyclic peptides has been studied by capillary electrochromatography (CEC) with a variety of different n-alkyl silica reversed-phase sorbents and also with mixed-mode phases containing both strong cation-exchange (sulfonic acid) and n-alkyl groups bonded onto the silica surface, using eluents ranging from pH 2.0 to pH 5.0. Depending upon the amino acid sequence, electrochromatographic retention of the peptides was strongly affected by the composition of the eluent, its pH value, and the choice of sorbent packed into the capillaries. The dominant separation processes operating with these charged analytes could be modulated inter alia by the content of organic modifier, acetonitrile, in the eluent, with peptide resolution predominantly arising from electrophoretic migration processes at high acetonitrile content. As the concentration of acetonitrile was decreased, chromatographic retention processes became more pronounced. With the n-alkyl silica CEC columns used in this study, silanophilic interactions between the sorbents and the charged peptides could be suppressed by increasing the molarity of the buffer and by adjusting the pH of the eluent to lower values. On the other hand, electrostatic interactions between basic peptides and the surface of strong cation-exchanger, mixed-mode materials can be suppressed at low pH values by using higher ionic strength conditions in the eluent. Different selectivity behavior was achieved with desmopressin and the other peptides with Spherisorb C18/SCX and Hypersil mixed-mode materials when an identical eluent composition of 60% (v/v) acetonitrile with 7.6 mM triethylammonium phosphate, pH 3.0, was used. These findings confirm that the surface charge density of the sorbent fulfills an important role in the modulation of peptide selectivity in CEC. These studies also confirm that the dependency of the logarithm of the CEC retention coefficients, i.e., log Kcec, of a peptide separated with n-octadecyl silica sorbents under CEC conditions, on the volume fraction, psi, of the organic solvent modifier, acetonitrile, within the range of 0.20 < or = psi < or = 0.60, can be approximated by a linear relationship. Moreover, these studies show that the selectivity differences of peptides separated by CEC with nonpolar sorbents in packed capillary systems can be discussed in terms of semiempirical dependencies that link peptide retention behavior with their molecular descriptor properties, e.g., their hydrophobicity, surface charge anisotropy, surface area, molecular mass and intrinsic charge, and thus to their corresponding linear free energy relationships.

Isolation and characterization of structurally novel antimutagenic flavonoids from spinach (Spinacia oleracea).

Thirteen compounds, isolated from spinach (Spinacia oleracea), acted as antimutagens against the dietary carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline in Salmonella typhimurium TA 98. The antimutagens were purified by preparative and micropreparative HPLC from a methanol/water (70:30, v/v) extract of dry spinach (commercial product) after removal of lipophilic compounds such as chlorophylls and carotenoids by solid-phase extraction (SPE). Pure active compounds were identified by instrumental analysis including FT-IR, (1)H and (13)C NMR, UV-vis spectroscopy, and mass spectrometry. All of these compounds were flavonoids and related compounds that could be attributed to five groups: (A, methylenedioxyflavonol glucuronides) 5,3'-dihydroxy-4'-methoxy-6,7-methylenedioxyflavonol 3-O-beta-glucuronide (compound 1), 5,2',3'-trihydroxy-4'-methoxy-6,7-methylenedioxyflavonol 3-O-beta-glucuronide (compound 2), 5-hydroxy-3',4'-dimethoxy-6,7-methylenedioxyflavonol 3-O-beta-glucuronide (compound 3); (B, flavonol glucuronides) 5,6,3'-trihydroxy-7,4'-dimethoxyflavonol 3-O-beta-glucuronide (compound 4), 5,6-dihydroxy-7,3',4'-trimethoxyflavonol 3-O-beta-glucuronide (compound 5); (C, flavonol disaccharides) 5,6,4'-trihydroxy-7,3'-dimethoxyflavonol 3-O-disaccharide (compound 6), 5,6,3',4'-tetrahydroxy-7-methoxyflavonol 3-O-disaccharide (compounds 7 and 8); (D, flavanones) 5,8,4'-trihydroxyflavanone (compound 9), 7,8,4'-trihydroxyflavanone (compound 10); (E, flavonoid-related compounds) compounds 11, 12, and 13 with incompletely elucidated structures. The yield of compound 1 was 0.3%, related to dry weight, whereas the yields of compounds 2-13 ranged between 0.017 and 0.069%. IC(50) values (antimutagenic potencies) of the flavonol glucuronides ranged between 24.2 and 58.2 microM, whereas the flavonol disaccharides (compounds 7 and 8), the flavanones (compounds 9 and 10), and the flavonoid-related glycosidic compounds 11-13 were only weakly active. The aglycons of compounds 7 and 8, however, were potent antimutagens (IC(50) = 10.4 and 13.0 microM, respectively).

Impact of the post-treatment conditions of parent silica on the silanization of n-octadecyl bonded silica packings in reversed-phase high-performance liquid chromatography.

Native mesoporous silica beads were subjected to a sequence of post-treatment procedure including hydrochloric acid treatment, calcination and subsequent rehydroxylation. The post-treated silica beads were converted into RP-18 silica by silanization with monochloro- and dimethoxy-n-octadecylsilanes, respectively. The influence of post-treatments and silanization conditions on the physico-chemical characteristics and on the chromatographic behaviour of the RP-silicas was studied. Also the changes of the pore structural parameters and the silanol group densities during the post-treatment and silanization were assessed.

Life-time studies with capillary electrochromatography columns operated under different conditions.

A test system has been established to permit the monitoring of the life-time performance of several reversed- phase capillary electrochromatography (CEC) columns. The retention factors, k(cec), peak symmetry coefficients, lambda(sym), and column efficiencies, N, of three neutral n-alkylbenzene analytes, namely ethyl-, n-butyl- and n-pentylbenzenes, were determined for Hypersil 3 microm n-octylsilica and n-octadecylsilica packed into CEC capillary columns of 100 microm I.D., with a packed length of 250 mm, and a total length of 335 mm. The performances of these CEC capillary columns were examined for a variety of eluents with pH values ranging between pH 2.0 - 8.0, similar to those employed to study the retention behaviour of peptides that we have previously reported. The relative standard deviation (RSD) of the retention factors (k(cec) values) of these n-alkylbenzenes, acquired with an eluent of (25 mM Tris-HCl, pH 8.0,)-acetonitrile (1:4, v/v), when the CEC capillary columns were used for the first time (virgin values), were 4% (based on data acquired with 4 CEC capillary columns) for the n-octyl bonded silica capillary columns, and 6% (based on 8 columns) for n-octadecyl bonded silica capillary columns. The RSD values of the k(cec) values of the n-alkylbenzenes for one set of replicates (n=6) with one CEC capillary column was < 0.5%. The theoretical plate numbers, N, for the virgin CEC capillary columns were ca. 60,000, whilst the observed N values for all new CEC capillary columns were > or = 40,000 for n-octyl bonded silica capillary columns and > or = 50,000 for n-octadecyl bonded silica capillary columns. The peak symmetry coefficients, lambda(sym), of the n-alkylbenzenes for virgin CEC capillary columns and for CEC capillary columns used for more than 1,000 injections were always in the range 0.95-1.05. The experimental results clearly document that the life-time performance of the CEC capillary columns depends on the eluent composition, as well as the nature of the analytes to which the CEC capillary columns are exposed.

Comparative study of capillary electroendosmotic chromatography and electrically assisted gradient nano-liquid chromatography for the separation of peptides.

Capillary electroendoendosmotic chromatography (CEC), being a hybrid of high-performance liquid chromatography (HPLC) and capillary electrophoresis, offers considerable changes to enhance column efficiency, speed of analysis and additional selectivity as compared to the parent methods. The analytes are driven by the electroendosmotic flow (EOF) and separated by surface-solute interactions as well as by differences in electromigration. In this paper on the separation of peptides on C18 reversed-phase and mixed-mode (sulphonic acid-n-alkyl) packings in CEC and electrically assisted reversed-phase gradient nano-LC are investigated. It is shown that mixed mode packings generate a higher EOF than reversed-phase packings that is scarcely dependent on the pH of the eluent. Applying a potential in gradient elution reversed-phase nano-LC of peptides shortens the analysis time as compared to separations without a potential. Electrically assisted reversed-phase gradient elution nano-LC is a powerful separation tool for analysis of tryptic digests. Peptides can be successfully resolved in acidic organic mobile phase at pH 2-3 with and without trifluoroacid as ion pairing reagent under isocratic conditions. It is demonstrated that CEC with mixed mode packing and an eluent of pH 2.3 with varying acetonitrile content can be applied to monitor impurities in a synthetic peptide.

Protein mapping by two-dimensional high performance liquid chromatography.

Current developments in drug discovery in the pharmaceutical industry require highly efficient analytical systems for protein mapping providing high resolution, robustness, sensitivity, reproducibility and a high throughput of samples. The potential of two-dimensional (2D) HPLC as a complementary method to 2D-gel electrophoresis is investigated, especially in view of speed and repeatability. The method will be applied for proteins of a molecular mass <20 000 which are not well resolved in 2D-gel electrophoresis. The 2D-HPLC system described in this work consisted of anion- or cation-exchange chromatography in the first dimension and reversed-phase chromatography in the second dimension. We used a comprehensive two-dimensional approach based on different separation speeds. In the first dimension 2.5 microm polymeric beads bonded with diethylaminoethyl and sulfonic acid groups, respectively, were applied as ion exchangers and operated at a flow-rate of 1 ml/min. To achieve very high-speed and high-resolution separations in the second dimension, short columns of 14 x 4.6 mm I.D. with 1.5 microm n-octadecyl bonded, non-porous silica packings were chosen and operated at a flow-rate of 2.5 ml/min. Two reversed-phase columns were used in parallel in the second dimension. The analyte fractions from the ion-exchange column were transferred alternatively to one of the two reversed-phase columns using a 10-port switching valve. The analytes were deposited in an on-column focusing mode on top of one column while the analytes on the second column were eluted. Proteins, which were not completely resolved in the first dimension can, in most cases, be baseline-separated in the second dimension. The total value of peak capacity was calculated to 600. Fully unattended overnight runs for repeatability studies proved the applicability of the system. The values for the relative standard deviation (RSD) of the retention times of proteins were less than 1% (n = 15), while the RSDs of the peak areas were less than 15% (n = 15) on average. The limit of detection was 300 ng of protein on average and decreased to 50 ng for ovalbumin. The 2D-HPLC system offered high-resolution protein separations with a total analysis time of less than 20 min, equivalent to the run time of the first dimension.

Influence of temperature on the behaviour of small linear peptides in capillary electrochromatography.

The influence of temperature, T, on the retention times, peak widths, peak symmetry coefficients and theoretical plate numbers of two small linear peptides, [Met5]enkephalin and [Leu5]enkephalin, has been studied with capillary electrochromatography (CEC) capillary columns of 100 microm I.D. and 250 mm packed length with a total length of 335 mm, containing 3 microm Hypersil n-octadecyl bonded silica. With increasing column temperature from 15 to 60 degrees C, the electroosmotic flow (EOF) and the column efficiencies increased, whereas the retention coefficients (Kcec) of both peptides decreased. A linear relationship was found between the EOF value and the square root of the temperature over this temperature range, with a linear regression correlation of 0.998. Non linear Van 't Hoff plots (In Kcec versus 1/T) were observed for these peptides between 15 and 60 degrees C, suggesting that a phase-transition occurred with the n-octadecyl chains bonded on the silica surface, affecting the CEC retention behaviour of these peptides. In CEC systems, the Kcec values of peptides incorporate contributions from both electrophoretic migration and chromatographic retention. Positive and negative Kcec values can, in principle, thus arise with these charged analytes. However, the Kcec values of the enkephalin peptides under all temperature conditions studied were positive with an eluent composed of water-50 mM NH4OAc/AcOH, pH 5.2-acetonitrile (5:2:3, v/v) and therefore the chromatographic component dominates the retention process with these small peptides under these conditions.

Synthesis of spherical porous silicas in the micron and submicron size range: challenges and opportunities for miniaturized high-resolution chromatographic and electrokinetic separations.

Classical silica technology has reached its limit with respect to an ultimate minimum particle size of about 2 microm in diameter. Here, a novel process is presented which allows one to synthesize porous silica beads and control their particle diameter in situ, within the range of 0.2-2.0 microm. As a result, no sizing is required and losses of silica are avoided. Furthermore, the process enables one to control in situ the pore structural parameters and the surface chemistry of the silica beads. Even though surface funtionalized silicas made according to this process can principally be applied in fast HPLC the column pressure drop will be high even for short columns. In addition, the column efficiency, expressed in terms of the theoretical plate height is about H-2d(p) in the best case and limited by the A and C term of the Van Deemter equation. In other words the gain in total plate number when using 1-2 microm silica beads in short columns is minimal as compared to longer columns packed with 5 microm particles. Capillary electrochromatography (CEC) as a hybrid method enables the application of micron size as well as submicron size particles. This consequently enhances column efficiency by a factor of 5-10 when compared to HPLC. The use of short CEC columns packed with submicron size silicas provides the basis for fast and efficient miniaturized systems. The most significant feature of CEC as compared to HPLC is that the former allows one to resolve polar and ionic analytes in a single run. An alternative method for miniaturization is capillary electrophoresis (CE) which generates extremely high efficiencies combined with fast analysis. Its application, however, is limited to ionic substances.

Analysis of neuropeptide Y and its metabolites by high-performance liquid chromatography-electrospray ionization mass spectrometry and integrated sample clean-up with a novel restricted-access sulphonic acid cation exchanger.

A novel restricted access cation exchanger with sulphonic acid groups at the internal surface was proven to be highly suitable in the sample clean up of peptides on-line coupled to HPLC-electrospray ionization (ESI)-MS. Neuropeptide Y (NPY) and several of its fragments in plasma were subjected to the sample clean-up procedure. The peptides were eluted by a step gradient from the restricted access column, applying 10 mM phosphate buffer pH 3.5 from 5 to 20% (v/v) of acetonitrile with 1 M NaCl and transferred to a Micra ODS II column (33x4.6 mm). The separation of the peptides and their fragments was performed by a linear gradient from 20 to 60% (v/v) acetonitrile in water with 0.1% formic acid and 0.01% trifluoroacetic acid in 4 min at a flow-rate of 0.75 ml/min. An integrated and completely automated system composed of sample clean up-HPLC-ESI-MS was used to analyze real life samples. The sample volumes ranged between 20 and 100 microl. Peaks due to the fragments NPY 1-36, 3-36 and 13-36 in porcine plasma were identified by ESI-MS. The limit of detection was in the 5 nmol/ml range. The total analysis required 21 min and allowed the direct injection of plasma.