Investigation of the novel mixed-mode stationary phase for capillary electrochromatography. II. Separation of amino acids and peptides on sulfonated naphthalimido-modified silyl silica gel

The potential of 3-(4-sulfo-1,8-naphthalimido)propyl-modified silyl silica gel (SNAIP) as a mixed-mode stationary phase for capillary electrochromatography (CEC) was investigated for the separation of charged analytes, taking four amino acids (tyrosine, phenylalanine, tryptophan, histidine) as model analytes. The elution process of these charged analytes in CEC with SNAIP was dominated by a combination of both electrophoretic process and chromatographic process involving hydrophobic as well as electrostatic interactions. In order to study the retention mechanism, the CEC retention factor k* and the velocity factor ke* were measured for the amino acids, which allowed the assessment of the respective contribution from the differential processes underlying the separation. Migration and retention could be mediated by changing various mobile phase compositions, including buffer pH, buffer concentration, and concentration of organic solvent. Based on the results obtained by separation of the amino acids, the separation of eight peptides (Gly-Val, Gly-Phe, Gly-Ile, Gly-His, Gly-Lys, Lys-Lys, Gly-Gly-Gly, Gly-Gly-His) was attempted. A good separation was achieved under an isocratic elution with a mobile phase consisting of 35 mM phosphate buffer (pH 3.8) and 40% methanol.     

Investigation of a novel mixed-mode stationary phase for capillary electrochromatography. Part III: Separation of nucleosides and nucleic acid bases on sulfonated naphthalimido-modified silyl silica gel

Capillary electrochromatography (CEC) with a novel stationary phase, 3-(4-sulfo-1,8-naphthalimido)propyl-modified silyl silica gel (SNAIP), proved useful for the separation of nucleosides and nucleic acid bases. The application scope of SNAIP, which is a relatively polar reversed-phase (RP)-type stationary phase, was successfully expanded to include the CEC separation of polar compounds although the combination of non-polar RP phase with highly aqueous mobile phase is often inadequate. Due to the permanently charged sulfonic acid groups and the naphthalimidopropyl moiety, the retention of charged and relatively polar nucleosides as well as bases on the SNAIP stationary phase was effected by electrostatic and hydrophobic interactions. This yielded a unique selectivity on SNAIP toward nucleosides and bases. The characteristic EOF on SNAIP, which was stronger at higher aqueous content in the mobile phase, proved suitable for the separation of polar compounds in reversed-phase mode with highly aqueous mobile phase. In addition, when a double stepwise gradient was employed to accelerate the latest peak (adenine), the elution time was shortened to less than half its original duration.     

Stepwise gradient of buffer concentration for capillary electrochromatography of peptides on sulfonated naphthalimido-modified silyl silica gel

The advantage of using a stepwise gradient of buffer concentration in CEC was demonstrated with the mixed-mode stationary phase, 3-(4-sulfo-1,8-naphthalimido)propyl-modified silyl silica gel (SNAIP). Before the application of a stepwise gradient, the effect of buffer concentration on the separations of six peptides and tryptic digests was investigated. Bubble formation caused by Joule heating at currents up to 95 microA was successfully suppressed by using SNAIP column even without pressurization, which contributed to a stepwise gradient of buffer concentration. Utilizing the stepwise gradient improved and shortened the separation of six peptides as compared to the separation under an isocratic elution.     

Preparation and characterization of mixed-mode monolithic silica column for capillary electrochromatography

A silica-based monolithic stationary phase with mixed-mode of reversed phase (RP) and weak anion-exchange (WAX) for capillary electrochromatography (CEC) has been prepared. The mixed-mode monolithic silica column was prepared using the sol–gel technique and followed by a post-modification with hexadecyltrimethoxysilane (HDTMS) and aminopropyltrimethoxysilane (APTMS). The amino groups on the surface of the stationary phase were used to generate a substantial anodic EOF as well as to provide electrostatic interaction sites for charged compounds at low pH. A cathodic EOF was observed at pH above 7.3 due to the full ionization of residual silanol groups and the suppression in the ionization of amino groups. A variety of analytes were used to evaluate the electrochromatographic characterization and column performance. The monolithic stationary phase exhibited RP chromatographic behavior toward neutral solutes. The model anionic solutes were separated by the mixed-mode mechanism, which comprised RP interaction, WAX, and electrophoresis. Symmetrical peaks can be obtained for basic solutes because positively charged amino groups can effectively minimize the adsorption of positively charged analytes to the stationary phase.     

Preparation of monolithic silica column with strong cation-exchange stationary phase for capillary electrochromatography

A monolithic silica based strong cation-exchange stationary phase was successfully prepared for capillary electrochromatography. The monolithic silica matrix from a sol-gel process was chemically modified by treatment with 3-mercaptopropyltrimethoxysilane followed by a chemical oxidation procedure to produce the desired function. The strong cation-exchange stationary phase was characterized by its substantial and stable electroosmotic flow (EOF), and it was observed that the EOF value of the prepared column remained almost unchanged at different buffer pH values and slowly decreased with increasing phosphate concentration in the mobile phase. The monolithic silica column with strong cation-exchange stationary phase has been successfully employed in the electrochromatographic separation of beta-blockers and alkaloids extracted from traditional Chinese medicines (TCMs). The column efficiencies for the tested beta-blockers varied from 210,000 to 340,000 plates/m. A peak compression effect was observed for atenolol with the mobile phase having a low phosphate concentration.     

Use of vancomycin silica stationary phase in packed capillary electrochromatography I. Enantiomer separation of basic compounds

Chiral separation of basic compounds was achieved by using 75 or 100 microm ID fused-silica capillaries packed with a vanoomycin-modified diol silica stationary phase. The capillary was firstly packed for about 12 cm with a slurry mixture composed of diolsilica (3:1) then with the vancomycin modified diol-silica (3:1) (23 cm), and finally with diol-silica (3:1) for about 2 cm. Frits were prepared by a heating wire at the two ends of the capillary; the detector window was prepared at 8.5 cm from the end of the capillary where vancomycin was not present. The influence of the mobile phase composition (pH and concentration, organic modifier type and concentration) on the velocity of the electroosmotic flow, chiral resolution and enantioselectivity was studied. Good enantiomeric resolution was achieved for atenolol, oxprenolol, propranolol, and venlafaxine using a mobile phase composition of 100 mM ammonium acetate solution (pH 6)/water/acetonitrile (5:5:90 v/v/v) while for terbutaline a mixture of 5:15:80 v/v/v provided the best separations. The use of methanol instead of acetonitrile caused a general increase of enantiomer resolution of the studied compounds together with a reduction of efficiency and detector response. However, the combination of acetonitrile and methanol in the mobile phase (as, e.g., 10% methanol and 80% acetonitrile) allowed to improve the enantiomer resolution with satisfactory detector response.     

Investigation of the ion-exchange properties of methacrylate-based mixed-mode monolithic stationary phases employed as stationary phases in capillary electrochromatography

The potential of methacrylate-based mixed-mode monolithic stationary phases bearing sulfonic acid groups for the separation of positively charged analytes (alkylanilines, amino acids, and peptides) by capillary electrochromatography (CEC) is investigated. The retention mechanism of protonated alkylanilines as positively charged model solutes on these negatively charged mixed-mode stationary phases is investigated by studying the influence of mobile phase and stationary phase parameters on the corrected retention factor which was calculated by taking the electrophoretic mobility of the solutes into consideration. It is shown that both solvophobic and ion-exchange interactions contribute to the retention of these analytes. The dependence of the corrected retention factor on (1) the concentration of the counter ion ammonium and (2) the number of methylene groups in the alkyl chain of the model analytes investigated shows clearly that a one-site model (solvophobic and ion-exchange interactions take place simultaneously at a single type of site) has to be taken to describe the retention behaviour observed. Comparison of the CEC separation of these charged analytes with electrophoretic mobilities determined by open-tubular capillary electrophoresis shows that mainly chromatographic interactions (solvophobic and ion-exchange interactions) are responsible for the selectivity observed in CEC, while the electrophoretic migration of these analytes plays only a minor role.     

Preparation and evaluation of a lysine-bonded silica monolith as polar stationary phase for hydrophilic interaction pressurized capillary electrochromatography

A silica-based monolith as polar stationary phase was described for hydrophilic interaction pressurized capillary electrochromatography (HI-pCEC). The polar monolithic column was prepared by on-column reaction of lysine with epoxy groups on a gamma-glycidoxypropyltrimethosysilane-modified silica monolith. The stationary phase yielded strong hydrophilic interaction due to the slightly polar hydroxyl groups, and the strong polar lysine ligand with amino groups and carboxylic groups contained on the surface of the monolith. In order to evaluate the hydrophilic character of lysine ligand, the chromatographic behaviors of epoxy monolith (before lysine bonded) and diol monolith (hydroxyl groups contained) were also investigated. Two groups of comparative experiment were developed in terms of the separation of typical neutral non-polar and polar compounds performed in a mobile phase of aqueous-acetonitrile solution. Results showed that the lysine monolith was much more hydrophilic than the diol monolith, which presented less hydrophobic than the epoxy monolith. For further study on its hydrophilic character, the lysine monolith was demonstrated in the HI-pCEC mode for the separations of various polar compounds such as phenols, nucleic acid bases and nucleosides.     

Investigation of mixed-mode monolithic stationary phases for the analysis of charged amino acids and peptides by capillary electrochromatography

The potential of N,N-dimethylacrylamide-piperazine diacrylamide-based monolithic stationary phases bearing sulfonic acid groups for electroosmotic flow generation is investigated for the separation of positively charged amino acids and peptides. The capillary columns were used under electrochromatographic but also under purely chromatographic (nano-HPLC) conditions and the separations interpreted as the result of possible chromatographic and electrophoretic contributions. The stationary phases were found to be mechanically stable up to pressures of 190 bar and chemically stable towards a wide variety of organic and hydro-organic mobile phases. In order to investigate the retention mechanism, the salt concentration and the organic solvent content of the (hydro-)organic mobile phase were varied in a systematic manner, taking three aromatic amino acids (phenylalanine, tryptophan, histidine) as model analytes. The respective contributions of electrostatic and hydrophobic and/or hydrophilic interactions were further investigated by varying the charge density and the hydrophobicity of the standard stationary phase. The former was done by varying the amount of charged monomer (vinylsulfonic acid) added during synthesis, the latter by (partially) replacing the interactive monomer (N,N-dimethylacrylamide) by other more hydrophobic monomers. A mixed mode retention mechanism based primarily on electrostatic interactions modified in addition by "hydrophilic" ones seems most suited to interpret the behavior of the amino acids, which stands in contradistinction to the previously investigated case of the behavior of neutral analytes on similar stationary phases. Finally the separation of small peptides was investigated. While the separation of Gly-Phe and Gly-Val was not possible, the separation of Phe-Gly-Phe-Gly and Gly-Phe but also of the closely related Gly-His and Gly-Gly-His could be achieved.     

Physically adsorbed chiral stationary phase of avidin on monolithic silica column for capillary electrochromatography and capillary liquid chromatography

The physical adsorption method proposed previously has been successfully applied to a monolithic silica column. By virtue of the physical adsorption, a chiral stationary phase of avidin was prepared onto the silica monolith. The phase ratio of resulting stationary phase was evaluated with frontal analysis. The method proved to be comparable in phase ratio to the chemical bonding methods used in high-performance liquid chromatography (HPLC). Enantiomer separations were carried out in capillary electrochromatography (CEC) and capillary liquid chromatography (CLC) modes. Due to its larger phase ratio, the resulting column showed more powerful separation capability as compared to open-tubular CEC (OTCEC). Twelve chiral compounds were baseline-resolved. The resulting column showed high separation efficiency, with average theoretical plate numbers of 66 000/m for CLC and 122 000/m for CEC. Good reproducibility was observed, with RSD value less than 1.3% for retention time, retention factor and separation factor, and less than 6.6% for plate counts and resolution (n = 40). Fast separations were achieved with a short column. The test enantiomers were baseline-resolved within 4 min under CLC and CEC modes. In addition, field-enhanced sample injection (FESI) was coupled to CLC as well as CEC to improve the detection sensitivity.     

Preparation and characterization ofp-tert-butyl-calix[6]arene-bonded silica gel stationary phase for high-performance liquid chromatography

Summary For the first time calix[6]arene has been chemically combined with silica gel via a longer spacer to prepare calix[6]arene-bonded silica gel stationary phase for high-performance liquid chromatography (HPLC). The separation of positional isomers and polycyclic aromatic hydrocarbons on the calix[6]arene-bonded phase was achieved with methanol-water as mobile phase. Some nucleosides were also separated on the bonded phase. The reversed-phase chromatographic performance of the bonded phase was studied. The results showed that the calix[6]arene-bonded phase was highly hydrophobic. A possible separation mechanism has been proposed.     

Preparation and evaluation of zirconia-coated silica monolith for capillary electrochromatography

Silica monoliths were fabricated inside fused-silica capillaries. Then the monolithic columns were coated with membrane-like zirconia. The zirconia-coated silica monoliths exhibited different EOF behavior comparing with that of bare silica monoliths. The magnitude and direction could be manipulated by changing the running buffers. Due to the amphoteric characteristic of zirconia, the silica monoliths with zirconia surface facilitate the separation of basic compounds. Aromatic amines and alkaloids were separated without obvious peak tailing. The zirconia surface was easily modified with octadecylphosphonic acid for the separation of neutral compounds. Column efficiency as high as 90,000 and 80,000 m⁻¹ was obtained for beberine and naphthalene, respectively. Furthermore, the zirconia coating increased the stability of the monolithic columns. Even after being exposed to severe condition, there was no apparently efficiency decrease for the test samples.     

Evaluation of ODS-AQ stationary phase for use in capillary electrochromatography

The aim of this study was to evaluate the applicability of ODS-AQ packing material as a stationary phase in capillary electrochromatography (CEC). The electroosmotic flow created on an ODS-AQ stationary phase was measured at different mobile phase compositions and at different column temperatures. It was observed that the electroosmotic flow generated in the column increased by 50% when the temperature of the system was raised from 20 degrees C to 60 degrees C, while all other conditions were kept constant. The electroosmotic flow produced by the ODS-AQ stationary phase was found to be comparable to the flow generated in a column packed with Nucleosil bare-silica material. In addition, a set of polar compounds (D-lysergic acid diethylamide derivatives) was utilized to determine the influence of temperature and mobile phase composition on their chromatographic behavior on an ODS-AQ stationary phase in a CEC mode. A linear relationship between the solute retention factor and column temperatures was seen over the temperature range studied (20 degrees C to 60 degrees C). A quadratic function was used to describe the changes in the solute retention factors with variation of acetonitrile concentration in the mobile phase.     

Separation of basic and acidic compounds by capillary electrochromatography using monolithic silica capillary columns with zwitterionic stationary phase

A novel monolithic silica column with zwitterionic stationary phase was prepared by in-situ covalent attachment of phenylalanine to a 3-glycidoxypropyltriethoxysilane-modified silica monolith. Due to the zwitterionic nature of the resulting stationary phase, the density and sign of the net surface charge, and accordingly the direction and magnitude of electroosmotic flow in this column during capillary electrochromatography could be manipulated by adjusting the pH values of the mobile phase. CEC separations of various acidic and basic compounds were performed on the prepared column in anodic and weakly cathodic EOF modes, respectively. The peak tailing of basic compounds in CEC on a silica column could be alleviated at optimized buffer compositions. Besides the electrophoretic mechanism and weak hydrophobic interaction, weak cation- and anion-exchange interactions are also involved in the separations of acids and bases, respectively, on the zwitterionic column.     

Capillary electrochromatography with novel stationary phases. IV. Retention behavior of glycosphingolipids on porous and non-porous octadecyl sulfonated silica

In this investigation, capillary electrochromatography (CEC) with a novel stationary phase proved useful for the separation of neutral and acidic glycosphingolipids (GSLs). Four different gangliosides, namely G(M1a), G(D1a), G(D1b) and G(T1b), served as the acidic GSLs model solutes. The following four GSLs: galactosylceramide (GalCer), lactosylceramide (LacCer), globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) served as the typical neutral GSLs. The stationary phase, octadecyl sulfonated silica (ODSS), consisted of octadecyl functions bonded to a negatively charged layer containing sulfonic acid groups. Porous and non-porous ODSS stationary phases were examined. The retention behavior of the acidic and neutral GSLs was examined over a wide range of elution conditions, including the nature of the electrolyte and organic modifier and the pH of the mobile phase. The porous ODSS stationary phase yielded the separation of the four different gangliosides using a hydro-organic eluent of moderate eluent strength whereas the non-porous ODSS stationary phase permitted the separation of the four neutral GSLs with a mobile phase of relatively high eluent strength.     

Histidine-functionalized silica and its copper complex as stationary phases for capillary electrochromatography

A histidine-functionalized silica was prepared by covalent bonding of the functional groups to silane-treated silica gel. Conversion of functional groups was confirmed by infrared (IR) spectra, elemental analysis, and potentiometry. The functionality of the silica gel is 0.293 mmol g(-1). The coordination behavior of the histidine-functionalized silica was investigated by metal capacity and electron paramagnetic resonance (EPR). EPR measurements at different copper loadings were made. The results showed that the copper histidine complex might be distorted tetragonal. Both histidine-functionalized silica and its copper complex were employed as stationary phases for packed capillary electrochromatography (CEC). Electrical current was found helpful for evaluating the properties of frit construction and the stationary phase packing. Test samples include neutral compounds, inorganic anions and organic anions. Factors influencing the separation behavior have been studied. With copper-histidine functionalized silica under the condition of citrate buffer (10 mM, pH 4.0) and applied voltage of -20 kV, the separation of benzoic acid, D- and L-mandelic acid, phthalic acid and salicylic acid could be achieved within 12 min. The column efficiency for these acids was more than 1.2 x 10(5) plates m(-1), except salicylic acid.     

Investigations on the behaviour of acidic, basic and neutral compounds in capillary electrochromatography on a mixed-mode stationary phase

This work describes the separation of acidic, basic and neutral organic compounds as well as inorganic anions in a single run by capillary electrochromatography employing a stationary phase which exhibits both strong anion-exchange and reversed-phase chromatographic characteristics. The positive surface charge of this stationary phase provided a substantial anodic electroosmotic flow. The analytes were separated by a mixed-mode mechanism which comprised chromatographic interactions (hydrophobic interactions, ion-exchange) as well as electrophoretic migration. The influence of ion-exchange and hydrophobic interactions on the retention/migration of the analytes could be manipulated by varying the concentration of a competing ion and/or the amount of organic modifier present in the background electrolyte. Additionally the effects of pH changes on both the chromatographic interactions as well as the electrophoretic migration of the analytes were investigated.     

高效液相色谱喹啉醚基键合硅胶固定相的制备及评价

采用固液相连续反应法,以γ-环氧丙氧基)丙基三甲氧基硅烷(KH560)为偶联剂,制备了一种喹啉醚基键合硅胶固定相(QBS),采用元素分析、漫反射红外光谱和热分析表征了固定相的结构。多种溶质为探针(包括非极性的烷基苯和多环芳烃、芳香族化合物位置异构体及极性的核苷和碱基等),较系统地研究了该新固定相的色谱性能。研究表明,新固定相与ODS相比,除具有弱的疏水性外,还能与溶质发生多种作用,如：氢键和π-π作用等。在分离非极性的多环芳烃时主要基于疏水作用;在分离极性的核苷和碱基时,氢键和络合作用较重要;在分离芳香族化合物位置异构体时,溶质极性取代基与喹啉醚基键合相的氢键作用。溶质苯环与喹啉基配体之间的π-π作用,两协同作用提高了QBS对位置异构体的分离选择性。     

Recent highlights in stationary phase design for open-tubular capillary electrochromatography

This review examines the most recent innovations made to achieve high performance in open-tubular capillary electrochromatography (OT-CEC) separations, focusing on the ingenious chemical and physical solutions made to increase the surface area and equip the stationary phase with exploitable selectivity. Among the approaches taken are chemically bonded ligands, etching with chemical bonding, sol-gels, molecularly imprinted polymers, porous layers, physically attached or adsorbed phases, and nanoparticle coatings. Particularly noteworthy are modern developments with macrocyclic receptor ligands, nanoparticles and open channel electrochromatography on-chip.     

Immobilisation and evaluation of a vancomycin chiral stationary phase for capillary electrochromatography

The macrocyclic antibiotic, vancomycin, is covalently bonded to LiChrospher diol silica packed columns and evaluated in capillary electrochromatography (CEC) both in the reversed-phase and polar organic mode. Initially, capillaries were packed with 5 microm LiChrospher 100 A diol silica and evaluated in CEC with a reversed-phase biphenyl-pyrene achiral test resulting in resolution and efficiency values of ca. 2.5 and 100000 plates meter(-1), respectively. Repeatability for this test (resolution and efficiency) was also examined and found to be acceptable for both run-to-run (n=5, 0.74% and 1.5%) and column-to-column (n=5, 3.4% and 9.0%), respectively. Similar results were obtained when the 10 microm LiChrospher 1000 A diol silica was examined with the exception of efficiency, where a reduced plate height value of four times lower was obtained compared to the 100 A material. A simple three step in-situ vancomycin immobilisation procedure was subsequently carried out on these packed diol columns. Selectivity was obtained for thalidomide enantiomers on this vancomycin chiral stationary phase in reversed-phase CEC with resolution and efficiency values of ca. 2.5 and 80000 plates meter(-1), with acceptable repeatability (n=8) 0.9% and 3.0%, respectively. Selectivity was also obtained for thalidomide enantiomers on this phase in the polar organic mode with resolution and efficiency values of ca. 2.5 and 120000 plates meter(-1), with acceptable repeatability (n=7) 0.9% and 2.0%, respectively. It was possible to deduce from a chemometric design carried out for evaluating the mobile phase component effects that organic modifier ratio, MeOH/MeCN, played a significant role in controlling both resolution and efficiency. It was also possible to separate a number of basic analytes including four beta-adrenergic blocking agents in the polar organic mode albeit with lower resolution and efficiency values, ca. 1.5 and 45000 plates meter(-1), respectively.