Capillary electrochromatography with novel stationary phases: II. Studies of the retention behavior of nucleosides and bases on capillaries packed with octadecyl-sulfonated-silica microparticles

An octadecyl-sulfonated silica (ODSS) stationary phase specially designed for performing capillary electrochromatography (CEC) at relatively strong electroosmotic flow (EOF) proved useful for the separations of some nucleosides and bases. The ODSS stationary phase is composed of a hydrophilic, negatively charged sublayer to which a nonpolar top layer containing octadecyl ligands is covalently attached. The charged sublayer contains sulfonic acid groups which ensure a relatively strong EOF. Due to the presence of permanently charged sulfonic acid groups in the sublayer, the hydrophilic nature of the sublayer and the hydrophobic character of the top octadecyl layer, retention and selectivity of charged and relatively polar nucleosides and bases on the ODSS stationary phase are based on electrostatic interaction, hydrophilic interaction, and reversed-phase mechanisms. This yielded for the ODSS stationary phase a unique selectivity towards the nucleosides and bases, thus allowing their rapid separation. To gain insight into the chromatographic behavior of nucleosides and bases on the ODSS stationary phase, the results were compared to those obtained on an octadecyl-silica (ODS) capillary under otherwise the same elution conditions. Due to the difference in the nature of the organic layers on the surface of the ODSS and ODS stationary phases, the elution order on both stationary phases differed significantly, and the ODSS capillary proved more suitable for the separation of the nucleosides and bases than the ODS capillary.     

On the structural diversity of Shiga toxin glycosphingolipid receptors in lymphoid and myeloid cells determined by nanoelectrospray ionization tandem mass spectrometry

Shiga toxin (Stx, synonymous to verotoxin, VT) binds with high and low affinity to the globo‐series neutral glycosphingolipids (GSLs), globotriaosylceramide (Gb3Cer or Galα4Galβ4Glcβ1Cer, also known as CD77) and globotetraosylceramide (Gb4Cer or GalNAcβ3Galα4Galβ4Glcβ1Cer), respectively, which represent the targets of Stxs on many different cell types. B‐cell‐derived Raji cells and THP‐1 cells of monocytic origin are widely used for the investigation of Stx‐mediated cellular response, because Stx is known to cause cell death in both cell lines. Despite their functional importance, the Stx receptors of Raji and THP‐1 cells have so far not been investigated. This prompted us to explore the structures of their GSL receptors in detail by means of nanoelectrospray ionization quadrupole time‐of‐flight mass spectrometry (nanoESI‐QTOF‐MS) with collision‐induced dissociation (CID) in conjunction with Stx1 as well as anti‐Gb3Cer and anti‐Gb4Cer antibodies. Using the combination of a thin‐layer chromatography (TLC) overlay assay and MS¹ and MS² analysis we identified Gb3Cer (d18:1, C24:1/C24:0) as the prevalent Stx1‐receptor accompanied by less abundant Gb3Cer (d18:1, C16:0) in the neutral GSL fraction of Raji cells. The same Gb3Cer species but with almost equal proportions of the C24:1/C24:0 and C16:0 variants were found in THP‐1 cells. In addition, unusual hydroxylated Gb3Cer (d18:1, C24:1/C24:0) and Gb3Cer (d18:1, C26:1) could be identified in trace quantities in both cell lines. As the most obvious difference between Raji and THP‐1 cells we observed the expression of Gb4Cer in THP‐1 cells, whereas Raji cells failed to express this elongation product of Gb3Cer. Both short‐ and long‐chain fatty acid carrying Gb4Cer (d18:1, C16:0) and Gb4Cer (d18:1, C24:1/C24:0), respectively, were the prevalent Gb4Cer variants. This first report on the differential expression of Gb3Cer and Gb4Cer and their structural diversity in lymphoid and myeloid cell lines supports the hypothesis that such heterogeneities might play a functional role in the molecular assembly of GSLs in membrane organization and cellular signaling of Stx‐susceptible cells. Copyright © 2010 John Wiley & Sons, Ltd.     

Application of combined high-performance thin-layer chromatography immunostaining and nanoelectrospray ionization quadrupole time-of-flight tandem mass spectrometry to the structural characterization of high- and low-affinity binding ligands of Shiga toxin 1

Shiga toxin 1 (Stx1) represents an AB5 toxin produced by enterohemorrhagic Escherichia coli, which cause gastrointestinal diseases in humans that are often followed by potentially fatal systemic complications, such as acute encephalopathy and hemolytic uremic syndrome. The expression of the preferential Stx1 receptor, Gb3Cer/CD77 (Gal alpha1-4Gal beta1-4Glc beta1-1Cer), is one of the primary determinants of susceptibility to tissue injury. Due to the clinical importance of this life-threatening toxin, a combined strategy of preparative high-performance thin-layer chromatography (HPTLC) overlay assay and mass spectrometry was developed for the detection and structural characterization of Stx1-binding glycosphingolipids (GSLs). A preparation of neutral GSLs from human erythrocytes, comprising 21.4% and 59.1% of the high- and low-affinity Stx1-binding ligands Gb3Cer/CD77 and Gb4Cer, respectively, was separated on silica gel precoated HPTLC plates and probed for the presence of Stx1 receptors. Stx1 positive on the one hand and anti-Gb3Cer/CD77 and anti-Gb4Cer antibody positive bands from parallel reference runs on the other hand were extracted with chloroform/methanol/water (30/60/8, v/v/v). These crude extracts were used without any further purification for a detailed structural analysis by nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS) in the negative ion mode. In all extracts investigated, neutral GSLs were detected as singly charged deprotonated molecular ions, [M-H]-, and neither buffer-derived salt adducts nor coextracted contaminants from the overlay assay procedure or the silica gel layer were observed. For the structural characterization of Stx1- and antibody-binding GSLs low-energy collision-induced dissociation (CID) was applied to high and low abundant receptor species of the crude extracts. All MS/MS spectra obtained contained full series of Y-type ions, B-type ions and additional ions generated by ring cleavages of the sugar moiety. Only analytical quantities in the microgram scale of a single GSL species within the complex GSL mixture were required for the structural MS characterization of Stx1 ligands as Gb3Cer/CD77 and Gb4Cer. This effective combined HPTLC/MS procedure offers a broad range of applications, not only for toxins of bacterial origin, but also for any GSL-binding agents such as plant-derived lectins or human proteins with yet unknown binding specificities.     

Enantiomeric and diastereomeric high-performance liquid chromatographic separation of cyclic beta-substituted alpha-amino acids on a teicoplanin chiral stationary phase

High-performance liquid chromatographic (HPLC) separation of stereomeric cyclic beta-substituted or-quaternary alpha-amino acids was performed on a chiral stationary phase based on the glycopeptide antibiotic teicoplanin. The investigated amino acids are the 1-amino-2-methylcyclohexanecarboxylic acids, the 1-amino-2-hydroxycyclohexanecarboxylic acids, Ala, Cha, Phe and Tle. The effects of the mobile phase composition (type and content of organic modifier, pH) and of the temperature on the enantio- and diastereoselectivity were studied and the conditions were optimised to resolve the four stereomers of one amino acid in a single chromatographic run. The influence of the modifier concentration and the pH of the mobile phase reveal two enantiomeric and diastereomeric discrimination mechanisms based on different interactions with the stationary phase. For optimal separation of diastereomers the column has to be conditioned with an acidic eluent.     

Preparation of a neutral porous monolith and its evaluation in pressurized capillary electrochromatography with neutral and charged solutes

A neutral, nonpolar monolithic capillary column was evaluated as a hydrophobic stationary phase in pressurized CEC system for neutral, acidic and basic solutes. The monolith was prepared by in situ copolymerization of octadecyl methacrylate and ethylene dimethacrylate in a binary porogenic solvent consisting of cyclohexanol/1,4‐butanediol. EOF in this hydrophobic monolithic column was poor; even the pH value of the mobile phase was high. Because of the absence of fixed charges, the monolithic capillary column was free of electrostatic interactions with charged solutes. Separations of neutral solutes were based on the hydrophobic mechanism with the pressure as the driving force. The acidic and basic solutes were separated under pressurized CEC mode with the pressure and electrophoretic mobility as the driving force. The separation selectivity of charged solutes were based on their differences in electrophoretic mobility and hydrophobic interaction with the stationary phase, and no obvious peak tailing for basic analytes was observed. Effects of the mobile phase compositions on the retention of acidic compounds were also investigated. Under optimized conditions, high plate counts reaching 82 000 plates/m for neutral compounds, 134 000 plates/m for acid compounds and 150 000 plates/m for basic compounds were readily obtained.     

Retention of C60 and C70 fullerenes on reversed-phase high-performance liquid chromatographic stationary phases.

The separation of C60 and C70 fullerenes on four different polysiloxane stationary phases was examined. It was determined that polar solvents can be used as mobile phases effectively for the separation of fullerene molecules. Unlike previously published work, a polymeric octadecyl siloxane (ODS) stationary phase provided higher separation factors for C70/C60 than did monomeric ODS stationary phases or phenyl substituted stationary phases. For example, for a methanol-diethyl ether (50:50, v/v) mobile phase and C60, k' approximately 5.0 separation factors, alpha = 3.3, were achieved with polymeric ODS compared to alpha = 2.2, with a monomeric ODS stationary phase. A linear solvation energy relationship (LSER) was used to model the importance of solvent interactions and stationary phase interaction to solute retention.     

Analysis of propantheline bromide and related quaternary ammonium drugs by reversed-phase,ion-pair liquid chromatography with two counterions in the eluent

Summary The addition of appropriate concentrations of an organic amine and an alkylsulphonate to the mobile phase in reversed-phase, ion-pair liquid chromatography can introduce unique selectivity in to the chromatographic system allowing separation of complex mixtures of basic, acidic and neutral compounds. As an example, the methodology for a specific stability-indicating determination of propantheline bromide, a quaternary ammonium anticholinergic agent, on several reversedphase stationary phases, was developed. The retention mechanism was studied and it was concluded that both ion-interaction processes and ion-exchange processes were involved in the separation technique developed with two counterions of opposite charge in the eluent.     

Superheated water as chromatographic eluent for parabens separation on octadecyl coated zirconia stationary phase

In this study, the use of pure water at superheated temperatures, between 100 and 200 degrees C, as a mobile phase for RP separation is explored. Instrumental parameters, such as temperature, flow rate, preheating and cooling, have shown significant effects on the quality of the chromatographic peaks. The properties of superheated water as an eluent were investigated by observing the chromatographic behaviour of four parabens on a carbon-clad zirconia (ZR) phase with covalently bonded octadecyl groups. Results were compared with those obtained at 30 degrees C on a silica-based phase with octadecyl groups, using water and ACN as mobile phase. The optimized method was finally applied to analyse parabens in a commercial body cream.     

Separation of small peptides by electrochromatography on silica-based reversed phases and hydrophobic anion exchange phases

Peptide separations are regarded as a promising application of capillary electrochromatography (CEC) and, at the same time, a suitable model to elucidate its mixed separation mechanism when charged analytes are involved. In this paper, studies on the separation of small peptides (2-4 amino acids) on a Spherisorb octadecyl silane (ODS) phase at acidic pH and on a strong anion exchange (SAX)/C18 mixed mode phase at weakly basic pH are reported. For the ODS phase a comparison of CEC, capillary zone electrophoresis (CZE) and high-performance liquid chromatography (HPLC) under identical buffer/eluent conditions is presented. The predicted retention factors for CEC under the assumption of simple superposition of HPLC retention and CZE migration matched the measured results for the peptides that had small retention factors in HPLC. For both types of stationary phases, a variation of the acetonitrile content in the mobile phase led to a wide range of retention factors, including negative values when co-electroosmotic migration was dominant. Though both the ODS and the SAX/C18 phase offer unique advantages, the SCX/C18 phase at pH 9 provides more flexibility to alter separation selectivity for the selected peptides.     

Fast separation of amino acid phenylthiohydantion derivatives by HPLC on a non-porous stationary phase

Summary A new method is described for the separation of phenylthiohydantoin (PTH) derivatives of the 20 common amino acids. The analysis requires approximately 7 minutes and good resolution is obtained by RP-HPLC on columns packed with a non-porous stationary phase (Kovasil-C14; 33×4.6 mm). Gradient elution was chosen with eluents containing either sodium acetate/ acetic acid buffers (moderately acidic conditions) or a heptafluorobutyric acid modifier (strongly acidic eluent). A slightly different elution order of the PTH-amino acids was found in the two systems. Low detection limits (in the femtomol range) were achieved with simple commercial HPLC equipment. Presented at the Balaton Symposium on High Peformance Separtion Methods, Siófok, Hungary, september 1–3, 1999     

Effect of mobile phase pH on the retention of nucleotides on different stationary phases for high-performance liquid chromatography

The main aim of the present investigation was to study the retention and separation of eight nucleotides with the use of seven stationary phases in RP HPLC mode. Two octadecyl columns were used; however, aminopropyl, alkylamide, cholesterol, alkyl-phosphate, and phenyl were also studied. Special attention was paid to the mobile phase buffer pH, since it appears that this factor is very influential in the case of chromatographic separation of nucleotides. The retention of nucleotides was greater for mobile phase pH?4.0 in comparison with pH?7.0 (except for octadecyl and phenyl packing). This is a consequence of protonization of polar groups present on the stationary phase surface. It was proved that aminopropyl, alkyl phosphate, alkylamide packing materials are not suitable for the resolution of nucleotides. On the other hand, cholesterol and phenyl stationary phases are alternatives for conventional octadecyl phases. Application of cholesterol packing allows separation of small, polar nucleotides, which is impossible to achieve in the case of octadecyl phase. Moreover, a phenyl support allows separation of nucleotides in a shorter time in comparison with octadecyl packing.     

Neutral octadecyl monolith for reversed phase capillary electrochromatography of a wide range of solutes

A neutral octadecyl monolithic (ODM) column for RP capillary electrochromatography (RP-CEC) has been developed. The ODM column was prepared by the in situ polymerization of octadecyl acrylate (ODA) as the monomer and trimethylolpropanetrimethacrylate (TRIM) as the crosslinker, in a ternary porogenic solvent containing cyclohexanol, ethylene glycol, and water. The ODM column exhibited cathodal EOF over a wide range of pH and ACN concentration in the mobile phase despite the fact that it was devoid of any fixed charges. It is believed that the EOF is due to the adsorption of ions from the mobile phase onto the surface of the monolith thus imparting to the neutral ODM column the zeta potential necessary to support the EOF required for mass transport across the monolithic column. Furthermore, the adsorption of mobile phase ions to the neutral monolith modulated solute retention and affected the separation selectivity. The wide applications of the neutral ODM column were demonstrated by its ability to separate a wide range of small and large solutes, both neutral and charged. While the separation of the neutral solutes was based on RP retention mechanism, the charged solutes were separated on the basis of their electrophoretic mobility and hydrophobic interaction with the C18 ligands of the stationary phase. As a typical result, the neutral monolithic column was able to separate peptides quite rapidly with a separation efficiency of nearly 200,000 plates/m, and this efficiency was exploited in tryptic peptide mapping of standard proteins, e. g., lysozyme and cytochrome C, by isocratic elution.     

The challenge of separating and determining biologically active electrostatically stabilized silanates using the high‐performance liquid chromatography technique

We present the results of research on the optimal conditions for the separation and determination of newly obtained hypercoordinated compounds, which belong to the group of electrostatically stabilized silanates. The research involved five stationary and four mobile phases. The best selectivity was obtained using the graphite phase and the mobile phase consisting of acetonitrile/water (80/20). The maximum selectivity of the determined electrostatically stabilized silanates was 1.13 and 1.06 for (1), (2), (3); 1.10 and 1.15 for (4), (5), (6); and 1.12 and 1.15 for (7), (8), (9). The octadecyl phase (which is recommended as standard) did not yield satisfactory results.     

Vitamin U-bonded stationary phase in capillary ion chromatography

A vitamin U-bonded stationary phase was prepared and the retention behavior of inorganic anions was examined using ion chromatography. Inorganic anions were retained on the vitamin U-bonded stationary phase under acidic as well as neutral eluent conditions in the ion-exchange mode. The elution order of the examined anions under neutral eluent conditions was nearly the same as that observed in common ion exchange mode, while the elution order observed under acidic eluent conditions was completely different from that observed in common ion exchange mode. The retention of the analyte anions under the neutral eluent conditions was due to the sulfonium groups of the vitamin U, while protonated primary amino groups caused retention of the analyte anions with different selectivity under acidic conditions. The retention factor of the analyte anions increased with decreasing eluent concentration under both eluent conditions. The present system was applied to the determination of bromide and nitrate contained in seawater.     

Novel imidazolium stationary phase for high-performance liquid chromatography

A new imidazolium anion-exchange phase immobilized on silica is synthesized. HPLC separations of common inorganic anions (IO3-, Cl-, NO2-, Br-, NO3-, I-, SCN-) have been performed using a HPLC column (200 mm x 4.6 mm I.D.) packed with this stationary phase, with a phosphate buffer solution as the mobile phase and UV detection at 200 nm. The effects of pH and concentration of eluent on the separation of anions have been studied. Chromatographic parameters are calculated and the results show that the new stationary phase is of significant potential for the analysis of these anions. Successful separations of some ordinary organic anions have also been achieved with the said stationary phase. Meaningfully, organic and inorganic anions can be determined simultaneously and satisfactorily with several neutral compounds using the column. The separation of some organic compounds including hydroxybenzenes, bases and amines by this stationary phase with only water as the eluent has been investigated.     

Two-dimensional liquid chromatography with mixed mode stationary phases

Mixed mode stationary phases with ion-pairing reagent (acidic or basic) as integral part of hydrophobic chain offers unique selectivity, and hence, are ideal for multidimensional separations. The retention of hydrophobic components is a function of organic content, whereas that of charged species is a function of organic content, ionogenic modifier and its level in the mobile phase. Hence, by controlling the parameters influencing component retention (stationary phase and mobile phase), the selectivity of chemical components in the two-dimensional plane can be manipulated to improve the separation. A two-dimensional liquid chromatograph has been developed by coupling similar and dissimilar mixed mode stationary phases in the two dimensions. This technique has immense potential in resolving co-eluting components as the retention mechanism in the two-dimensions are complementary. However, with only part of the primary column eluent sampled into the secondary column, the technique is limited to qualitative analysis.     

Investigation of the novel mixed-mode stationary phase for capillary electrochromatography. I. Preparation and characterization of sulfonated naphthalimido-modified silyl silica gel

A novel packing material, 3-(4-sulfo-1,8-naphthalimido)propyl-modified silyl silica gel (SNAIP), was prepared for the use as a stationary phase of capillary electrochromatography (CEC). The sulfonic acid groups on SNAIP stationary phase contributed to the generation of electroosmotic flow (EOF) at low pH and served as a strong cation-exchanger. In CEC with SNAIP, a mixed-mode separation was predicted, comprising hydrophobic and electrostatic interactions as well as electrophoretic migration process. In order to understand the retention mechanism on SNAIP, effects of buffer pH, concentration, and mobile phase composition on EOF mobility and the retention factors of barbiturates and benzodiazepines were systematically investigated. Moreover, the retention behavior of barbiturates on SNAIP was investigated and compared with those on octadecyl silica (ODS), phenyl-bonded silica, and 3-(1,8-naphthalimido)propyl-modified silyl silica gel to confirm the presence of pi-pi interaction on its retention mechanism. It was observed that a column efficiency was more than 85,000 N/m for retained compounds and the relative standard deviations for the retention times of EOF marker, thiourea, and five barbiturates were below 2.5% (n = 4). Under an applied voltage of 20 kV and a mobile phase consisted of 5 mM phosphate (pH 3.8) and 40% methanol, the baseline separation of five barbiturates was achieved within 3 min.     

High performance liquid chromatography separation of structurally related enkephalins on quaternary ammonium-embedded stationary phase in isocratic mode

Separation of twelve enkephalins was investigated on a quaternary ammonium-embedded stationary phase (Stability BS-C23). Variation of buffer pH of the mobile phase highlighted the complex relationship between repulsive/attractive electrostatic interactions and the reversed-phase partitioning mechanism. The effect of three different anions employed as additives (phosphate, chloride and perchlorate) was examined at various concentrations and two pH values (acidic and neutral). At pH 2.5, an increase in the anion eluent concentration resulted in a higher retention factors of positively charged enkephalins. This effect was more pronounced when perchlorate ions were added to the mobile phase rather than phosphate and chloride ions, due to chaotropic and ion-pairing effects. In contrast, at pH 7.5, retention factors of negatively charged enkephalins decreased when these salts were added, due to an anion-exchange mechanism. Perchlorate caused a sharper decrease than chloride and phosphate anions did. The results presented here provide insight into the possible adjustment of retention and separation of peptides on a mixed-mode stationary phase (BS-C23) by a careful control of the buffer pH, the nature and concentration of anions, added to the buffer, and organic modifier content.     

Polyethyleneimine Immobilized on Silica Endcapped with Octadecyl Groups as a Stationary Phase for RP-LC

A new reversed stationary phase was prepared, based on thermal immobilization of trimethoxysilylpropyl modified polyethyleneimine onto silica particles endcapped with octadecyl molecules. The physicochemical and morphological properties of the stationary phase were characterized by solid state cross-polarization and magic angle spinning ²⁹Si nuclear magnetic resonance, infrared spectroscopy, porosimetry, and elemental analysis. For the studies on reversed phase high-performance liquid chromatography (HPLC) retention, separation of the established Tanaka and Engelhardt test mixtures was performed. The stationary phase showed a typical partition mechanism for the reversed phase; however, the low hydrophobicity required a low organic content solvent in the mobile phase for chromatographic separation of more hydrophobic compounds. The stationary phase also showed low residual silanol activity for the elution of basic compounds due to the protection offered by octadecyl endcapped molecules and the competition provided by the imine groups of the polymeric layer. The proposed stationary phase possesses interesting selectivity and is convenient for applications requiring the separation of more retentive compounds in conventional HPLC columns using more aqueous mobile phases.     

Polyacrylamide-based monolithic capillary column with coating of cellulose tris(3,5-dimethylphenyl-carbamate) for enantiomer separation in capillary electrochromatography

A hydrophilic chiral capillary monolithic column for enantiomer separation in CEC was prepared by coating cellulose tris(3,5-dimethylphenyl-carbamate) (CDMPC) on porous hydrophilic poly(acrylamide-co-N,N'-methylene-bisacrylamide) (poly(AA-co-MBA)) monolithic matrix with confine of a fused-silica capillary. The coating conditions were optimized to obtain a stable and reproducible chiral stationary phase for CEC. The effect of organic modifier of ACN in aqueous mobile phase for the enantiomer separation by CEC was investigated, and the significant influence of ACN on the enantioresolution and electrochromatographic retention was observed. Twelve pairs of enantiomers including acidic, neutral, and basic analytes were tested and nine pairs of them were baseline-enantioresolved with acidic and basic aqueous mobile phases. A good within-column repeatability in retention time (RSD = 2.4%) and resolution (RSD = 3.2%) was obtained by consecutive injections of a neutral compound, benzoin, on a prepared chiral monolithic column, while the between-column repeatability in retention time (RSD = 6.4%) and resolution (RSD = 9.6%) was observed by column-to-column examination. The prepared monolithic stationary phase showed good stability in either acidic or basic mobile phase.