Separation of selected peptides by capillary electroendoosmotic chromatography using 3 microns reversed-phase bonded silica and mixed-mode phases.

The retention behaviour and selectivity of selected basic, neutral and acidic peptides have been studied by capillary electroendoosmotic chromatography (CEC) with Hypersil C8, C18, Hypersil mixed-mode, and Spherisorb C18/SCX columns, 250 (335) mm x 100 microns, packed with 3 microns particles, and eluted with mobile phases composed of acetonitrile-triethylamine-phosphoric acid (TEAP) at pH 3.0 using a Hewlett-Packard Model HP3DCE capillary electrophoresis system. The selected peptides were desmopressin (D), two analogues (A and B) of desmopressin, oxytocin (O) and carbetocin (C). The peptides eluted either before or after the electroendoosmotic flow (EOF) marker, depending on the concentration of acetonitrile used and the buffer ionic strength. The retention and selectivity of these peptides under CEC conditions were compared to their behaviour in free zone capillary electrophoresis (CZE), where the separation mode was based on the electrophoretic migration of the analytes due to their charge and Stokes radius properties. In addition, their retention behaviour in RP-HPLC was also examined. As a result, it can be concluded that the elution process of this group of synthetic peptides in CEC with a TEAP buffer at pH 3.0 is mediated by a combination of both electrophoretic migration processes and retention mechanisms involving hydrophobic as well as silanophilic interactions. This CEC method when operated with these 3 microns reversed-phase and mixed-mode sorbents with peptides is thus a hybrid of two well-known analytical methods, namely CZE and RP-HPLC. However, the retention behaviour and selectivity of the selected peptides differs significantly in the CEC mode compared to the RP-HPLC or CZE modes. Therefore this CEC method with these peptides represents an orthogonal analytical separation procedure that is complimentary to both of these alternative techniques.     

Application of hydrophobic anion-exchange phases in capillary electrochromatography

Capillary electrochromatography (CEC) requires stationary phases that enable appropriate electroosmotic propel under various conditions. Analyte retention can be controlled through hydrophobic or electrostatic interaction with the packing material. The development and characterization of new strong anion-exchange materials with additional hydrophobic moieties (SAX/C18 mixed-mode phases) is described. The synthesis was based on polymer encapsulation of porous silica. The phases were systematically characterized by means of elemental analyses, HPLC frontal analyses and CEC experiments. The studies focused on the influence of various parameters (e.g., pH, kind of buffer, capillary wall) on the electroosmotic flow (EOF). Phases with high anion-exchange capacity generated a fast and constant EOF over a wide pH range. Long-time stability of EOF and hydrophobic retention under CEC conditions were demonstrated within the course of 100 consecutive injections. The applicability of the SAX/C18 phases in appropriate buffer systems is demonstrated for neutral, acidic and basic compounds.     

Chromatographic behaviour of peptides on a mixed-mode stationary phase with an embedded charged group by capillary electrochromatography and high-performance liquid chromatography

Retention behaviour of biological peptides was investigated on a stationary phase bearing an embedded quaternary ammonium group in a C21 alkyl chain by both high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). In HPLC experiments, variation of acetonitrile (ACN) content in the mobile phase showed that peptides are mainly separated by RP mechanism. The weak or negative retention factors observed as compared to C18 silica stationary phase suggested the involvement of an electrostatic repulsion phenomenon in acidic conditions. Comparison of HPLC and CEC studies indicated that (i) ion-exclusion phenomenon is more pronounced in HPLC and (ii) higher ACN percentage in mobile phase induce for some peptides an increase of retention in CEC, pointing out the existence of mechanisms of retention other than partitioning mainly involved in chromatographic process. This comparative study demonstrated the critical role of electric field on peptide retention in CEC and supports the solvatation model of hydrolytic pillow proposed by Szumski and Buszewski for CEC using mixed mode stationary phase in CEC.     

Rapid separation of peptides and proteins by isocratic capillary electrochromatography at elevated temperature

The use of capillary electrochromatography (CEC) for the separation by isocratic elution of synthetic peptides, proteins as well as the tryptic digest of cytochrome c has been demonstrated. The monolithic porous stationary phase was prepared from silanized fused-silica capillaries of 75 microm I.D. by in situ copolymerization of vinylbenzyl chloride and ethylene glycol dimethacrylate in the presence of propanol and formamide as the porogens. The chloromethyl groups at the surface of the porous monolith were reacted with N,N-dimethylbutylamine to form a positively charged chromatographic surface with fixed n-butyl chains. Results of studies on the influence of temperature and mobile phase composition on the retention and selectivity of separation by CEC demonstrated the feasibility of rapid polypeptide analysis and tryptic mapping at elevated temperature with high resolution and efficiency. Typically the chromatography of a tryptic digest of cytochrome c took about 5 min at 55 degrees C and 75 kV/m with hydro-organic mobile phases containing acetonitrile in 50 mM phosphate buffer, pH 2.5. For peptides and proteins plots of logarithmic k'cec against acetonitrile concentration were nonlinear, whereas Arrhenius plots for the mobilities were nearly linear. Comparison of the separation of such samples under conditions of CEC and capillary zone electrophoresis (CZE) indicates that the mechanism of separation in CEC is unique and leads to a chromatographic profile different from that obtained by CZE.     

Capillary electrochromatography of proteins and peptides with a cationic acrylic monolith

For the separation of proteins and peptides by capillary electrochromatography (CEC), columns with a monolithic stationary phase were prepared from silanized fused-silica capillaries of 50 microm I.D. by in situ copolymerization of glycidyl methacrylate, methyl methacrylate and ethylene glycol dimethacrylate in the presence of propanol and formamide as porogens. The epoxide groups at the surface of the porous monolith were reacted with N-ethylbutylamine to form fixed tertiary amino functions with ethyl- and butyl-chains. A mixture of ribonuclease A, insulin, alpha-lactalbumin and myoglobin was separated isocratically by counterdirectional CEC with hydro-organic mobile phases containing acetonitrile and sodium phosphate buffer, pH 2.5. The separation of four angiotensin type peptides by CEC was also achieved under similar conditions. The elution order of proteins was similar to that obtained in reversed-phase chromatography. Plots of the migration factors for proteins and peptides against the acetonitrile concentration exhibit opposite trends. This is most likely due to the greater chromatographic retention and lower electrophoretic migration velocity of proteins than that of peptides in the counterdirectional CEC system. From this it is concluded that the separation is governed by a dual mechanism that involves the complex interplay between selective chromatographic retention and differential electrophoretic migration.     

Properties and Applications of Mixed Packing Capillary Electrochromatography

Mixed packing capillary electrochromatography (MP CEC) with the stationary phase comprising a physical mixture of strong cation exchange (SCX) phase and octadecysilyl (ODS) phase was developed. With the existence of a sulfonic acid group on the surface of SCX, not only could the electroosmotic flow (EOF) remain high at low pH, but also the hydrophilicity of the stationary phase was increased greatly, leading to broad adaptable ranges of both pH and organic modifier concentration in the mobile phase. At the same time, with the coexistence of C₁₈ on the surface of ODS, both the retention and the resolution of samples were improved. Accordingly, MP CEC combined the advantages of both SCX and ODS columns. Effects of operation parameters on EOF and the capacity factors of solutes as well as the retention mechanism of such a column were studied systematically. In addition, MP CEC columns were used in the analysis of strong polar solutes as well as for the high speed separation of acidic, basic, and neutral compounds in a single run.     

Capillary electrochromatography of peptides on a neutral porous monolith with annular electroosmotic flow generation

A new kind of monolithic capillary column was prepared for capillary electrochromatography (CEC) with a positively charged polymer layer on the inner wall of a fused-silica capillary and a neutral monolithic packing as the bulk stationary phase. The fused-silica capillary was first silanized with 3-glycidoxypropyltrimethoxysilane (GPTMS). Polyethyleneimine (PEI) was then covalently bonded to the GPTMS coating to form an annular positively charged polymer layer for the generation of electroosmotic flow (EOF). A neutral bulk monolithic stationary phase was then prepared by in situ copolymerization of vinylbenzyl chloride (VBC) and ethylene glycol dimethacrylate in the presence of 1-propanol and formamide as porogens. Benzyl chloride functionalities on the monolith were subsequently hydrolyzed to benzyl alcohol groups. Effects of pH on the EOF mobility of the column were measured to monitor the completion of reactions. Using a column with this design, we expected general problems in CEC such as irreversible adsorption and electrostatic interaction between stationary phase and analytes to be reduced. A peptide mixture was successfully separated in counter-directional mode CEC. Comparison of peptide separations in isocratic monolithic CEC, gradient HPLC and capillary zone electrophoresis (CZE) indicated that the separation in CEC is governed by a dual mechanism that involves a complex interplay between selective chromatographic retention and differential electrophoretic migration.     

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.     

Separation of peptides on mixed mode of reversed-phase and ion-exchange capillary electrochromatography with a monolithic column

The mixed mode of reversed phase (RP) and strong cation-exchange (SCX) capillary electrochromatography (CEC) based on a monolithic capillary column has been developed. The capillary monolithic column was prepared by in situ copolymerization of 2-(sulfooxy)ethyl methacrylate (SEMA) and ethylene dimethacrylate (EDMA) in the presence of porogens. The sulfate group provided by the monomer SEMA on the monolithic bed is used for the generation of the electroosmotic flow (EOF) from the anode to the cathode, but at the same time serves as a SCX stationary phase. A mixed-mode (RP/SCX) mechanism for separation of peptides was observed in the monolithic column, comprising hydrophobic and electrostatic interaction as well as electrophoretic migration at a low pH value of mobile phase. A column efficiency of more than 280,000 plates/m for the unretained compound has been obtained on the prepared monoliths. The relative standard deviations observed for t(0) and retention factors of peptides were about 0.32% and less than 0.71% for ten consecutive runs, respectively. Effects of mobile phase compositions on the EOF of the monolithic column and on the separation of peptides were investigated. The selectivity on separation of peptides in the monolithic capillary column could be easily manipulated by varying the mobile phase composition.     

Effects of G-quartet DNA stationary phase destabilization on fibrinogen peptide resolution in capillary electrochromatography

The migration of fibrinogen peptides in capillaries coated with G-quartet-forming DNA oligonucleotides in open-tubular CEC (OTCEC) was studied, in order to investigate factors affecting the retention of peptides on G-quartet DNA stationary phases. At 25 degrees C, the peptides eluted in the same order in OTCEC using a two-plane G-quartet DNA stationary phase as in CZE, including two peptides that were completely overlapped. It was found that baseline resolution of the coeluting peptides could be achieved in the OTCEC experiment, but not in CZE, at run temperatures of 35-40 degrees C. A stationary phase formed by a scrambled-sequence oligonucleotide that does not form a G-quartet did not provide any resolution of the two coeluting peptides, even at the higher temperatures, indicating that some destabilization of the G-quartet enhances resolution but that some degree of G-quartet structure is necessary. The effects of destabilization were further explored through variation of the cations (sodium or potassium) used in attachment of the G-quartet oligonucleotide to the capillary surface and in the mobile-phase buffer. Resolution was lower when a more stable, four-plane G-quartet stationary phase was used, supporting the conclusion that some flexibility in the G-quartet structure facilitates differential interactions that resolve otherwise coeluting peptides. The increase in peptide resolution upon destabilization of the G-quartet structure could prove to be an important factor in the application of G-quartet DNA stationary phases for nonaffinity-based separation of native proteins and peptides.     

Separation and identification of etodolac and its urinary phase I metabolites using capillary electrochromatography and on-line capillary electrochromatography-electrospray ionisation mass spectrometry coupling

Capillary high-performance liquid chromatography (capillary HPLC), pressure-assisted capillary electrochromatography (pCEC) and capillary electrochromatography (CEC) were performed in the same capillary packed with 5 microm octadecylsilica (C18) as stationary phase. These three separation modes were compared from the viewpoint of peak efficiency and separation selectivity in order to critically evaluate the advantages which CEC may offer compared to capillary HPLC for the solution of practical biomedical problems. The separation of the non-steroidal anti-inflammatory drug etodolac (ET, 1) and its phase I metabolites, 6-hydroxy etodolac (6-OH-ET, 2), 7-hydroxy etodolac (7-OH-ET, 3) and 8-(1'-hydroxyethyl) etodolac (8-OH-ET, 4) was selected as an example. Baseline separation of all compounds was achieved in different modes and conditions. The effect of pure electrophoretic separation mechanism on the overall separation selectivity observed in CEC has been shown. A high electroosmotic flow (EOF) was observed in C18 packed capillary even at pH 2.5 in various buffers. Furthermore, these separations were coupled on-line with electrospray ionisation mass spectrometry (ESI-MS) and the parent drug and its metabolites were identified in urine. For the coupling of CEC with ESI-MS a laboratory-made electrophoretic device was used in order to overcome some technical disadvantages of commercial instrumentation.     

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.     

Peptide separation in hydrophilic interaction capillary electrochromatography

Separation of small peptides by hydrophilic interaction capillary electrochromatography (HI-CEC) has been investigated. The negative surface charge of a hydrophilic, strong-cation-exchange stationary phase (PolySULFOETHYL A) provided a substantial cathodic electroosmotic flow (EOF). The influence of acetonitrile content, ionic strength, mobile phase pH as well as applied voltage on the migration of the peptides was studied. Possible retention mechanisms of the peptides in HI-CEC were discussed. It was found that hydrophilic interaction between the solutes and the stationary phase played a major role in this system, especially when mobile phases with high acetonitrile content were used. However, an ion-exchange mechanism and electrophoretic mobility also affect the migration of the peptides in HI-CEC. Elution order and selectivity was proved to be different in HI-CEC and capillary zone electrophoresis (CZE), thus revealing the potential of HI-CEC as a complementary technique to CZE for the separation of peptides. Efficiency and selectivity of HI-CEC for the separation of peptides were demonstrated by baseline separating nine peptides in 6 min.     

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.     

Use of mixed-mode sorbents for the electrochromatographic separation of thrombin receptor antagonistic peptides

In this study, the thrombin receptor antagonistic peptide TRAP-1 and its alanine-scan analogues, TRAP 2-6, have been employed as probes to characterise the performance of C18/SCX mixed-mode capillary electrochromatographic (CEC) columns. It was found that the resolution of this group of peptides could only be achieved in a narrow pH range with phosphate-based running electrolytes. The influence of the running electrolyte composition, e.g. the buffer choice, the ionic strength, the pH and the organic solvent content, on the electroosmotic flow (EOF) of these mixed-mode CEC columns was investigated. In addition, the retention mechanism for this group of peptide probes in the electrochromatographic process was studied by examining the effect of varying the running electrolyte composition. As a result, it can be concluded that the electrochromatographic separation of this set of peptides was mediated by a combination of electrophoretic migration and chromatographic retention involving both hydrophobic as well as ion exchange interactions. By modulating the running electrolyte composition, the hydrophobic or ion exchange components of the interaction process could be made to dominate the chromatographic retention of the peptides. Based on this strategy, a high-resolution separation of six closely related synthetic peptides was demonstrated with this mixed-mode CEC system.     

Fast separation of pyrimidine derivatives by capillary electrochromatography on ion-exchange/reversed-phase mixed-mode stationary phases

This work describes the use of mixed-mode stationary phases which exhibit both strong ion-exchange (either cation-exchange, SCX, or anion-exchange, SAX) and reversed-phase chromatographic characteristics in capillary electrochromatographic separations of pyrimidine derivatives. Different packing materials, namely C6, SCX/C6 and SAX/C6, were compared and the influence of the composition of the carrier electrolyte (concentration of acetonitrile and pH) on the retention behavior of the selected solutes was investigated. A separation of all eight pyrimidine derivatives could be obtained on a 6.5 cm column packed with the SAX/C6 stationary phase in less than 3 min, with good peak shapes and efficiencies in the range 39,000 to 81,000 plates per meter.     

Use of electrokinetic measurements for characterization of columns used in capillary electrochromatography

The separation mechanism in capillary electrochromatography (CEC) is a hybrid differential migration process, which entails the features of both high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE), i.e., chromatographic retention and electrophoretic migration. The focus of this paper is on the use of electrokinetic data, such as current, electroosmotic flow (EOF) and column efficiency measurements, that are readily available, for an improved understanding of CEC separations. A framework is presented here for the use of this data for evaluation of a variety of performance parameters including, conductivity ratio, interstitial EOF mobility, porosity, and zeta potential. This framework is applied for characterization of two monolithic columns with different chemistry that were manufactured in-house. The above-mentioned performance parameters were calculated for the two columns and it is found that the poly(VBC-EGDMA-SWNT) monolithic column with the GPTMS-PEI coating offers a significantly improved flow distribution in comparison to the poly(VBC-EGDMA) monolithic column. This observation is confirmed by performing separation of peptides on the two columns and height equivalent of a theoretical plate (HETP) measurements on the resulting peaks. It is shown that following our approach leads to an improved understanding of the separations achieved with the columns and to better column design.     

Capillary electrochromatographic evaluation of vitamin E-active oil constituents: tocopherols and tocotrienols

Separations of lipid antioxidants, tocopherols (T) and tocotrienols (T3), on octylsilica (OS), octadecylsilica (ODS), phenylsilica, or silica were studied by capillary electrochromatography (CEC)-UV detection. The homologues and isomers of the vitamin E-active compounds were best separated with an OS column. CEC with an ODS column tended to yield broad peaks with poor resolution. Among the various mobile phases evaluated, [acetonitrile-methanol (64:36)]-[25 mM tris(hydroxymethyl)aminomethane, pH 8] (95:5) eluent systems produced the most satisfactory results. Under these conditions, a baseline separation of an 11-component mixture was obtained with elution order similar to that observed in reversed-phase HPLC: deltaT3 > (gamma+beta)T3 > alphaT3 > epsilonT > (delta+zeta2)T > (gamma+beta)T > alphaT > alphaT-acetate. CEC of the antioxidant acetates led to separations inferior to those of the parent compounds. Effects of CEC experimental variables (e.g., mobile phase solvents and buffers, stationary phases and electric field) on analyte separations were assessed in the context of resolution factors and retention factors.     

Succinyl methacrylate polymerized in porous-layered phases for open-tubular capillary electrochromatography: Comparison with silica hydride monolayered phases

A polymer phase, which was constructed with butyl methacrylate (BMA), an ionizable monomer (mono-(2-(methacryloyloxy)ethyl) succinate (MES)), and a crosslinking agent (ethylene dimethacrylate), was first formed in a porous-layered open-tubular (PLOT) capillary. The PLOT capillary was characterized with SEM and electrophoretic flow as the pH level, ionic strength and addition of organic modifiers in the running buffers changed. In addition, a bare capillary and a silica hydride based capillary (SiH-MES), which bore a monolayered MES phase on it, were used to compared with the BMA-MES capillary. Besides optimizing the capillary electrochromatographic (CEC) conditions for each group of analytes, which were a mixture of nucleosides and thymine, flavonoids, and phenolic acids,comparison of the separation selectivity among analytes between the BMA-MES and SiH-MES capillaries was done according to the velocity and retention factors obtained from the CEC data. Overall, the polymeric phase formed in the PLOT mode was capable of preventing blockage of the columns and was superior to the monolayered phase bonding with the same ionizable ligands for application in CEC as well as to the bare silica phase in CE.     

Capillary Electrochromatography as a Method Development Tool for the Liquid Chromatographic Separation of DUP 654 and Related Substances

Capillary Electrochromatography (CEC) offers a rapid, economical, and efficient means for resolving nonionic compounds in the reversed phase mode on octadecylsilane (ODS) columns. A CEC optimization on a Hypersil ODS capillary column was employed to identify a suitable mobile phase for the pressure-driven (reversed phase ODS) separation of the anti-inflammatory 2-phenylmethyl-1-naphthol (DUP 654), and its related substances. The proportions of mobile phase modifiers methanol, acetonitrile, and water as well as pH were employed as variables in a stacked mixture design. Comparable response surface profiles were obtained for the CEC separations at pH 4 and pH 8. However, subtle differences were evident in the quality of separations obtained in the liquid chromatographic (LC) mode when using a specially-prepared column packed with exactly the same stationary phase as used in the CEC experiments. A mapping of the response surface for separations on a commercially available Hypersil ODS LC column revealed obvious differences. The differences indicate that the transfer of ODS based separation methods between CEC and LC involves more than simply transferring the conditions from one mode to the other.