Stationary Phases For Capillary Electrochromatography Research Articles

Evaluation of a methacrylate-bonded cyclodextrins as a monolithic chiral stationary phase for capillary electrochromatography (CEC)-UV and CEC coupled to mass spectrometry.

Glycidyl methacrylate-bonded β-cyclodextrin (GMA-β-CD) is synthesized as a new chiral monomer by direct chemical bonding with GMA using a fast and simple alternative procedure. Next, rigid and homogenous monolithic columns were prepared by polymerization of GMA-β-CD monomer with ethylene dimethacrylate (EDMA), in the presence of commonly used porogens and a charged achiral monomer to form a versatile chiral monolith. This is the first report in which a preparation procedure for a methacrylate-bonded CD is introduced for chiral separations in CEC. The degree of substitution of GMA-β-CD monomer and mobile-phase parameters were optimized to achieve the highest enantioselectivity and plate number. To evaluate the GMA-β-CD monolithic column, different classes of chiral compounds were screened. Under the optimized β-CD monolith phase and the optimum mobile-phase conditions, 30 neutral and basic chiral compounds and two acidic compounds could be separated. The high chemical and mechanical stability, homogenous microflow and no loss of material at the interface allows for the first time the feasibility of applying this polymer-based monolithic column for CEC coupled to ESI-MS. Compared with CEC-UV, CEC-ESI-MS showed higher sensitivity and lower resolution. However, resolution greater than 1.0 can still be obtained for majority of the select tested compound in CEC-ESI-MS with at least three out of seven compound providing Rs≥1.5. The results reinforce the potential of GMA-β-CD monolithic columns for chiral separations with high sensitivity in CEC-ESI-MS. Finally, using hexobarbital as the model chiral analyte, the monolithic column demonstrated excellent stability and reproducibility of retention time and enantioselectivity.

Synthesis of ionic liquid‐bonded organic‐silica hybrid monolithic column for capillary electrochromatography

An ionic liquid (IL) was introduced into the organic-silica hybrid monolithic column as the stationary phase for capillary electrochromatography (CEC). The monolithic silica matrix containing chloropropyl functional group was prepared by the in situ co-condensation of tetramethoxysilane and (3-chloropropyl)-trimethoxysilane via a sol-gel process and chemical modification with N-methylimidazole. The electroosmotic flow of the IL-modified hybrid monolithic column was reversed at acidic pH and the morphology of the column was characterized by scanning electron microscope. Four aromatic hydrocarbons were completely separated with 40% acetonitrile phosphate buffer as the mobile phase and seven inorganic ions were efficiently separated with the phosphate buffer on the column in CEC. Reproducibilities of migration time for four aromatic hydrocarbons (benzene, naphthalene, anthracene, chrysene) were acceptable on IL-modified hybrid monolithic columns. Relative standard deviations of run-to-run (n=5), peak area-to-peak area (n=5), day-to-day (n=3) and column-to-column (n=3) were in the range of 0.72-0.88, 1.47-5.40, 2.44-4.99 and 3.01-8.11%, respectively.

Preparation and characterization of silica monolith modified with bovine serum albumin‐gold nanoparticles conjugates and its use as chiral stationary phases for capillary electrochromatography

This paper describes the development of silica monolith modified with bovine serum albumin-gold nanoparticles (BSA-GNPs) conjugates as chiral stationary phases for capillary electrochromatography (CEC). The bare monolithic silica column was prepared by a sol-gel process and has been modified chemically with 3-mercaptopropyltrimethoxysilane to provide thiol groups, followed by immobilization of gold nanoparticles via the formation of an Au-S bond and modification with BSA as the chiral selector via the nitrogen lone pair of electrons. It has been demonstrated that the monolithic chiral stationary phases can be used for the enantioseparation of a number of phenylthiocarbamyl amino acids (PTC-D/L-AAs) by CEC. Ten pairs of tested amino acids enantiomers were successfully resolved within 18 min under optimized conditions, and the resolution values were in the range of 1.486-2.083. With PTC-D/L-tryptophan used as the probe solute, the influences of applied voltage, organic modifier and buffer pH in mobile phase on apparent retention factor, enantioselectivity and resolution factor were also investigated.

MIXED-MODE MONOLITHIC SILICA AS A CHROMATOGRAPHIC SEPARATION MEDIUM

Capillary electro chromatography (CEC) is a separation technique that combines the dual properties of capillary zone electrophoresis (CZE) and micro-liquid chromatography (μ-LC). A hybrid-type silica monolith capillary column was prepared from a mixture of tetramethoxysilanes (TMOS) and methyltrimethoxysilanes (MTMS). Surface modification with octadecyldimethyl-(N-N-diethylamino)silane (ODS-DEA) and N-3[-(dimethylamino)propyl]acrylamide methyl] chloride-quaternary salt, (DMAPAA-Q) was carried out to form a mixed-mode of C18 and a strong anion exchange stationary phase. The synthesized mixed-mode silica monolithic column was then characterized physically with a scanning electron microscope (SEM) and shown to have an average through-pore size and skeleton size of 2.2 µm and 2.0 µm, respectively. This new column with mixed-mode characteristic of reversed-phase (RP) and strong anion exchange (SAX) functionalities was then applied as the stationary phase in CEC for the separation of inorganic and organic ions using a 50 mM phosphate electrolyte of pH 6.9. The inorganic and organic anions were base-line separated within 10 min with column efficiency up to 114 900 plates/min.

Sulfoalkylbetaine‐based monolithic column with mixed‐mode of hydrophilic interaction and strong anion‐exchange stationary phase for capillary electrochromatography

A novel monolithic stationary phase with mixed mode of hydrophilic and strong anion exchange (SAX) interactions based on in situ copolymerization of pentaerythritol triacrylate (PETA), N,N-dimethyl-N-methacryloxyethyl N-(3-sulfopropyl) ammonium betaine (DMMSA) and a selected quaternary amine acrylic monomer was designed as a multifunctional separation column for CEC. Although the zwitterionic functionalities of DMMSA and hydroxy groups of PETA on the surface of the monolithic stationary phase functioned as the hydrophilic interaction (HI) sites, the quaternary amine acrylic monomer was introduced to control the magnitude of the EOF and provide the SAX sites at the same time. Three different quaternary amine acrylic monomers were tested to achieve maximum EOF velocity and highest plate count. The fabrication of the zwitterionic monolith (designated as HI and SAX stationary phase) was carried out when [2-(acryloyloxy)ethyl]trimethylammonium methylsulfate was used as the quaternary amine acrylic monomer. The separation mechanism of the monolithic column was discussed in detail. For charged analytes, a mixed mode of HI and SAX was observed by studying the influence of mobile phase pH and salt concentration on their retentions on the poly(PETA-co-DMMSA-co-[2-(acryloyloxy)ethyl]trimethylammonium methylsulfate) monolithic column. The optimized monolith showed good separation performance for a range of polar analytes including nucleotides, nucleic acid bases and nucleosides, phenols, estrogens and small peptides. The column efficiencies greater than 192 000 theoretical plates/m for estriol and 135 000 theoretical plates/m for charged cytidine were obtained.

Poly(divinylbenzene-alkyl methacrylate) monolithic stationary phases in capillary electrochromatography

In this study, a series of poly(divinylbenzene-alkyl methacrylate) monolithic stationary phases, which were prepared by single step in situ polymerization of divinylbenzene and various alkyl methacrylates (butyl-, octyl-, or lauryl-methacrylate), were developed as separation columns of benzophenone compounds for capillary electrochromatography (CEC). In addition to the presence of plenty of benzene moieties, the stationary phases contained long and flexible alkyl groups on the surface. With an increase in the molecular length of alkyl methacrylate, the polymeric monolith, which had higher hydrophobicity, effectively reduced the peak tailing of benzophenones, but a weaker retention was observed. The unusual phenomenon was likely due to the π–π interaction between the aromatic compound and the polymeric material. The usage of longer alkyl methacrylate as reaction monomer limited the retention of aromatic compounds on the stationary phase surface, thus the π–π interaction between them was possibly reduced. Consequently, the retention time of aromatic compounds was markedly decreased with an increase in carbon length of alkyl methacrylate that was carried on the polymeric monolith. Compared to previous reports on polystyrene-based columns in which the peak-tailing problem was reduced by decreasing the benzene moieties on the stationary phase, this study demonstrated that the undesirable retention (peak-tailing) could also be improved by the inclusion of long alkyl methacrylate to the polystyrene-based columns.

Enantioseparation of α-amino acids on an 18-crown-6-tetracarboxylic acid-bonded silica by capillary electrochromatography

(-)-(18-Crown-6)-2,3,11,12-tetracarboxylic acid-bonded silica was used as the chiral stationary phase in capillary electrochromatography (CEC) for enantioseparation of some alpha-amino acids. Separation data in CEC were measured in mobile phases of varying pH, and composition of methanol and buffer, and compared with those in capillary liquid chromatography (CLC). In CEC better enantioseparation was generally obtained in the eluent of lower pH, higher buffer concentration and intermediate MeOH content, usually at the expense of analysis time. CEC showed generally better enantioselectivity and resolutions than CLC for the amino acids investigated.

CEC column behaviour of butyl and lauryl methacrylate monoliths prepared in non‐aqueous media

Polymeric monolithic stationary phases for capillary electrochromatography were prepared using two bulk monomers, butyl methacrylate (BMA) and lauryl methacrylate (LMA), by in situ polymerization in non-aqueous media. The effect of 1,4-butanediol/1-propanol ratio on porous properties was investigated separately for each monomer, keeping the proportion of monomers to pore-forming solvents fixed at 40:60 wt:wt. Also, mixtures of BMA and LMA at different 1,4-butanediol/1-propanol ratios were studied for tailoring the morphological features of the monolithic columns. The chromatographic performance of the different columns was evaluated by means of van Deemter plots of polycyclic aromatic hydrocarbons. Mercury-intrusion porosimetry, SEM, and nitrogen-adsorption measurements were also performed in order to understand their retention behaviour and porous properties. A comparison of these features was also performed for monoliths made with one bulk monomer (BMA or LMA) and with mixtures of both. These mixed monoliths showed satisfactory efficiencies and analysis times compared with those made with one bulk monomer; thus, the BMA-LMA monoliths constitute an attractive alternative to manipulate the electrochromatographic properties of methacrylate beds in CEC.

Sulfated and sulfonated polysaccharide as chiral stationary phases for capillary electrochromatography and capillary electrochromatography–mass spectrometry

The applications of polysaccharide phenyl carbamate derivatives as chiral stationary phases (CSPs) for capillary electrochromatography (CEC) are often hindered by longer retention times, especially using a normal-phase (NP) eluent due to very low electroosmotic flow (EOF). Therefore, in this study, we propose an approach for the aforementioned problems by introducing two new types of negatively charged sulfate and sulfonated groups for polysaccharide CSPs. These CSPs were utilized to pack CEC columns for enantioseparation with a NP eluent. Compared to conventional cellulose tris(3,5-dimethylphenyl carbamate) or CDMPC CSPs, the sulfated CDMPC CSP (sulfur content 4.25%, w/w) shortened the analysis time up to 50% but with a significant loss of enantiomeric resolution (∼60%). On the other hand, the sulfonated CDMPC CSP (sulfur content 1.76%, w/w) not only provided fast throughput but also maintained excellent resolving power. In addition, its synthesis is much more straightforward than the sulfated one. Furthermore, we studied several stationary phase parameters (CSP loading and silica gel pore size) and mobile phase parameters (including type of mobile phase and its composition) to evaluate the throughput and enantioselectivity. Using the optimized conditions, a chiral pool containing 66 analytes was screened to evaluate the enantioselectivity under three different mobile phase modes (i.e., NP, polar organic phase (POP) and reversed-phase (RP) eluents). Among these mobile phase modes, the RP mode showed the highest success rate, whereas some degree of complementary enantioselectivity was observed with NP and POP. Finally, the feasibility of applying this CSP for CEC–MS enantioseparation using internal tapered column was evaluated with NP, POP and RP eluents. In particular, the NP-CEC–MS provided significantly enhanced sensitivity when methanol was replaced with isopropanol in the sheath liquid. Using aminoglutethimide as model chiral analyte, all three modes of CEC–MS demonstrated excellent durability as well as excellent reproducibility of retention time and enantioselectivity.

Carboxylic multi‐walled carbon nanotubes as immobilized stationary phase in capillary electrochromatography

Carboxylic multi-walled carbon nanotubes (c-MWNT) have been immobilized into a fused-silica capillary for capillary electrochromatography. The c-MWNT were successfully incorporated after the silanization and coupling with glutaraldehyde on the inner surface of the capillary. The electrochromatographic features of the c-MWNT immobilized stationary phase have been evaluated for the analysis of different compounds of pharmaceutical interest. The results indicated high electrochromatographic resolution, good capillary efficiency and retention factors. In addition, highly reproducible results between runs, days and capillaries were obtained.

Synthesis of a polyrotaxane‐based macroporous polymer as stationary phase for capillary electrochromatography via host–guest complexation of N,N ′‐ethylenedianilinediacrylamide with statistically methylated β‐cyclodextrin

A synthetic route to acrylamide-based monolithic stationary phases for CEC with rotaxane-type immobilized derivatized beta-CD was explored. N,N'-Ethylenedianilinediacrylamide was synthesized as the water-insoluble crosslinker forming water-soluble inclusion complexes with statistically methylated beta-CD. Mixed-mode stationary phases were synthesized by free radical copolymerization of the bisacrylamide-CD host-guest complex with water-soluble monomers and an additional water-soluble crosslinker in aqueous solution. Complex formation in solution and inclusion of the pseudorotaxane into the polymeric network (formation of a polyrotaxane architecture) were studied by means of (1)H-NMR chemical shift analysis, CD modified micellar EKC (CD-MEKC), 2D-NOESY spectroscopy, and solid state( 13)C-NMR spectroscopy. The presence of a mixed-mode selectivity of the stationary phase based on hydrophobic and hydrophilic interaction was confirmed by CEC with neutral polar and nonpolar solutes.

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.

Surfactant-Assisted Ethane-Bridged Silica Coating for Capillary Electrochromatography

With surfactant P123 as structure directing reagent, 1, 2-bis (trimethoxysilyl) ethane was hydrolyzed under acid condition. The resulting ethane-bridged silica was coated onto the inner walls of fused silica capillaries and used as the stationary phase for capillary electrochromatography. The bridged ethyl silica provided hydrophobic groups for reversed-phase separation. A comparative coated capillary was fabricated without the use of surfactant in the preparation of the bonded silica. Separation of model neutral compounds was compared between these two kinds of capillaries. Surfactant-assisted organosilica-coated capillaries displayed much superior retention efficiency without obviously decreased electroosmotic flow. The existence of surfactant in the synthesis of the sol results in higher surface areas of the coating. Such ethane-bridged organosilica stationary phases can be used under basic conditions.

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.

Monolithic enantiomer‐selective stationary phases for capillary electrochromatography

Monolithic materials have become a well-established format for stationary phases in the field of capillary electrochromatography. Four types of monoliths, namely particle-fixed, silica-based, polymer-based, and molecularly imprinted monoliths, have been utilized as enantiomer-selective stationary phases in CEC. This review summarizes recent developments in the area of monolithic enantiomer-selective stationary phases for CEC. The preparative procedure and the characterization of these columns are highlighted. In addition, the disadvantages and limitations of different monolithic enantiomer-selective stationary phases in CEC are briefly discussed.

Evaluation of poly{‐N‐isopropylacrylamide‐co‐[3‐(methacryloylamino)propyl]trimethylammonium} as a stationary phase for capillary electrochromatography

A cationic polyacrylamide-based stationary phase was synthesized and characterized for CEC. The stationary phase was prepared by radical copolymerization of N-isopropylacrylamide (NIPAAm) and (3-(methacryloylamino)propyl)trimethylammonium chloride (MAPTA), producing a copolymer attached to 5 microm porous silica particles. Fourier transform infrared spectroscopy and thermogravimetric analysis were used to characterize the copolymer. Under capillary electrochromatographic conditions, the poly-NIPAAm-co-MAPTA stationary phase showed to be stable in a wide pH range. The amino groups in the MAPTA provided an anodic EOF for CEC separation. The electroosmotic mobility changed less than 10% when the pH of the mobile phase was changed from 2 to 12. The run-to-run RSD of analyte migration time was less than 1.5% (n = 3), and the RSD of peak area was less than 3% (n = 3). The day-to-day RSD for migration time was less than 2% (n = 3). The polar groups present in the stationary phase contributed to the selectivity of the phase providing for hydrophilic interactions. In the separation of a series of neutral and acidic compounds, the stationary phase shows a mixed-mode separation mechanism with both hydrophobicity and hydrophilicity contributing to the separation.

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.

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.

Recent developments in the field of monolithic stationary phases for capillary electrochromatography

This review summarizes the contributions to the rapidly growing area of monolithic columns based on both silica and synthetic polymers for capillary electrochromatography and chip electrochromatography, with a focus on those published during the year 2004. A wide variety of both modified approaches to the "old" monoliths and new monoliths have been reported despite the very short period of time covered. This demonstrates that monolithic stationary phases have become a well-established format in the field of electrochromatography. The simplicity of their preparation as well as the good control over their porous properties and surface chemistries make the monolithic separation media an attractive alternative to capillary columns packed with particulate materials.

Investigation of a novel mixed-mode stationary phase for capillary electrochromatography. Part III1): Separation of nucleosides and nucleic acid bases on sulfonated naphthalimido-modified silyl silica gel

Investigation of a novel mixed-mode stationary phase for capillary electrochromatography. Part III1): Separation of nucleosides and nucleic acid bases on sulfonated naphthalimido-modified silyl silica gel