Open-tubular Capillary Electrochromatography Research Articles

Separation of monoclonal antibody charge state variants by open tubular capillary electrochromatography with immobilised protein as stationary phase

Monoclonal antibodies (mAbs) are highly heterogeneous and complex glycoproteins requiring powerful analytical tools for characterization and quality control. In this work, we utilize adsorbed bovine serum albumin (BSA) as a stationary phase in open tubular (OT) capillary electrochromatography for separation of charge state variants of mAbs. Poly(diallydimethylammonium chloride) (PDDA) was used to assist fabrication of BSA coated OT column by electrostatic self-assembly. Scanning electron microscopy and electroosmotic flow measurement were carried out to characterize the as-prepared BSA coated PDDA OT columns. The electrochromatographic performance of the OT columns was evaluated by separation of basic proteins and different charge state variants of mAbs. The effects of background solution pH and concentration on separation were investigated. A rapid separation of charge state variants of mAbs was successfully achieved in the BSA coated PDDA OT column. Separation of seven variants of the mAb cetuximab was achieved using the prepared column. Two basic variants and one acidic variant of rituximab, and two basic variants and four acidic variants of trastuximab were successfully distinguished from the main forms. In addition, the columns demonstrated good repeatability and stability with the run-to-run, day-to-day and batch-to-batch relative standard deviations of migration times less than 3.7%.

High Efficiency Robust Open Tubular Capillary Electrochromatography Column for the Separation of Peptides

High Efficiency Robust Open Tubular Capillary Electrochromatography Column for the Separation of Peptides

Layer-by-layer assembly of zeolite imidazolate framework-8 as coating material for capillary electrochromatography.

In this work, open-tubular capillary column coated with zeolite imidazolate framework-8 (ZIF-8) nanocrystals was prepared by a layer-by-layer method. The coating was formed by growing ZIF-8 nanocrystals on either bare fused silica capillary wall or the capillary column premodified with amino groups. The shape and the thickness of the coating formed by using these two methods were almost the same. However, the coverage of the ZIF-8 crystals on the bare fused silica capillary wall was higher than that on the capillary column premodified with amino groups. The ZIF-8 coated capillary column was evaluated for open-tubular capillary electrochromatography. The effect of pH value, buffer concentration, and applied voltage on the separation of phenols was investigated. Good separation of nine phenolic isomers was achieved because of the strong interaction between unsaturated Zn sites and phenols. The column performance for o-nitrophenol was as high as 208 860 plates m(-1) . The run-to-run, day-to-day, and column-to-column reproducibility of retention time and resolution for p-nitrophenol and o-nitrophenol were very good with RSDs of less than 6.5%.

Novel Zn-based MOFs stationary phase with large pores for capillary electrochromatography.

Metal-organic frameworks (MOFs) are attractive stationary phases in the field of separation sciences for their unique properties such as large specific surface areas, high porosity, and diverse structures. However, there are few MOFs, which have ultrahigh porosities and gas uptake capacities. In this work, MOF-180 with exceptional porosity, a cage size of 15 × 23 Å, was grown on inner wall of capillary as a novel stationary phase for open-tubular CEC. It has been demonstrated that the MOF-180-modified capillary column exhibited good performance for separation of acidic, basic, and neutral analytes. As compared with MOF-199-modified column, MOF-180-modified column exhibited specific recognition and size selectivity to the tested compounds. The characteristics observed by SEM and FTIR indicated that MOF-180 was successfully grafted on the inner wall of the capillary. The precisions (RSDs) of retention time, peak area, and W1/2 for methylbenzene, ethylbenzene, n-propylbenzene, chlorobenzene, o-dichlorobenzene, and 1,2,4-trichlorobenzene were 0.50-0.54%, 3.31-4.13%, 0.35-1.61%, 1.73-4.22%, 2.67-4.37%, and 1.60-3.38%, respectively. Besides, the run-to-run, day-to-day, and column-to-column RSDs of EOF were 0.6%, 4.19%, and 4.31%, respectively.

Graphene oxide-SiO2 hybrid nanostructure as coating material for capillary electrochromatography.

Graphene oxide (GO) has been considered as a promising stationary phase for chromatographic separation. However, the very strong adsorption of the analytes on the GO surface lead to the severe peak tailing, which in turn resulting in decreased separation performance. In this work, GO and silica nanoparticles hybrid nanostructures (GO/SiO2 NPs@column) were coated onto the capillary inner wall by passing the mixture of GO and silica sol through the capillary column. The successful of coating of GO/SiO2 NPs onto the capillary wall was confirmed by SEM and electroosmotic flow mobilities test. By partially covering the GO surface with silica nanoparticles, the peak tailing was decreased greatly while the unique high shape selectivity arises from the surface of remained GO was kept. Consequently, compared with the column modified with GO (GO@column), the column modified with GO and silica nanoparticles through layer-by-layer method (GO-SiO2 NPs@column), or the column modified with silica nanoparticles (SiO2 NPs@column), GO/SiO2 NPs@column possessed highest resolutions. The GO/SiO2 NPs@column was applied to separate egg white and both acidic and basic proteins as well as three glycoisoforms of ovalbumin were separated in a single run within 36 min. The intra-day, inter-day, and column-to-column reproducibilities were evaluated by calculating the RSDs of the retention of naphthalene and biphenyl in open-tubular capillary electrochromatography. The RSD values were found to be less than 7.1%.

Polydopamine-supported immobilization of covalent-organic framework-5 in capillary as stationary phase for electrochromatographic separation

Covalent-organic frameworks (COFs) are attractive materials for their fascinating properties, such as rigid structures, exceptional thermal stabilities, low densities, and permanent porosity with specific surface areas, which indicate potential for application in chromatography similar to related metal-organic frameworks (MOFs). However, the utilization of COFs in analytical chemistry is far behind as compared to that of the MOFs due to the challenging work of their immobilization. Here, we have successfully demonstrated the growth of the boron COF-5 on the inner wall of the fused silica capillary by a developed polydopamine-supported method. Combined with the layer-by-layer strategy, multilayer COF-5-coated capillary was obtained. The formation of COF-5 on polydopamine-coated substrate has been confirmed by scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction. A novel stationary phase of COF-5 was developed on the basis of successful growth of COF-5 on polydopamine for open-tubular capillary electrochromatography (OT-CEC). Baseline separation of neutral, acidic and basic analytes was achieved on multilayer COF-5-coated capillary column. The fabricated capillary columns showed high column efficiency (154,060 plates/m for methylbenzene), excellent stability and repeatability. The precision (relative standard deviation (RSD), n=3) of retention time, peak height, and peak area for tested neutral compounds were in the range of 1.2–1.3%, 1.8–4.2%, and 0.9–2.4%, respectively. To the best of our knowledge, it was the first demonstration that COF-5 was developed as a novel stationary phase.

An immobilized carboxyl containing metal-organic framework-5 stationary phase for open-tubular capillary electrochromatography

A novel capillary column with metal-organic framework-5 (MOF-5) as the stationary phase was prepared for open-tubular capillary electrochromatography (OT-CEC). To grow MOF-5, the fused-silica capillary was functionalized firstly using 3-aminopropyltriethoxysilane and glutaraldehyde as covalent linkers; and then MOF-5 would be immobilized on the inner wall of COOH-terminated capillary by epitaxial growth, to produce a MOF-5-modified capillary. The successful growth of MOF-5 has been characterized and confirmed by scanning electron microscopy, X-Ray diffraction and Fourier transform infrared spectra. The influence of pH value and methanol on electroosmotic flow (EOF) of the MOF-5-modified capillary column was investigated. The EOF showed a pH-dependent from anode to cathode. The immobilization of MOF-5 improved the interactions between analytes and layer on inner wall of the capillary. Excellent separations of substituted benzenes and acidic and basic analytes were obtained on the fabricated capillary columns. The MOF-5-modified capillary columns exhibited good repeatability, with relative standard deviations for intra-day, inter-day runs and column-to-column less than 1.87%, 3.53%, and 8.49%, respectively. Our successful application of MOF-5 paved the way for introducing series of dicarboxylate-based isoreticular MOFs to OT-CEC as novel stationary phase.

Application of pH-responsive poly(2-dimethyl-aminoethylmethacrylate)-block-poly(acrylic acid) coatings for the open-tubular capillary electrochromatographic analysis of acidic and basic compounds

A new type of stimuli-responsive polymeric (SRP) coating has been prepared for use in open tubular capillary electrochromatography (OT-CEC), by grafting poly(2-dimethylaminoethylmethacrylate)-block-poly(acrylic acid) (PDMAEMA-b-PAA) as a Y-shaped block copolymer with two dissimilar chain compositions onto the inner walls of aminopropyl-modified silica capillaries. The grafting process introduced weakly charged functional groups from the PAA and PDMAEMA, enabling the generation of electroendosmotic flow with magnitude and direction adjustable by changing the pH of the running buffer electrolyte. This stimuli-responsive PDMAEMA-b-PAA block copolymer was found to provide excellent resolution of various acidic and basic compounds, leading to efficient analyte separation. When operated in the OT-CEC mode, separation selectivities could be readily manipulated via differential contributions from chromatographic and electrophoretic mechanisms, simply by changing the pH or the ionic strength of the running buffer electrolyte.

Coatings of molecularly imprinted polymers based on polyhedral oligomeric silsesquioxane for open tubular capillary electrochromatography

Polyhedral oligomeric silsesquioxane (POSS) was successfully applied, for the first time, to prepare imprinted monolithic coating for capillary electrochromatography. The imprinted monolithic coating was synthesized with a mixture of PSS-(1-Propylmethacrylate)-heptaisobutyl substituted (MA 0702), S-amlodipine (template), methacrylic acid (functional monomer), and 2-methacrylamidopropyl methacrylate (crosslinker), in a porogenic mixture of toluene–isooctane. The influence of synthesis parameters on the imprinting effect and separation performance, including the amount of MA 0702, the ratio of template to monomer, and the ratio of monomer to crosslinker, was investigated. The greatest resolution for enantiomers separation on the imprinted monolithic column prepared with MA 0702 was up to 22.3, about 2 times higher than that prepared in absence of the POSS. Column efficiency on the POSS-based MIP coatings was beyond 30,000platem−1. The comparisons between MIP coating synthesized with the POSS and without the POSS were made in terms of selectivity, column efficiency, and resolution. POSS-based MIP capillaries with naproxen or zopiclone was also prepared and separation of enantiomers can be achieved.

Separation of small organic molecules using covalent organic frameworks-LZU1 as stationary phase by open-tubular capillary electrochromatography

Covalent organic frameworks (COFs) have attracted much attention because of their permanent nanoscale porosity and higher surface area compared to zeolites as well as robustness. COFs have great potential in several fields such as hydrogen storage, gas separation, and catalysis. However, COFs have not yet been applied in capillary electrochromatography. Herein, covalent organic frameworks-LZU1 (COF-LZU1) was used as the stationary phase in open-tubular capillary electrochromatography for the first time. Compared to the monoliths used in electrochromatography, the preparation technique of a COF-LZU1-coated capillary was simple and practical. The baseline separation of model analytes including alkylbenzenes, polyaromatic hydrocarbons, and anilines by the COF-LZU1-coated capillary was achieved based on the size selectivity of COF-LZU1 porous structure and hydrophobic interactions between the model analytes and organic ligands of COF-LZU1. The load capacity of the COF-LZU1-coated capillary for naphthalene was 0.6mg/mL. For three consecutive runs, the intraday relative standard deviations (RSDs) were 1.4–2.6% for the migration time and 2.7–8.7% for the peak area. The interday RSDs were 1.3–3.9% for the migration time and 3.7–9.7% for the peak area. The column-to-column reproducibility of migration time was in the range 1.0–3.9%. Moreover, the coated capillary was used for >300 runs with no changes in the separation efficiency. Thus, COFs have great potential in capillary electrochromatography and may provide a new method for chromatographic separation.

Preparation of a β-Cyclodextrin-Based Open-Tubular Capillary Electrochromatography Column and Application for Enantioseparations of Ten Basic Drugs.

An open-tubular capillary electrochromatography column was prepared by chemically immobilized β-cyclodextrin modified gold nanoparticles onto new surface with the prederivatization of (3-mercaptopropyl)-trimethoxysilane. The synthesized nanoparticles and the prepared column were characterized by transmission electron microscopy, scanning electron microscopy, infrared spectroscopy and ultraviolet visible spectroscopy. When the column was employed as the chiral stationary phase, no enantioselectivity was observed for ten model basic drugs. So β-cyclodextrin was added to the background electrolyte as chiral additive to expect a possible synergistic effect occurring and resulting in a better separation. Fortunately, significant improvement in enantioselectivity was obtained for ten pairs of drug enantiomers. Then, the effects of β-cyclodextrin concentration and background electrolyte pH on the chiral separation were investigated. With the developed separation mode, all the enantiomers (except for venlafaxine) were baseline separated in resolutions of 4.49, 1.68, 1.88, 1.57, 2.52, 2.33, 3.24, 1.63 and 3.90 for zopiclone, chlorphenamine maleate, brompheniramine maleate, dioxopromethazine hydrochloride, carvedilol, homatropine hydrobromide, homatropine methylbromide, venlafaxine, sibutramine hydrochloride and terbutaline sulfate, respectively. Further, the possible separation mechanism involved was discussed.

Simultaneous Determination of Indole-3-Acetic Acid and Indole-3-Butyric Acid in Plant by Field-Amplified Sample Stacking Open-Tubular Capillary Electrochromatography Based on Solid-Phase Extraction with Calixarene Sorbent

This paper describes the preparation and application of methyldiethanolamine amination polychloromethyl styrene nanolatex (MDEAPL) and MDEAPL coated capillary column (ccc-MDEAPL). The ccc-MDEAPL column was characterized with steady EOF values from −14.6 × 10−5 to −19.8 × 10−5 at pH 7.08–7.56. A method using tetraazacalix[2]arene[2]triazine-modified silica gel (NCS) as solid-phase extraction (SPE) sorbent was developed for concentrating and purifying the two phytohormones, indole-3-acetic acid (IAA) and beta-indolebutyric acid (IBA) of different plants. The determination conditions of IAA and IBA for field-amplified sample stacking open-tubular capillary electrochromatography based on SPE with NCS sorbent (NCS-SPE-FASS-OT-CEC) were optimized as follows: background electrolyte, 30 mmol L−1 Tris + 24 mmol L−1 HClO4 (30 % acetonitrile, pH 7.24); ccc-MDEAPL, 50 μm i.d. × 50 cm (effective length 41.5 cm); separation voltage, −25 kV; column temperature, 25 °C; UV detection wavelength, 230 nm; water-plug injection, 30 mbar × 20 s; sample electrokinetic injection, −20 kV × 20 s. Under the optimized conditions, IAA and IBA were well separated within 6 min (Rs > 1.5). The NCS-SPE-FASS-OT-CEC method was validated in terms of linearity, sensitivity, selectivity, precision and accuracy. The detection limits were 8 ng mL−1 for IAA and 25 ng mL−1 for IBA (S/N = 3), respectively. The overall recoveries ranged from 81.0 to 89.0 %. The developed method was successfully applied for the determination of IAA and IBA in different positions of various plants.

Open tubular-capillary electrochromatography: Developments and applications from 2013 to 2015.

Open tubular CEC (OT-CEC) separates analyte mixtures by a combination of electrophoretic, electro-osmotic, and/or chromatographic effects. OT-CEC research is an active and growing field, with studies encompassing a wide range of investigations related to new strategies for chemical modification of the inner surface of the capillary, leading to the introduction of novel stationary phase coatings. This review has examined the literature on OT-CEC from 2013 to August 2015 and highlights the developments in the fabrication of highly selective stationary phases, based on materials that include cyclodextrin chiral selectors, graphene and graphene oxide, metal-organic frameworks, molecularly imprinted polymers, nanoparticles, nanolatex particles, nanocomposites, in situ generated polymers, block polymers, tentacle-type polymers, polyelectrolyte multilayers, polysaccharides, phospholipids, and proteins. This review, while considering the development of novel OT-CEC coating materials, specifically examines different immobilization or coating methodologies and approaches and also discusses the separation mechanisms that occur with these new materials. These OT-CEC coatings are intended mainly to separate low molecular weight molecules relevant to the pharmaceutical, agricultural, and food industries as well as for use in environmental monitoring.

Three chiral metal organic frameworks used as stationary phases for the separation of racemates by open tubular capillary electrochromatography

Three chiral metal organic frameworks used as stationary phases for the separation of racemates by open tubular capillary electrochromatography

In situ rapid preparation of homochiral metal-organic framework coated column for open tubular capillary electrochromatography

Fabricating metal-organic frameworks (MOFs) with the use of nucleating agents in the microenvironment have attracted increasing attention recently. Herein, a simple and rapid synthesis method was developed to in situ fabricate homochiral MOF [Zn(s-nip)2]n in the capillary inner wall by using ZnO nanoparticles as efficient nucleating agents for open tubular capillary electrochromatography (OT-CEC) separation of monoamine neurotransmitters enantiomers of epinephrine, isoprenaline and synephrine, the diastereoisomers of ephedrine and pseudoephedrine, the isomers of nitrophenols and analogues of bisphenols with good resolution. The relative standard deviations (RSDs) for the analytes migration time of intra-day, inter-day and column-to-column were in the range of 0.8–2.1% (n=10), 0.3–3.2% (n=3) and 3.2–9.3% (n=3), respectively. Additionally, the homochiral MOF [Zn(s-nip)2]n coated capillary column could be successively used over 260 runs without observable change in the separation efficiency.

Open tubular capillary column for the separation of cytochrome C tryptic digest in capillary electrochromatography.

A silica capillary of 50 μm internal diameter and 500 mm length (416 mm effective length) was chemically modified with 4-(trifluoromethoxy) phenyl isocyanate in the presence of dibutyl tin dichloride as catalyst. Sodium diethyl dithiocarbamate was reacted with the terminal halogen of the bound ligand to incorporate the initiator moiety, and in situ polymerization was performed using a monomer mixture of styrene, N-phenylacrylamide, and methacrylic acid. The resultant open tubular capillary column immobilized with the copolymer layer was used for the separation of tryptic digest of cytochrome C in capillary electrochromatography. The sample was well eluted and separated into many components. The elution patterns of tryptic digest of cytochrome C were studied with respect to pH and water content in the mobile phase. This preliminary study demonstrates that open tubular capillary electrochromatography columns with a modified copolymer layer composed of proper nonpolar and polar units fabricated by reversible addition-fragmentation transfer polymerization can be useful as separation media for proteomic analysis.

Determination of trace nitrites and nitrates in human urine and plasma by field-amplified sample stacking open-tubular capillary electrochromatography in a nano-latex coated capillary

The associations of nitric oxide (NO) with many diseases are still being discovered, and sometimes, NO levels in the body fluids are too low to be detected. Thus, the determination methods for trace nitrites and nitrates in urine and plasma of healthy volunteers and rheumatoid arthritis (RA) patients are necessary. Using trimethylamine aminated polychloromethyl styrene nano-latex coated capillary column (ccc-TMAPL) and field-amplified sample stacking (FASS) injection, the sensitive open-tubular capillary electrochromatography (OT-CEC) methods for determining trace nitrites and nitrates in urine and plasma of healthy volunteers and RA patients were set up. The limits of detection for both nitrite and nitrate were 1 ng/mL. The intra- and inter-day relative standard deviations (RSDs) of the methods were less than 1.9%, and recoveries were between 89 and 96%. Plasma nitrate (diluted 10 times) levels were 93–301 ng/mL for healthy volunteers and 222–421 ng/mL for RA patients, respectively. Meanwhile, plasma nitrite levels were 7–18 and 40–82 ng/mL, respectively. The urinary nitrite levels were significantly elevated in RA patients. The proposed sensitive FASS-OT-CEC methods for trace nitrites and nitrates hold the promise of practical applications in a wide array of biological analyses, clinical and pathophysiology studies of the diseases related with NO hyperproduction and metabolites.

Preparation and characterization of tentacle-type polymer stationary phase modified with graphene oxide for open-tubular capillary electrochromatography

In this research, a novel organic polymer coated stationary phase modified with inorganic nanomaterial was prepared for open-tubular capillary electrochromatography (OT-CEC). The capillary was developed by covalent attaching graphene oxide (GO) onto the tentacle-type polymer chains grafted capillary which was derivatized with ammonium hydroxide. Scanning electron microscopy (SEM) and Raman spectroscopy provided evidences that the tentacle-type polymer and GO were coated to the capillary inner wall. This novel GO-polymer modified capillary column generated a strong and stable electroosmotic flow (EOF) from anode to cathode with the running electrolytes of pH ranging from 4.0 to 9.0, and showed excellent separation ability toward neutral small molecules, amino acids and ephedrine–pseudoephedrine isomers with resolution higher than 2.30 and column efficiency up to over 170,000plates/m. The repeatabilities of the GO-GMA@capillary were satisfying with relative standard deviations for intra-day and inter-day runs less than 3.27%, and column-to-column runs less than 7.49%. Additionally, the separation performance of GO-polymer modified capillary was compared with that of the polymer coated capillary and GO coated capillary.

Tunable thick porous silica coating fabricated by multilayer-by-multilayer bonding of silica nanoparticles for open-tubular capillary chromatographic separation

A simple coating procedure employing a multilayer-by-multilayer process to modify the inner surface of bare fused-silica capillaries with silica nanoparticles was established. The silica nanoparticles were adsorbed onto the capillary wall via a strong electrostatic interaction between amino functional groups and silica particles. The thickness of the coating could be tuned from 130 to 600nm by increasing the coating cycles from one to three. Both the retention factor and the resolution were greatly increased with increasing coating cycles. The loading capacity determined by naphthalene in the column with three coating cycles is 152.1pmol. The effects of buffer concentration and pH value on the stability of the coating were evaluated. The retention reproducibility of the separation of toluene was 0.8, 1.2, 2.3, and 4.5%, respectively, for run-to-run, day-to-day, column-to-column, and batch-to-batch, respectively. The chromatographic performance of these columns was evaluated by both capillary liquid chromatography and open-tubular capillary electrochromatography (OT-CEC). Separation of aromatic hydrocarbons in the column with three coating cycles provided high theoretical plate numbers (up to 269,280 platesm−1 for toluene) and short separation time (<15min) by using OT-CEC mode. The method was also used to separate egg white proteins. Both acidic and basic proteins as well as four glycoisoforms were separated in a single run.

Chiral ligand-exchange separation and determination of malic acid enantiomers in apple juice by open-tubular capillary electrochromatography

This study describes the application of an open tubular capillary column for chiral ligand-exchange separation and determination of malic acid enantiomers in apple juice by open-tubular capillary electrochromatography (OT-CEC). The open tubular column was prepared by in-situ grafting polymerization of 3-chloro-2-hydroxypropyl methacrylate (HPMA-Cl) and followed by l-Histidine (l-His) modification. l-His was used as a chiral ligand-exchange selector and copper (II) as a central ion. The electrochromatographic characterization of the open tubular column was performed with the use of thiourea as an electroosmotic flow (EOF) marker. Factors affecting electrochromatographic enantioseparation of malic acid were also studied. The running buffer conditions for optimum enantioseparation of malic acid were found to be ACN/5.0mM CuSO4, 20.0mM (NH4)2SO4 (60/40%, v/v) adjusted to pH 3.0. The separation and determination of the enantiomers of malic acid in the apple juice solution diluted 10- to 40-folds were successfully achieved.