Open tubular capillary electrochromatography with an N‐phenylacrylamide‐styrene copolymer‐based stationary phase for the separation of anomers of glucose and structural isomers of maltotriose

A ligand with a terminal halogen (4‐chloromethylphenyl isocyanate) was chemically bound on the inner surface of pretreated silica capillary with 50 μm internal diameter and 58 cm total and 50 cm effective length in the presence of dibutyl tin dichloride as a catalyst through isocyanate‐hydroxyl reaction. Attachment of initiator (sodium diethyl dithiocarbamate) to the bound ligand was carried out and followed by in situ polymerization. Reversible addition‐fragmentation chain transfer polymerization was used for the immobilization of N‐phenylacrylamide‐styrene copolymer on the inner surface of capillary column. The resultant open tubular column showed excellent separation performance for derivatized saccharide isomers in capillary electrochromatography. d ‐Glucose was separated into α‐ and β‐anomers while five structural isomers were separated for derivatized maltotriose with separation efficiency above one million theoretical plates per meter. The effects of pH and acetonitrile composition on the electrochromatographic performance of the derivatized saccharides were studied and the optimized elution condition was found to be 90:10 v/v% acetonitrile/30 mM sodium acetate at pH 6.6. UV absorption at 214 nm was used as detection mode in open tubular capillary electrochromatography separations.     

Growth of two-layer copolymer as the stationary phase with very high separation efficiency for separating peptides in capillary electrochromatography

Open tubular CEC (OT-CEC) column with a very high separation efficiency was prepared for peptides separation. A pretreated silica-fused capillary was reacted with 3-(methacryloxy) propyltrimethoxysilane followed by vinylbenzyl chloride and divinylbenzene to produce first thin monolithic monolayer. The second copolymer layer was formed on thin monolithic monolayer of the capillary by reversible addition-fragmentation transfer polymerization of N-phenylacrylamide and styrene. The key parameters including buffer pH value and organic modifier were systematically evaluated to provide the optimal chromatographic condition. The resultant OT-CEC columns were validated by separating a synthetic mixture of peptides and cytochrome C tryptic digest in capillary electrochromatography. The number of theoretical plates as high as 2.4 million per column was achieved for synthetic mixture peptides. In addition, the fabricated OT-CEC column also resolved more than 18 high-efficiency digestion peptides from a mixture containing tryptic digest of cytochrome C. The column to column and inter- to intraday repeatabilities of OT-CEC column through RSD% were found better than 3.0%, exhibiting satisfactory stability and repeatability of the two-layer deposited OT-CEC column. The results reveal that the open tubular capillary column modified with two-layer copolymer shows the great prospect for the separation of proteins in capillary electrochromatography.     

Open tubular layer of S‐ofloxacin imprinted polymer fabricated in silica capillary for chiral CEC separation

An open tubular molecule imprinted polymer (OT‐MIP) capillary column has been prepared for chiral separation of ofloxacin enantiomers in CEC. The S‐ofloxacin imprinted OT column was fabricated by thermally initiated non‐covalent polymerization procedure inside a pretreated and silanized fused silica capillary. The template molecule was incorporated with methacrylic acid (MAA), ethylene glycol dimethacrylate (EDMA) and 4‐styrenesulfonic acid (4‐SSA) and dissolved in a porogen mixture of ACN/2‐propanol (9:1). The separation efficiency of the 4‐SSA MIP column was found quite better than that of the MIP column without 4‐SSA. It has been demonstrated that our OT‐MIP column can separate ofloxacin enantiomers with excellent chiral separation efficiency after tuning the various chromatographic conditions. The optimized chromatographic eluent was 85:15, v/v%, ACN/60 mM sodium acetate at pH 7. The separation efficiency and selectivity of chiral separation of this study were far better than those obtained by previous methods for chiral separation of R‐ and S‐ofloxacin.     

Preparation and evaluation of open‐tubular capillary column combining a metal–organic framework and a brush‐shaped polymer for liquid chromatography

In this work, an open‐tubular capillary liquid‐phase column was prepared by modifying chain polymer on the inner surface of capillary and chemical bonding of metal organic frameworks, NH₂‐UiO‐66, to the brushes of chain polymer (poly(glycidyl methacrylate)). Besides advantages of facial preparation and good permeability, the chain polymer effectively increases the modification amount of NH₂‐UiO‐66 nanoparticles to increase the phase ratio of open‐tubular capillary column and enhance the interactions with analytes. The results of scanning electron microscope energy‐dispersive X‐ray spectra indicated that NH₂‐UiO‐66 nanoparticles were successfully bonded to the chain polymer. Because of the hydrophobic interaction and hydrogen bonding interaction between the analytes and the ligand of NH₂‐UiO‐66, different analytes were well separated on the NH₂‐UiO‐66‐modified poly(glycidyl methacrylate) capillary (1.12 m × 25 μm id × 365 μm od) with the high absolute column efficiency reaching 121 477 plates, benefiting from an open‐tubular column and low mass transfer resistance provided by polymer brush and metal–organic framework crystal. The relative standard deviations of the retention time for run‐to‐run, day‐to‐day, and column‐to‐column (n = 3) runs are below 4.28%, exhibiting good repeatability. Finally, the column was successfully applied to separation of flavonoids in licorice.     

Preparation of open tubular capillary column covalently coated with polystyrene sulfonate with 4,4′-Azobis(4-cyanopentanoyl chloride) as polymerization initiator for electrochromatographic separation of alkaloids,sulfonamides, and peptides

An open tubular capillary electrochromatography column covalently bonded with polystyrene sulfonate was prepared via in situ polymerization using functionalized Azo-initiator 4,4′-Azobis(4-cyanopentanoyl chloride). Scanning electron, fluorescence, and atomic force microscopy techniques showed the formation of a relatively rough layer of polymer. In addition, –CN and C = O stretching vibrations from infrared spectroscopy proved the successful immobilization of the azo-initiator through covalent bonding and X-ray photoelectron spectroscopy confirmed the elemental composition of the formed polymer layer. The prepared column was found to be appropriate for small and medium-sized molecules separation. Compared to bare fused silica capillary column higher selectivity and resolution were obtained for the separation of alkaloids, sulfonamides, and peptides as a result of the electrostatic and pi-pi stacking interactions between the small organic molecules and the coated column without compromising the electroosmotic flow mobility. Separation efficiency was also increased compared to the bare capillary for the separation of alkaloids (about 1.5 times). Moreover, intraday, inter-day, intra-batch, and inter-batch relative standard deviation values of retention time and peak area of peptides were within 2% and 10%, respectively, indicating good repeatability of the column preparation procedure. The developed method for the covalent bonding of polymers through a functionalized azo-initiator could represent a promising stable method for the preparation of an open tubular column.     

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.     

新型聚合物多孔涂层毛细管开管柱的制备及电色谱研究

以甲基丙烯酸缩水甘油酯(GMA)和乙二醇二甲基丙烯酸酯(EDMA)为前驱体制备了新型聚合物多孔涂层毛细管开管(PLOT)柱固定相。通过优化聚合反应时间、致孔剂比例及交联剂比例获得了色谱性能良好的PLOT柱,扫描电镜结果显示毛细管柱内的多孔涂层厚度适中且均匀。在毛细管电色谱模式下,PLOT柱以反相色谱分离机理有效分离了中性、酸性和碱性小分子。人血清白蛋白(HSA)共价结合的蛋白亲和PLOT柱对5对手性对映体实现了较好的分离,且其分离度远高于HSA修饰的单层聚合物毛细管开管柱。PLOT柱分离烷基苯的日内、日间和柱间的相对标准偏差分别小于1.7%、4.8%和7.8%。     

Robust open tubular layer of S-ketoprofen imprinted polymer for chiral LC separation

This study is about the preparation of an open tubular capillary column of molecularly imprinted polymer (MIP) and its application to chiral separation by microLC. A non-covalent in-situ molecular imprinting polymerization protocol was used to synthesize the S-ketoprofen MIP. A special procedure was employed to secure formation of an open tubular and rigid MIP layer in a silica capillary of 100 microm id. The capillary was filled with the reaction mixture, sealed, and placed in a water bath at 50 degrees C for 3 h. Then it was flushed with a 0.5 MPa nitrogen flow for 5 min, and was again placed in the water bath for 2 h to complete MIP formation. Methacrylic acid (MAA) has been known to be an inefficient functional monomer in preparation of MIP of an acid molecule. However, MAA was used with ethylene glycol dimethacrylate in preparation of the S-ketoprofen MIP in this study. The open tubular structure and the microLC mode of separation enabled free optimization without any restriction, thus a very good resolution (R=4.7) of ketoprofen enantiomers was achieved when a mobile phase composed of 30% acetonitrile and 70% acetate buffer at pH 4.5 was used with 5 mbar inlet pressure. This may be partially attributed to the open tubular structure of our MIP, enabling low column back-pressure and free optimization of eluent composition, as well as to the small capillary dimensions. Our MIP capillary column also showed some versatility in chiral separation, thus a good chiral separation was observed for naproxen, ibuprofen, and fenoprofen enantiomers.     

Separation of polystyrenes by means of open tubular capillary chromatography

Simulating polymer separation in flow-through channels of monolithic columns, separation of a mixture of polystyrene standards was investigated using open tubular capillary column of 2 μm inner diameter. High column efficiency was observed for polymers of molar mass ranged from few tens to few hundred kDas. Column efficiency significantly decreased for polymers with molar mass larger than 500 kDa nevertheless preserving value of few tens of thousands theoretical plates. Calibration curve observed for open capillary column is rather steep and can be well described by simple equation without quadratic term. In spite of low selectivity, capillary columns were able in separating wide range of polystyrene standards due to column high efficiency and in such a way supported an idea of hydrodynamic mechanism of polymer separation in flow-through channel of monolithic packings.     

Triamine‐bonded stationary phase for open tubular capillary electrochromatography

A multi‐functional separation column modified with 3‐[2‐(2‐aminoethylamino)ethylamino] propyl‐trimethoxysilane was developed for open tubular capillary electrochromatography. This functional hydrophilic triamine‐bonded open tubular column could generate both anodic and cathodic EOF. When the pH of the running buffer was below 5.3 (30% 3‐[2‐(2‐aminoethylamino)ethylamino] propyl‐trimethoxysilane, v/v), the anodic EOF was exhibited, which greatly prevented the undesired adsorptions of basic proteins on the capillary inner wall. Favorable separation of four basic proteins (viz. trypsin, ribonuclease A, lysozyme and cytochrome c) was successfully achieved at pH 3.5 of 10 mmol/L phosphate buffer. The column efficiencies of proteins were in the range from 87 000 to 110 000 plates/m, and the RSD values for migration time of four proteins were less than 1.2% (run‐to‐run, n=5). The ionic analytes were also separated efficiently in the co‐electroosmotic mode. The average efficiencies ranged from 81 000 to 190 000 plates/m for seven aromatic acids and 186 000–245 000 plates/m for four nucleoside monophosphates, respectively, and good capillary column repeatability was gained with RSD of the migration time not more than 3.0%. The triamine‐bonded open tubular capillary column is favorable to be an alternative functional medium for the further analysis of basic proteins and anionic analytes.     

A novel open‐tubular capillary electrochromatography using carboxymethyl‐β‐cyclodextrin functionalized gold nanoparticles as chiral stationary phase

Enantioselective open tubular capillary electrochromatography with carboxymethyl‐β‐cyclodextrin conjugated gold nanoparticles as stationary phase was developed. This novel open tubular column was fabricated through layer‐by‐layer self‐assembly of gold nanoparticles on a 3‐mercaptopropyl‐trimethoxysilane‐modified fused‐silica capillary and subsequent surface functionalization of the gold nanoparticles through self‐assembly of 6‐mercapto‐β‐cyclodextrin. The 6‐mercapto‐β‐cyclodextrin was firstly synthesized and determined by extensive spectroscopic data. Scanning electron microscopy, energy dispersive X‐ray analysis spectroscopy, and electroosmotic flow experiments were carried out to characterize the prepared open tubular column. Then, the separation effectiveness of the open tubular column was verified by two pairs of ɑ‐tetralones derivatives enantiomers and two pairs of basic drug enantiomers (tramadol hydrochloride and zopiclone) as mode analytes. Factors that influence the enantioseparation were optimized, and under the optimized conditions, satisfactory separation results were obtained for the four enantiomers: compound A, compound B, tramadol hydrochloride, and zopiclone with resolutions of 3.79, 1.56, 1.03, 1.60, respectively. For the combination of gold nanoparticles and negatively charged carboxymethyl‐β‐cyclodextrin, the open tubular column exhibited wider separation range for neutral and basic drugs. Moreover, the repeatability and stability of the column were studied through the run‐to‐run and day‐to‐day investigations.     

窄内径多孔层开管柱的制备及在液相色谱中的应用

近年来,微纳分离技术由于其内在的优势而受到越来越多的关注.多孔层开管柱是一种重要的微分离柱形式,与粗内径的多孔层开管柱(>25μm)相比,窄内径的多孔层开管柱具有更高的分离效率和更低的试剂消耗量.本文综述了内径≤25μm的窄内径多孔层开管毛细管柱的制备方法、与质谱检测联用技术以及在液相色谱中的应用研究进展,对其发展前景进行了展望.     

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%.     

Recent developments of monolithic and open‐tubular capillary electrochromatography (2017–2019)

Capillary electrochromatography, which combined the high selectivity of high‐performance liquid chromatography and the high separation efficiency of capillary electrophoresis, is an attractive separation tool. In this review, the developments on monolithic and open tubular capillary electrochromatography during 2017 to August 2019 are summarized. Considering the development of novel stationary phases is the most active research field in capillary electrochromatography, monolithic capillary electrochromatography is classified according to the polymer‐based and hybrid monolithic columns, while open‐tubular capillary electrochromatography is categorized by cyclodextrin, silica, polymer, nanomaterials, microporous materials, and biomaterials‐based open tubular columns.     

The fabrication of poly (polyethylene glycol diacrylate) monolithic porous layer open tubular (mono‐PLOT) columns and applications in hydrophilic interaction chromatography and capillary gas chromatography for small molecules

The easy shrinkage and swelling of polymer monolithic column when exposed to mobile phase with different polarity is a problem that cannot be ignored. To overcome this drawback, a convenient aqueous two‐phase polymerization approach was used to prepare poly (polyethylene glycol diacrylate, PEGDA) monolithic porous layer open tubular (mono‐PLOT) columns (150 μm). The poly(PEGDA) mono‐PLOT column with homogeneous polymer porous layer was synthesized successfully. A maximum plate number of 41,500 plates per meter for allyl thiourea was obtained under a velocity of 1.8 mm/s. Several kinds of polar molecule were separated on the proposed mono‐PLOT column and a typical hydrophilic interaction retention mechanism was observed. High speed separation of benzoic acids was also carried out, baseline separation of five benzoic acids was successfully achieved within 5 min with a 70 cm mono‐PLOT column at 50°C. Furthermore, the resulting PLOT column was also successfully applied to separate standard analytes of three DNA oxidative damage products and RNA‐modified nucleosides and four chlorophenols. At last, the column could separate alcohols, alkanes, and aromatic isomers via GC. It had more than 20,000 plates per meter for butanol – higher than commercial coatings open tubular columns.     

An optimized mixed‐mode stationary phase based on silica monolith particles for the separation of peptides and proteins in high‐performance liquid chromatography

A phase with both hydrophobic and hydrophilic functionalities has been synthesized by modification of ground silica monolith particles with C18 and 1‐[3‐(trimethoxysilyl)propyl] urea ligands. A series of phases was prepared by changing the ratio of the two ligands to determine the optimal ratio in view of separation efficiency. The resultant optimized stationary phase was packed in narrow‐bore glass‐lined stainless‐steel columns (1 × 300 mm and 2.1 × 100 mm) and used for the separation of synthetic peptides and proteins. The average numbers of theoretical plates (N) of 52 100/column (174 000/m, 5.75 µm plate height) and 35 500/column (118 000/m, 8.47 µm plate height) were achieved with the 300 mm column at a flow rate of 25 µL/min (0.86 mm/s) in 60:40 v/v acetonitrile/30 mM aqueous ammonium formate for the mixture of peptides (Thr‐Tyr‐Ser, Val‐Ala‐Pro‐Gly, angiotensin I, isotocin, and bradykinin) and for the mixture of proteins (myoglobin, human serum albumin, and insulin), respectively. Fast analysis of the peptides and proteins was also carried out at a flow rate of 0.9 mL/min (6.88 mm/s) with the 100 mm column and all the analytes were eluted within 2 min with good separation efficiency.     

Preparation and evaluation of monodispersed,submicron, non‐porous silica particles functionalized with β‐CD derivatives for chiral‐pressurized capillary electrochromatography

Submicron, non‐porous, chiral silica stationary phase has been prepared by the immobilization of functionalized β‐CD derivatives to isocyanate‐modified silica via chemical reaction and applied to the pressurized capillary electrochromatography (pCEC) enantio‐separation of various chiral compounds. The submicron, non‐porous, cyclodextrin‐based chiral stationary phases (sub_μm‐CSP2) exhibited excellent chiral recognition of a wide range of analytes including clenbuterol hydrochloride, mexiletine hydrochloride, chlorpheniramine maleate, esmolol hydrochloride, and metoprolol tartrate. The synthesized submicron particles were regularly spherical and uniformly non‐porous with an average diameter of around 800 nm and a mean pore size of less than 2 nm. The synthesized chiral stationary phase was packed into 10 cm × 100 μm id capillary columns. The sub_μm‐CSP2 column used in the pCEC system showed better separation of the racemates and at a higher rate compared to those used in the capillary liquid chromatography mode (cLC) system. The sub_μm‐CSP2 possessed high mechanical strength, high stereoselectivity, and long lifespan, demonstrating rapid enantio‐separation and good resolution of samples. The column provided an efficiency of up to 170 000 plates/m for n‐propylbenzene.     

Surface initiated polymerization of a cationic monomer on inner surfaces of silica capillaries: Analyte separation by capillary electrophoresis versus polyelectrolyte behavior

[2‐(Methacryloyl)oxyethyl]trimethylammonium chloride was successfully polymerized by surface‐initiated atom transfer radical polymerization method on the inner surface of fused‐silica capillaries resulting in a covalently bound poly([2‐(methacryloyl)oxyethyl]trimethylammonium chloride) coating. The coated capillaries provided in capillary electrophoresis an excellent run‐to‐run repeatability, capillary‐to‐capillary and day‐to‐day reproducibility. The capillaries worked reliably over 1 month with EOF repeatability below 0.5%. The positively charged coated capillaries were successfully applied to the capillary electrophoretic separation of three standard proteins and five β‐blockers with the separation efficiencies ranging from 132 000 to 303 000 plates/m, and from 82 000 to 189 000 plates/m, respectively. In addition, challenging high‐ and low‐density lipoprotein particles could be separated. The hydrodynamic sizes of free polymer chains in buffers used in the capillary electrophoretic experiments were measured for the characterization of the coatings.     

Sol‐gel technique for the preparation of β‐cyclodextrin gold nanoparticles as chiral stationary phase in open‐tubular capillary electrochromatography

A novel open‐tubular capillary electrochromatography column coated with β‐cyclodextrin was prepared using the sol‐gel technique. In the sol‐gel approach, owing to the three‐dimensional network of sol‐gel and the strong chemical bond between the stationary phase and the surface of capillary columns, good chromatographic characteristics and unique selectivity in separating enantiomers were shown. The influences of capillary inner diameter, coating time, organic modifier, buffer pH, and buffer concentration on separation were investigated. The sol‐gel‐coated β‐cyclodextrin column has shown improved enantioseparation efficiency of chlorphenamine, brompheniramine, pheniramine, zopiclone in comparison with the sol‐gel matrix capillary column. The migration time relative standard deviation of the separation of the enantiomers was less than 0.89% over five runs and 2.9% from column to column. This work confirmed that gold nanoparticles are promising electrochromatographic support to enhance the phase ratio of open‐tubular capillary electrochromatography column in capillary electrochromatography.