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.     

Fast reversed‐phase liquid chromatographic separation of proteins by flow‐through poly(styrene‐co‐divinylbenzene) microspheres

With the explosive growth of the bioscience and biopharmaceuticals, the demand for high efficient analysis and separation of proteins is urgent. High‐performance liquid chromatography is an appropriate technology for this purpose, and the stationary phase is the kernel to the separation efficiency. In this study, flow‐through poly(styrene‐co‐divinylbenzene) microspheres characteristic of the binary pores, i.e. flow‐through pores and mesopores, were synthesized; this special porous structure would benefit the convective mass transfer while guarantee the high specific surface area. Owing to the hydrophobic nature, poly(styrene‐co‐divinylbenzene) microspheres were suitable as the reversed‐phase stationary phase for separation of proteins. For the high permeability of the poly(styrene‐co‐divinylbenzene) microspheres packed column, fast separation of the studied six proteins in ～2 min was achieved. The recoveries of studied proteins were acceptable in the range of 79.0–99.4%. The proposed column had good pH stability of 1–13 and repeatability. Moreover, the column was applied for egg white fast separation, further demonstrating its applicability for complex bio‐sample separation. The flow‐through poly(styrene‐co‐divinylbenzene) microspheres were promising for fast separation of large molecules.     

Separation of natural product using columns packed with Fused‐Core particles

Three HPLC columns packed with 3 μm, sub‐2 μm, and 2.7 μm Fused‐Core (superficially porous) particles were compared in separation performance using two natural product mixtures containing 15 structurally related components. The Ascentis Express^TM C18 column packed with Fused‐Core particles showed an 18% increase in column efficiency (theoretical plates), a 76% increase in plate number per meter, a 65% enhancement in separation speed and a 19% increase in back pressure compared to the Atlantis T3^TM C18 column packed with 3 μm particles. Column lot‐to‐lot variability for critical pairs in the natural product mixture was observed with both columns, with the Atlantis T3 column exhibiting a higher degree of variability. The Ascentis Express column was also compared with the Acquity^TM BEH column packed with sub‐2 μm particles. Although the peak efficiencies obtained by the Ascentis Express column were only about 74% of those obtained by the Acquity BEH column, the 50% lower back pressure and comparable separation speed allowed high‐efficiency and high‐speed separation to be performed using conventional HPLC instrumentation.     

Preparation of high‐selective HPLC packing materials based on alternating copolymer‐grafted silica

Three alternating copolymer‐grafted silica stationary phases for use in high‐selective RP‐HPLC were prepared from two vinyl monomers selected from styrene, N‐methylmaleimide, N‐octadecylmaleimide, and octadecyl acrylate; they were characterized by elemental analyses, thermogravimetric analysis, diffuse reflectance infrared Fourier transform spectroscopy, and ¹³C cross‐polarization magic‐angle spinning NMR spectroscopy. Aspects of molecular‐shape selectivity were evaluated for three different columns using Standard Reference Material 869b, Column Selectivity Test Mixture for Liquid Chromatography. The best selectivity for isomer separations was obtained for the stationary phase prepared with a copolymer of octadecyl acrylate and N‐octadecylmaleimide, which was able to separate 16 polycyclic aromatic hydrocarbons (Standard Reference Material 1647e) in an isocratic elution. In this paper, the effectiveness of this phase is also demonstrated by separation of tocopherol isomers.     

Improved efficiency of reversed‐phase carbon/nanodiamond/polymer core–shell particles for HPLC using carbonized poly(divinylbenzene) microspheres as the core materials

Here, we report efficiencies up to 112 000 plates per meter (a reduced plate height, h, of 2.22) for RP, carbon/nanodiamond/aminopolymer particles using conventional injection conditions in HPLC. This efficiency greatly exceeds our best previously reported value of 71 000 N/m (h = 3.52). The carbon cores used in this study were derived from carbonized poly(divinylbenzene) spheres that were either made in‐house by a two‐step polymerization procedure or obtained commercially. The resulting particles showed good uniformity and were oxidized in nitric acid to increase their dispersability. X‐ray photoelectron spectroscopy confirms particle oxidation and subsequent aminopolymer deposition. Layer‐by‐layer (LbL) growth of poly(allyamine) and nanodiamond was demonstrated to produce core–shell particles. After LbL growth, the particles were functionalized, sieved, and packed into columns. The column functionalization and packing were reproducible. Van Deemter curves indicated that the commercially obtained poly(divinylbenzene) spheres outperformed those synthesized in our laboratory. The columns appear to be stable at 120°C in a pH 11.3 mobile phase. Longer columns (2.1 × 50 mm) than previously reported were packed. Four essential oils were separated by gradient elution.     

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.     

Comparison of a new high‐resolution monolithic column with core‐shell and fully porous columns for the analysis of retinol and α‐tocopherol in human serum and breast milk by ultra‐high‐performance liquid chromatography†

The reduction of analysis time, cost, and improvement of separation efficiency are the main requirements in the development of high‐throughput assay methods in bioanalysis. It can be achieved either by ultra‐high‐performance liquid chromatography (UHPLC) using stationary phases with small particles (<2 μm) at high back pressures or by using opposite direction—monolithic stationary phases with low back pressures. The application of new types of monolithic stationary phases for UHPLC is a novel idea combining these two different paths. The aim of this work was to test the recently introduced second‐generation of monolithic column Chromolith® HighResolution for UHPLC analysis of liposoluble vitamins in comparison with core‐shell and fully porous sub‐2 μm columns with different particle sizes, column lengths, and shapes. The separation efficiency, peak shape, resolution, time of analysis, consumption of mobile phase, and lifetime of columns were calculated and compared. The main purpose of the study was to find a new, not only economical option of separation of liposoluble vitamins for routine practice.     

Preparation of chitosan‐graft‐(β‐cyclodextrin) based sol‐gel stationary phase for open‐tubular capillary electrochromatography

A novel open‐tubular CEC column coated with chitosan‐graft‐(β‐CD) (CDCS) was prepared using sol‐gel technique. In the sol‐gel approach, owing to the 3D 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 isomers were shown. The column efficiencies of 55 000～163 000 plates/m for the isomeric xanthopterin and phenoxy acid herbicides using the sol‐gel‐derived CDCS columns were achieved. Good stabilities were demonstrated that the RSD values for the retention time of thiourea and isoxanthopterin were 1.3 and 1.4% (run to run, n = 5), 1.6 and 2.0% (day to day, n = 3), 2.9 and 3.1% (column to column, n = 3), respectively. The sol‐gel‐coated CDCS columns have shown improved separations of isomeric xanthopterin in comparison with CDCS‐bonded capillary column.     

Continuous‐Bed Columns Containing Sol‐Gel Bonded Packing Materials for Capillary Electrochromatography

Sol‐gel bonded packing materials in continuous‐bed columns have been prepared for capillary electrochromatography (CEC). Three packing materials were investigated: small‐pore Spherisorb ODS1 (3 μm, 80 Å) with octadecyl as stationary phase, small‐pore mixed‐mode Spherisorb ODS/SCX (3 μm, 80 Å) with octadecyl and propyl sulfonic acid as stationary phases, and large‐pore Nucleosil ODS (7 μm, 1 400 Å) with octadecyl as stationary phase. The characteristics of these columns were compared in terms of electroosmotic flow, efficiency, inertness, and retention factors. In contrast to columns containing sol‐gel bonded ODS, columns containing sol‐gel bonded mixed‐mode ODS/SCX generated nearly pH independent electroosmotic flow (EOF) over pH 2–9. Columns containing sol‐gel bonded large‐pore ODS produced nearly three times lower reduced plate height than those containing small‐pore ODS. Efficiencies of 220,000 plates per meter and 175,000 plates per meter were obtained from columns containing sol‐gel bonded 7 μm, 1 400 Å ODS and columns containing sol‐gel bonded 3 μm, 80 Å ODS, respectively, which are among the highest reported efficiencies for continuous‐bed columns. In CEC, over one million plates per meter and pH independent EOF are expected from continuous‐bed columns containing sol‐gel bonded 1.5 μm particles with large pores and mixed‐mode stationary phases.     

Synthesis and chromatographic evaluation of phenyl/tetrazole bonded stationary phase based on thiol‐epoxy ring opening reaction

A silica‐based reversed‐phase stationary phase bonding with phenyl and tetrazole groups was synthesized by thiol‐epoxy ring opening reaction. The bonded groups could not only provide hydrophobic interaction, but also π–π, hydrogen bonding, electrostatic interactions, and so on. The results of characterization with elemental analysis and solid‐state ¹³C cross‐polarization magic‐angle‐spinning NMR spectroscopy indicated the successful preparation of phenyl/tetrazole sulfoether bonded stationary phase. Chromatographic evaluation revealed that phenyl/tetrazole sulfoether bonded stationary phase behaved well under the reversed‐phase mode. The column parameters (H, S*, A, B, and C) showed different selectivity compared with some typical commercial columns, and it was validated by the separation of estrogen, ginsenoside, alkaloid samples. Based on the different selectivity between phenyl/tetrazole sulfoether bonded stationary phase and C18 columns, phenyl/tetrazole sulfoether bonded stationary phase also showed potential to construct a 2D reversed‐phase liquid chromatography system with C18. And it was verified by the separation of corydalis tuber and curcuma zedoary extracts.     

Comparison between core–shell and totally porous particle stationary phases for fast and green LC determination of five hepatitis‐C antiviral drugs

The performances of core–shell 2.7 μm and fully porous sub‐2 μm particles packed in narrow diameter columns were compared under the same chromatographic conditions. The stationary phases were compared for fast separation and determination of five new antiviral drugs; daclatasvir, sofosbuvir, velpatasvir, simeprevir, and ledipasvir. The gradient elution was done using ethanol as green organic modifier, which is more environmentally friendly. Although both columns provided very good resolution of the five drugs, core–shell particles had proven to be of better efficiency. Under gradient elution conditions, core–shell particles exhibited faster elution, better peak shape, and enhanced resolution adding to lower system backpressure. The column backpressure on sub‐2 μm particles was more than twice that on core–shell particles. This gives a chance to use conventional high‐performance liquid chromatography conditions without needing special instrumentation as that required for ultra‐high performance liquid chromatography. The method was validated for determination of the five drugs by gradient elution using mobile phase composed of organic modifier ethanol and aqueous part containing 0.75 g sodium octane sufonate and 3.0 g sodium dihydrogen phosphate per liter at pH of 6.15. Detection was done using UV‐detector set at 210 nm. The linearity, accuracy, and precision were found very good within the concentration range of 2–200 μg/mL.     

Fast fabrication of a hybrid monolithic column containing cyclic and aliphatic hydrophobic ligands via photo‐initiated thiol‐ene polymerization

Three monomers, octakis (3‐mercaptopropyl) octasilsesquioxane, 1,2,4‐trivinylcyclohexane and isophytol were employed to synthesize a novel monolithic stationary phase via photo‐initiated thiol‐ene click polymerization for reversed‐phase liquid chromatography. Several factors such as porogenic system, reaction time and the molar ratio of functional groups were investigated in detail. The resulting poly(POSS‐co‐TVCH‐co‐isophytol) monolithic column exhibited suitable permeability for fast separation and outstanding thermal stability. Five alkylbenzenes were employed to evaluate the ability of chromatographic separation of the resulting monolithic columns at different flow rates, and showed the highest column efficiencies of 90,200–93,100 N/m (corresponding to 10.4–10.6 μm of plate height) at a velocity of 0.41 mm/s. The baseline separations of five anilines and eight phenols further proved the applicability of poly(POSS‐co‐TVCH‐co‐isophytol) monolithic column in the separation of small molecules.     

Preparation and evaluation of diblock copolymer‐grafted silica by sequential surface initiated‐atom transfer radical polymerization for reverse‐phase/ion‐exchange mixed‐mode chromatography

A novel approach that involved the grafting of diblock copolymer with two types of monomer onto substrate by sequential surface initiated‐atom transfer radical polymerization was proposed to prepare a mixed‐mode chromatographic stationary phase. The distinguishing feature of this method is that it can be applied in the preparation of various mixed‐mode stationary phases. In this study, a new reverse‐phase/ion‐exchange stationary phase was prepared by grafting hydrophobic styrene and cationic sodium 4‐styrenesulfonate by the proposed approach onto silica surface. The chromatographic properties of the prepared stationary phase were evaluated by the separation of benzene derivatives, anilines, and β‐agonists, and by the effect of pH values and acetonitrile content on the retention. Compared with typical RP columns, the prepared stationary phase achieved the better resolution and higher selectivity at a shorter separation time and lower organic content. Moreover, the application of the prepared column was proved by separating widely distributed polar and charged compounds simultaneously.     

Ultra‐high performance separation of basic compounds on reversed‐phase columns packed with fully/superficially porous silica and hybrid particles by using ultraviolet transparent hydrophobic cationic additives

The use of the tetrabutylammonium additive was investigated in the ultra‐high performance reversed‐phase liquid chromatographic elution of basic molecules of pharmaceutical interest. When added to the mobile phase at low pH, the hydrophobic tetrabutylammonium cation interacts with the octadecyl chains and with the residual silanols, thus imparting a positive charge to the stationary phase, modulating retention and improving peak shape of protonated basic solutes. Two sources of additive were tested: a mixture of tetrabutylammonium hydroxide/trifluoroacetic acid and tetrabutylammonium hydrogen sulfate. Retention and peak shape of 11 basic pharmaceutical compounds were evaluated on commercially available ultra‐fast columns packed with octadecyl stationary phases (Ascentis Express C18 2.0 µm, Acquity BEH C18 1.7 µm, Titan C18 1.9 µm). All columns benefit from the use of additive, especially tetrabutylammonium hydrogen sulfate, providing very symmetric peaks with reasonable retention times. Focusing on the probe compounds amitriptyline and sertraline, efficiency and asymmetry values were investigated at increasing retention factor. The trend is very different to that obtained in reversed‐phase conditions and the effect lies in the complex molecular interaction mechanisms based on hydrophobic and ion exchange interactions as well as electrostatic repulsion.     

Characterization and kinetic performance of 2.1 × 100 mm production columns packed with new 1.6 μm superficially porous particles

The overall kinetic performance of three production columns (2.1 mm × 100 mm format) packed with 1.6 μm superficially porous CORTECS‐C₁₈+ particles was assessed on a low‐dispersive I‐class ACQUITY instrument. The values of their minimum intrinsic reduced plate heights (h_min = 1.42, 1.57, and 1.75) were measured at room temperature (295 K) for a small molecule (naphthalene) with an acetonitrile/water eluent mixture (75:25, v/v). These narrow‐bore columns provide an average intrinsic efficiency of 395 000 plates per meter. The gradient separation of 14 small molecules shows that these columns have a peak capacity about 25% larger than similar ones packed with fully porous BEH‐C₁₈ particles (1.7 μm) or shorter (50 mm) columns packed with smaller core–shell particles (1.3 μm) operated under very high pressure (>1000 bar) for steep gradient elution (analysis time 80 s). In contrast, because their permeabilities are lower than those of columns packed with larger core–shell particles, their peak capacities are 25% smaller than those of narrow‐bore columns packed with standard 2.7 μm core–shell particles.     

Temperature‐programmed multicapillary gas chromatograph microcolumn for the analysis of odorous sulfur pollutants

We report the fabrication and performance of a silicon‐on‐glass micro gas chromatography eight‐capillary column based on microelectromechanical systems technology that is 50 cm long, 30 μm wide, and 300 μm deep. According to the theory of a gas chromatography column, an even gas flow among different capillaries play a vital role in the peak broadening. Thus, a flow splitter structure is designed by the finite element method through the comparison of the velocity distributions of the eight‐capillary columns with and without splitter as well as an open tubular column. The simulation results reveal that eight‐capillary column with flow splitters can receive more uniform flow velocity in different capillaries, hence decreases the peak broadening and in turn increases the separation efficiency. The separation experiment results show that the separation efficiency of about 22 000 plates/m is achieved with the chip column temperature programmed for analysis of odorous sulfur pollutants. This figure is nearly two times higher than that of the commercial capillary column coated the similar stationary phase. And the separation time of all the components in the microcolumn is less than 3.8 min, which is faster than the commercial capillary 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.     

Fabrication of ZIF‐8@SiO2 Core–Shell Microspheres as the Stationary Phase for High‐Performance Liquid Chromatography

The unique features of high porosity, shape selectivity, and multiple active sites make metal–organic frameworks (MOFs) promising as novel stationary phases for high‐performance liquid chromatography (HPLC). However, the wide particle size distribution and irregular shape of conventional MOFs lead to lower column efficiency of such MOF‐packed columns. Herein, the fabrication of monodisperse MOF@SiO₂ core–shell microspheres as the stationary phase for HPLC to overcome the above‐mentioned problems is reported. Zeolitic imidazolate framework 8 (ZIF‐8) was used as an example of MOFs due to its permanent porosity, uniform pore size, and exceptional chemical stability. Unique carboxyl‐modified silica spheres were used as the support to grow the ZIF‐8 shell. The fabricated monodisperse ZIF‐8@SiO₂ packed columns (5 cm long × 4.6 mm i.d.) show high column efficiency (23 000 plates m^?1 for bisphenol A) for the HPLC separation of endocrine‐disrupting chemicals (bisphenol A, β‐estradiol, and p‐(tert‐octyl)phenol) and pesticides (thiamethoxam, hexaflumuron, chlorantraniliprole, and pymetrozine) within 7 min with good relative standard deviations for 11 replicate separations of the analytes (0.01–0.39, 0.65–1.7, 0.70–1.3, and 0.17–0.91 % for retention time, peak area, peak height, and half peak width, respectively). The ZIF‐8@SiO₂ microspheres combine the advantages of the good column packing properties of the uniform monodisperse silica microspheres and the separation ability of the ZIF‐8 crystals.     

Enantioseparation of tryptophan and its unnatural derivatives by nano‐LC on CSP‐teicoplanin silica based

This work deals with the potentiality of nano liquid chromatography (Nano‐LC) for the chiral separation of racemic mixture of tryptophan and some selected derivatives by using 100 µm i.d. fused silica capillary packed with teicoplanin bonded to 5 µm diol silica stationary phase. The experiments were carried out by using a cheap and laboratory‐assembled nano‐LC–UV system. Elution was done in an isocratic mode using a polar organic mobile phase. In order to find the optimum chiral separation of the studied enantiomers, some chromatographic experimental parameters were systematically studied and optimized. Among them, mobile phase composition, namely organic modifier type and concentration, buffer type and pH and aqueous content and sample solvent dilution on retention time, retention factor and enantioresolution factor were studied. Baseline enantioresolution and good peak shape was achieved utilizing the mobile phase containing 40 mM ammonium formate at pH pH 2.5 in ACN/water/acetone (60:30:10, v/v/v) at 520 nL/min in less than 8 min analysis time.