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.     

Epoxide‐derived mixed‐mode chromatographic stationary phases for separation of active substances in fixed‐dose combination drugs

A method for the preparation of novel mixed‐mode reversed‐phase/strong cation exchange stationary phase for the separation of fixed‐dose combination drugs has been developed. An epoxysilane bonded silica prepared by vapor phase deposition was used as a starting material to produce diol, octadecyl, sulfonate, and mixed octadecyl/sulfonate groups bonded silica phases. The chemical structure and surface coverage of the functional groups on these synthesized phases were confirmed by fourier‐transform infrared and solid‐state ¹³C NMR spectroscopy and elemental analysis. Alkylbenzene homologs, basic drugs, nucleobases and alkylaniline homologs were used as probes to demonstrate the reversed‐phase, ion exchange, hydrophilic interaction and mixed‐mode retention behaviors of these stationary phases. The octadecyl/sulfonate bonded silica exhibits pronounced mixed‐mode retention behavior and superior retentivity and selectivity for alkylaniline homologs. The mixed‐mode retention is affected by either ionic or solvent strength in the mobile phase, permiting optimization of a separation by fine tuning these parameters. The mixed‐mode stationary phase was applied to separate two fixed‐dose combination drugs: compound reserpine tablets and compound methoxyphenamine capsules. The results show that simultaneous separation of multiple substances in the compound dosage can be achieved on the mixed‐mode phase, which makes multi‐cycles of analysis for multiple components obsolete.     

Imidazolium‐embedded iodoacetamide‐functionalized silica‐based stationary phase for hydrophilic interaction/reversed‐phase mixed‐mode chromatography

A novel imidazolium‐embedded iodoacetamide‐functionalized silica‐based stationary phase has been prepared by surface radical chain‐transfer polymerization. The stationary phase was characterized by Fourier transform infrared spectrometry, thermogravimetric analysis, and element analysis. Fast and efficient separations of polar analytes, such as nucleosides and nucleic acid bases, water‐soluble vitamins and saponins, were well achieved in hydrophilic interaction chromatography mode. Additionally, a mixed mode of hydrophilic interaction and reversed‐phase could be also obtained in the analysis of polar and nonpolar compounds, including weak acidic phenols, basic anilines and positional isomers, with high resolution and molecular‐planarity selectivity, outperforming the commercially available amino column. Moreover, simultaneous separation of polar and nonpolar compounds was also achieved. In conclusion, the multimodal retention capabilities of the imidazolium‐embedded iodoacetamide‐functionalized silica‐based column could offer a wide range of retention behavior and flexible selectivity toward hydrophilic and hydrophobic compounds.     

Chiral high‐performance liquid and supercritical fluid chromatographic enantioseparations of limonene‐based bicyclic aminoalcohols and aminodiols on polysaccharide‐based chiral stationary phases

Enantioseparation of limonene‐based bicyclic 1,3‐aminoalcohols and 1,3,5‐ and 1,3,6‐aminodiols was performed by normal‐phase high‐performance liquid chromatographic and supercritical fluid chromatographic (SFC) methods on polysaccharide‐based chiral stationary phases. The effects of the composition of the mobile phase, the column temperature and the structures of the analytes and chiral selectors on retention and selectivity were investigated by normal‐phase LC and SFC technique. Thermodynamic parameters derived from selectivity–temperature‐dependence studies were found to be dependent on the chromatographic method applied, the nature of the chiral selector and the structural details of the analytes. Enantiorecognition in most cases was enthalpically driven but an unusual temperature behavior was also observed: decreased retention times were accompanied by improved separation factors with increasing temperature, i.e. some entropically driven separations were also observed. The elution sequence was determined in all cases. The separation of the stereoisomers was optimized in both chromatographic modalities.     

Morphology and thermal properties of poly(L‐lactide)/poly(butylene succinate‐co‐butylene adipate) compounded with twice functionalized clay

Poly(L ‐lactide) (PLLA)/poly(butylene succinate‐co‐butylene adipate) (PBSA) blends were compounded with Cloisite 25A® (C25A) and C25A functionalized with epoxy groups, respectively. Epoxy groups on the surface of C25A were introduced by treating C25A with (glycidoxypropyl)trimethoxy silane (GPS) to produce so called Twice Functionalized Organoclay (TFC). Variation of morphology and properties of PLLA/PBSA/C25A composites was investigated before and after the treatment with GPS. The morphological structure of the composites was analyzed by using X‐ray diffractometry (XRD) and transmission electron microscopy (TEM). The silicate layers of PLLA/PBSA/TFC were exfoliated to a larger extent than PLLA/PBSA/C25A. Incorporation of the epoxy groups on C25A improved significantly elongation at break as well as tensile modulus and tensile strength of PLLA/PBSA/C25A. The larger amount of exfoliation of the silicate layers in PLLA/PBSA/TFC as compared with that in PLLA/PBSA/C25A was attributed to the increased interfacial interaction between the polyesters and the clay due to chemical reaction. Thermo gravimetric analysis revealed that both T_5%, which was the temperature corresponding to 5% weight loss, and activation energy of thermal decomposition of PLLA/PBSA/TFC were far superior to those of PLLA/PBSA/C25A as well as to those of PLLA/PBSA, indicating that the composites with exfoliated silicate layers were more thermally stable than those with intercalated silicate layers. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 478–487, 2005     

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.     

High‐performance liquid chromatographic enantioseparation of isoxazoline‐fused 2‐aminocyclopentanecarboxylic acids on a chiral ligand‐exchange stationary phase

The application of a chiral ligand‐exchange column for the direct high‐performance liquid chromatographic enantioseparation of unusual β‐amino acids with a sodium N‐((R)‐2‐hydroxy‐1‐phenylethyl)‐N‐undecylaminoacetate‐Cu(II) complex as chiral selector is reported. The investigated amino acids were isoxazoline‐fused 2‐aminocyclopentanecarboxylic acid analogs. The chromatographic conditions were varied to achieve optimal separation. The effects of temperature were studied at constant mobile phase compositions in the temperature range 5–45°C, and thermodynamic parameters were calculated from plots of lnk or lnα versus 1/T. Δ(ΔH°) ranged from –2.3 to 2.2 kJ/mol, Δ(ΔS°) from –3.0 to 7.8 J mol^?1 K^?1 and –Δ(ΔG°) from 0.1 to 1.7 kJ/mol, and both enthalpy‐ and entropy‐controlled enantioseparations were observed. The latter was advantageous with regard to the shorter retention and greater selectivity at high temperature. Some mechanistic aspects of the chiral recognition process are discussed with respect to the structures of the analytes. The sequence of elution of the enantiomers was determined in all cases.     

基于聚倍半硅氧烷微球的亲水/反相混合模式色谱填料的制备与评价

亲水/反相混合模式色谱应用广泛,但pH使用范围有限,不利于碱性药物的分离。该工作利用巯基-烯基点击化学合成了单分散多孔的半胱氨酸改性乙烯基功能化聚甲基倍半硅氧烷(C-V-PMSQ)微球。元素分析表明半胱氨酸成功键合在微球表面。C-V-PMSQ微球为介孔结构,单分散性好且具有优良的化学稳定性。以几种常见的核苷和核酸碱基作为测试样品,考察其色谱保留行为,溶质的保留因子随流动相中水相含量的变化呈现典型的U型曲线,表明C-V-PMSQ固定相具有亲水/反相的双重保留特征。使用该固定相可以分离苯的同系物及一系列亲水性与疏水性化合物。另外在高碱性流动相条件下利用亲水和反相模式成功分离了中药苦参中的3种主要活性成分,表明它在分离碱性药物方面具有较大的优势。     

Preparation of a stationary phase with s‐triazine ring embedded group for reversed phase high‐performance LC

This paper describes the synthesis and chromatographic evaluation of a new polar‐embedded stationary phase, which utilized 2,4,6‐trichloro‐1,3,5‐triazine as the spacer. The resulting materials were characterized by elemental analysis, IR, and solid‐state ¹³C NMR. Empirical test mixtures were utilized to evaluate the column, and showed that it had good performance for basic compounds and high selectivity for polyaromatic hydrocarbons. Moreover, the novel stationary phase has unique property, especially in the separation of “homologous alkaloids” from natural products.     

Chiral separation of novel diazenes on a polysaccharide‐based stationary phase in the reversed‐phase mode

Chiral high‐performance liquid chromatography separation of two recently synthesized liquid crystalline materials C1 and C2 was studied in the reversed‐phase mode. Both materials have an azo‐moiety and one chiral center in their molecular structures. They were available in racemic and pure S forms. For the enantiomeric separations, a Chiralpak AY‐RH stationary phase based on amylose tris(5‐chloro‐2‐methylphenylcarbamate) coated on 5 μm silica was used. The compounds were analyzed in both of their possible forms, the more thermodynamically stable E form and the labile Z form. The conditions and time scale of the UV‐induced E to Z transition were briefly evaluated. Under the optimized conditions, we were able to baseline separate S and R enantiomers of both of the studied materials not only in their E forms, but also in their Z forms. In comparison to the separation in the normal‐phase mode, which we have reported recently, the resolution in the reversed‐phase mode is significantly better. Interestingly, peak reversal was noticed for the S and R enantiomers when the separation was carried out with E versus Z forms of both compounds.     

Anionic synthesis and characterization of ω‐carboaldehyde‐functionalized polybutadienes and polyisoprenes

Attempted preparation of ω‐formyl‐functionalized polydienes by termination of poly(butadienyl)lithium and poly(isoprenyl)lithium with 4‐morpholinecarboaldehyde resulted in 73 and 38% dimer formation (SEC), respectively, under conditions that quantitativey produced ω‐formyl‐functionalized polystyrene. Dimer formation was attributed to postfunctionalization, base‐catalyzed, aldol‐type condensation based on FTIR and ¹H‐NMR analysis of the dimer products. High yields (>97%) of ω‐formyl‐functionalized polydienes were formed by workup using acidic methanol; quantitative functionalization resulted from end capping the polymeric organolithium chain ends with 1,1‐diphenylethylene prior to the functionalization reaction. The ω‐formylpolydienes were characterized by hydroxylamine end‐group titration, FTIR, and both ¹H‐ and ¹³C‐NMR spectroscopy. © 1999 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 37: 1143–1156, 1999     

Piperidine‐functionalized silica: an efficient and environmentally benign catalyst for Claisen–Schmidt condensation

Piperidine‐functionalized silica as a basic heterogeneous catalyst was synthesized via a simple protocol by condensing silica chloride with piperidine. The catalyst was characterized with various techniques (FT‐IR, solid state NMR, scanning electron microscopy, energy‐dispersive X‐ray, thermogravimetric, elemental, and NH₃ and CO₂ temperature‐programmed desorption analyses). Surface area was also evaluated through Brunauer–Emmett–Teller analysis. Its catalytic activity was evaluated for Claisen–Schmidt condensation under solvent‐free conditions. The catalyst was easily recovered and reused up to five cycles without considerable loss of activity and was not deactivated due to amide formation. Also, this method has attractive advantages such as short reaction time, mild reaction conditions, good to excellent yield of products, easy handling of the catalyst and simple operational procedure. Copyright © 2014 John Wiley & Sons, Ltd.     

Preparation of functionalized block copolymers based on a polysiloxane backbone by anionic ring‐opening polymerization

Polysiloxane diblock copolymers containing a pure polysiloxane backbone were prepared by the functionalization of poly(dimethylsiloxane)‐b‐poly(methylvinylsiloxane) copolymers. The copolymers were obtained by the sequential anionic copolymerization of either 1,3,5,7‐tetramethyl‐1,3,5,7‐tetravinylcyclotetrasiloxane or 1,3,5‐trimethyl‐1,3,5‐trivinylcyclotrisiloxane with hexamethylcyclotrisiloxane. The two vinyl monomers showed large differences in the propagation rates, but both could be used for the formation of polysiloxane block copolymers. Differences in the polymerization sequences were investigated and revealed that better control was obtained if the slower propagating monomer was polymerized first. The method permitted the synthesis of block copolymers with molecular weight distributions around 1.4 and lower and high block purities. The vinyl groups of the block copolymers were quantitatively and selectively functionalized by hydrosilation or epoxidation reactions. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 1539–1551, 2002     

Synthesis and characterization of water‐soluble barbiturate‐ and thiobarbiturate‐functionalized polystyrene

The synthesis and characterization of barbiturate‐ and thiobarbiturate‐functionalized polystyrene from polystyrene homopolymer by polymer‐modification reactions is discussed. Polystyrene homopolymer quantitatively functionalized at the para postion with diethyl oxomalonate functionality was subjected to a condensation reaction with urea and thiourea in the presence of sodium methoxide in methanol. This reaction proceeded essentially to quantitative conversion to the barbiturate‐ (BAPS) and thiobarbiturate‐functionalized polystyrenes (TBAPS) as estimated by ¹H NMR, UV, and IR spectroscopies. Thus, several copolymers of styrene with barbiturate‐ and thiobarbiturate‐functionalized styrene were synthesized. The detailed characterizations of quantitatively functionalized polystyrene using gel permeation chromatographic, IR, UV, and ¹H NMR spectroscopic techniques as well as thermogravimetric analysis are discussed. An application of the newly synthesized polymer in removing Cu(II) ions from aqueous solution is demonstrated. This is the first report on the synthesis of BAPS and TBAPS by the polymer‐modification route or otherwise. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 731–737, 2002; DOI 10.1002/pola.10154     

Liquid chromatographic resolution of racemic rasagiline and its analogues on a chiral stationary phase based on (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid

A liquid chromatographic chiral stationary phase based on (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid was applied to the resolution of 15 analytes, including racemic rasagiline, a chiral drug for the treatment of Parkinson's disease, and its analogues. The composition of mobile phase was optimized to be ethanol/acetonitrile/acetic acid/triethylamine (80:20:0.2:0.3, v/v/v/v) by evaluating the chromatographic results for the resolution of five selected analytes under various mobile phase conditions. Under the optimized mobile phase conditions, racemic rasagiline was resolved quite well with a separation factor of 1.48 and resolution of 2.71 and its 14 analogues were also resolved reasonably well with separation factors of 1.06–1.54 and resolutions of 0.54–2.11. Among 15 analytes, racemic rasagiline was resolved best except for just one analyte. The analyte structure–enantioselectivity relationship indicated that racemic rasagiline has the most appropriate structural characteristics for resolution on the chiral stationary phase.     

Surface functionalization with phosphazene substrates—part VII. Silicon‐based materials functionalized with hexachlorocyclophosphazene

This paper investigates by means of XPS analysis the surface functionalization of soda‐lime and fused quartz glass substrates with hexachlorocyclophosphazene (HCCP), obtained by simple immersion in solutions of phosphazene in anhydrous solvents. Several experimental parameters like reaction solvent, temperature and treatment duration were studied, together with the influence of physisorbed water on the surface of the substrates. Phosphazene‐derived deposits resistant to tetrahydrofuran (THF) washing and vacuum drying were obtained. They presented P:N:Cl stoichiometric ratios close to 1:1:1 when the reaction and the final drying of the samples were performed at room temperature; use of higher temperatures resulted in lower nitrogen and chlorine content. The chemical nature of the films was studied by XPS peaks deconvolution which enabled us to propose a possible, temperature‐enhanced hydrolytical degradation mechanism. Copyright © 2008 John Wiley & Sons, Ltd.     

Novel functionalized poly(glycidyl methacrylate‐co‐ethylene dimethacrylate) microspheres for the solid‐phase extraction of glycopeptides/glycoproteins

Novel 3‐aminophenylboronic acid functionalized poly(glycidyl methacrylate‐co‐ethylene dimethacrylate) microspheres were prepared for the solid‐phase extraction of glycopeptides/glycoproteins. The adsorption efficiency, maximum adsorption capacity, and specific recognition of the microspheres to glycoprotein were investigated. The results indicated excellent adsorption of glycoproteins by the microspheres, which are attributed to the well‐defined boronic acid brushes on the microsphere surfaces. Furthermore, a solid‐phase extraction microcolumn filled with the microspheres was used to efficiently enrich glycopeptides from enzymatic hydrolysates from human serum samples. The mass spectrometry results demonstrated that the method is suitable for the separation and enrichment of glycopeptides/glycoproteins from complex biological samples.     

Cinnamate‐functionalized poly(ester‐carbonate): Synthesis and its UV irradiation‐induced photo‐crosslinking

A functionalized cyclic carbonate monomer containing a cinnamate moiety, 5‐methyl‐5‐cinnamoyloxymethyl‐1,3‐dioxan‐2‐one (MC), was prepared for the first time with 1,1,1‐tri(hydroxymethyl) ethane as a starting material. Subsequent polymerization of the new cyclic carbonate and its copolymerization with L ‐lactide (LA) were successfully performed with diethyl zinc (ZnEt₂) as initiator/catalyst. NMR was used for microstructure identification of the obtained monomer and copolymers. Differential scanning calorimetry (DSC) was used to characterize the functionalized poly(ester‐carbonate). The results indicated that the copolymers displayed a single glass transition temperature (T_g) and the T_g decreased with increasing carbonate content and followed the Fox equation, indicative of a random microstructure of the copolymer. The photo‐crosslinking of the cinnamate‐carrying copolymer was also demonstrated. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 161–169, 2009     

A new access to polypyrrole‐based functionalized magnetic core‐shell nanoparticles

Magnetic nanoparticles (NP) have found various important applications in nanotechnology and nanomedicine, because they can be manipulated by external magnetic field and can be functionalized on their surface. Although a variety of magnetic core shell NP are known present research focuses on new NP with better properties (reduced toxicity, high colloidal and chemical stability, wide scope of application) and more straightforward and reproducible syntheses. In this work, we report the synthesis of azido‐functionalized polypyrrole (PPy)‐based superparamagnetic core shell NP by surface initiated polymerization wherein miniemulsion technique have been applied in this field for the first time. The new NP are attractive for biomedical applications because the PPy is biocompatible, the shell can easily be functionalized by Cu‐catalyzed click‐reaction as shown by the introduction of biotin and the material exhibits superparamagnetic behavior. The surface initiated polymerization is carried out at new magnetite NP, which are stabilized by pyrrol‐containing fatty acids. Although these starting NP lack a polymer shell, they show a remarkable stability and thus have the potential for further functionalization. The magnetic NP are characterized by various methods such as FTIR, X‐ray photoelectron spectroscopy, magnetic measurements, thermal gravimetric analysis, and dynamic light scattering. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Alkaloid purification using rosin‐based polymer‐bonded silica stationary phase in HPLC

Alkaloids are important natural products that exhibit a wide spectrum of pharmacological activities. To efficiently separate and purify them, a rosin‐based polymer‐bonded silica stationary phase in high‐performance liquid chromatography was synthesized via the surface radical polymerization of ethylene glycol maleic rosinate acrylate and methacrylic acid onto functionalized silica. The stationary phases, columns, optimization of chromatographic conditions for alkaloids, and thermodynamic behavior of the analytes on the column were fully studied. Under the optimized conditions, the prepared column efficiently purified natural camptothecine, caffeine, and evodiamine with the corresponding purities of 92, 96, and 97%. With this work, we have developed an efficient approach to isolate alkaloids and promoted the research on rosin‐based materials in biomedicine and analytical chemistry.