Preparation of imidazole-functionalized silica by surface-initiated atom transfer radical polymerization and its application for hydrophilic interaction chromatography

A novel imidazole-functionalized stationary phase for hydrophilic interaction chromatography (HILIC) was prepared via surface-initiated atom transfer radical polymerization (SI-ATRP). 1-Vinylimidazole as a monomer was polymerized on the surface of initiator-immobilized silica by SI-ATRP using CuCl and 2,2'-bipyridyl as a catalyst. The graft chain length and polymer grafting density were controlled by varying the ratio of monomer to initiator. The resulting materials were characterized by elemental analysis and thermogravimetric analysis. Then, high-performance liquid chromatography separation of eight nucleobases/nucleosides was performed on the imidazole-functionalized chromatographic column in HILIC mode. The effects of mobile phase composition, buffer pH, and column temperature on the separation of nucleobases/nucleosides were investigated, and the retention mechanisms were studied. Chromatographic parameters were calculated, and the results showed that surface adsorption through hydrogen bonding and electrostatic interaction dominated the retention behavior of the solutes in HILIC mode. Lastly, the stationary phase was successfully used to determine the nucleobases and nucleosides from Cordyceps militaris.     

原子转移自由基聚合制备PDMAEMA亲水作用色谱固定相及其性能评价

以甲基丙烯酸二甲氨乙酯为单体,2-溴异丁酰溴为引发剂,CuBr/五甲基二乙烯基三胺(PMDETA)为催化剂,通过原子转移自由基聚合(ATRP)反应,将甲基丙烯酸二甲氨乙酯(DMAEMA)接枝到5μm大孔硅胶表面上,得到了接枝聚合物(PDMAEMA)亲水作用色谱固定相.通过改变反应体系中单体的量,制备了三种不同接枝量的亲水作用色谱固定相,利用元素分析对所制备的固定相进行了表征.详细考察了该固定相的分离性能以及流动相中盐浓度、水含量对溶质保留行为的影响,并将该固定相用于宁心宝胶囊中核苷类化合物的分离和测定.在亲水模式下,该固定相可以基线分离7种核苷类化合物,保留时间随着接枝量的增加而增大,与氨基亲水作用色谱柱相比,该合成柱的分离效率高,溶质在该填料上的保留符合分配作用保留机理.实验结果表明,该填料具有良好的分离性能.     

Effect of electric field on the partitioning behavior of solutes in entropic interaction chromatography

In this study, a novel column design with a round cross‐section was proposed to be suitable for a transverse electric field (EF). Additionally, two beads for entropic interaction chromatography (EIC) were prepared by grafting glycidyl methacrylate onto Toyopearl HW‐65F (T65F) beads. Solute partitioning was then investigated to elucidate the role of graft polymerization with and without an EF. In a T65F column, solute partitioning was attributed to the distinct pore structure in the beads and was governed by pore flow. Under EF, partition coefficients (K_p) for solutes decreased with increasing EF strength. In the two EIC columns, a decrease of K_p was also observed without an EF while the fractionation windows were extended. It was more pronounced in the EIC column with a high grafting density (T65F‐H). This was explained by the decrease in the effective pore size of solutes caused by the steric hindrance of polymer chains. Under an EF, the solutes showed different partitioning behaviours in the T65F‐H column. With increasing EF strength, K_p for vitamin B₁₂ and myoglobin was decreased. In contrast, K_p for large solutes increased as a result of concentration polarization on the bead surface. Both behaviors were related to the modulation of graft polymerization to residual charge on the matrix and the pore size of the solutes.     

Inhibitory effect of hydrophilic polymer brushes on surface-induced platelet activation and adhesion

Poly(N,N-dimethylacrylamide) (PDMA) brushes are successfully grown from unplasticized poly(vinyl chloride) (uPVC) by well-controlled surface-initiated atom transfer radical polymerization (SI-ATRP). Molecular weights of the grafted PDMA brushes vary from ≈ 35,000 to 2,170000 Da, while the graft density ranges from 0.08 to 1.13 chains · nm(-2). The polydispersity of the grafted PDMA brushes is controlled within 1.20 to 1.80. Platelet activation (expression of CD62) and adhesion studies reveal that the graft densities of the PDMA brushes play an important role in controlling interfacial properties. PDMA brushes with graft densities between 0.35 and 0.50 chains · nm(-2) induce a significantly reduced platelet activation compared to unmodified uPVC. Moreover, the surface adhesion of platelets on uPVC is significantly reduced by the densely grafted PDMA brushes. PDMA brushes that have high molecular weights lead to a relatively lower platelet activation compared to low-molecular-weight brushes. However, the graft density of the brush is more important than molecular weight in controlling platelet interactions with PVC. PDMA brushes do not produce any significant platelet consumption in platelet rich plasma. Up to a seven-fold decrease in the number of platelets adhered on high graft density brushes is observed compared to the bare PVC surface. Unlike the bare PVC, platelets do not form pseudopodes or change morphology on PDMA brush-coated surfaces.     

Surface interaction of well‐defined,concentrated poly(2‐hydroxyethyl methacrylate) brushes with proteins

The interaction of concentrated polymer brushes with proteins was chromatographically investigated. By the use of surface‐initiated atom transfer radical polymerization, a low‐polydispersity poly(2‐hydroxyethyl methacrylate) (PHEMA) was densely grafted onto the inner surfaces of silica monoliths with mesopores of about 50 and 80 nm in mean size. The graft density reached 0.4–0.5 chains/nm². The 80‐nm‐mesopore monolithic column with the concentrated PHEMA brush was characterized through the elution of low‐polydispersity pullulans with different molecular weights, clearly showing two modes of size exclusion, that is, one by the mesopores and the other by the brush phase. The latter mode gave a sharp separation with a critical molecular weight (size‐exclusion limit) of about 1000. This molecular size of pullulan was comparable to the distance between the nearest‐neighbor graft points. The elution behaviors of five proteins of different sizes (bovine serum thyroglobulin, bovine serum immunoglobulin G, bovine serum albumin, horse heart myoglobin, and bovine serum aprotinin) were studied with this PHEMA‐grafted column. The smallest protein, aprotinin, with a pullulan‐reduced molecular weight slightly larger than the critical value of 1000, was eluted much behind the corresponding pullulan, and this indicated that it barely got into the brush layer, suffering from a strong affinity interaction within the brush. On the other hand, the other four larger proteins were eluted at the same elution volumes as the equivalent pullulans, and this meant that they were perfectly excluded from the brush layer and separated only in the size‐exclusion mode by the mesopores without an affinity interaction with the brush surface. This excellent inertness of the concentrated brush in the interaction with the large proteins should afford the system long‐term stability against biofouling. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 4795–4803, 2007     

A New Stationary Phase for Hydrophilic Interaction Chromatography: Polyacrylate-Based Hydrophilic,Monosized-Porous Beads with Zwitterionic Molecular Brushes

Hydrophilic, polyacrylate-based, monosized-porous beads with zwitterionic molecular brushes were synthesized as a new stationary medium for hydrophilic interaction chromatography. Monosized-porous poly(glycerol-1,3-diglycerolate diacrylate-co-glycerol dimethacrylate), poly(GDGDA-co-GDMA), beads 5 μm in size were obtained by a staged-shape template polymerization. As an initiator for surface-initiated atom transfer radical polymerization (SI-ATRP), bromine functionality was obtained on the beads by reacting their hydroxyl groups with 3-(aminopropyl)triethoxysilane and α-bromoisobutyryl bromide, respectively. Zwitterionic molecular brushes on the hydrophilic poly(GDGDA-co-GDMA) beads were generated by SI-ATRP of a sulfobetaine monomer, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl) ammonium hydroxide (MESH). Poly(MESH)-grafted poly(GDGDA-co-GDMA), poly(MESH)g-poly(GDGDAco-GDMA), beads were slurry packed into the microbore columns with 2 mm i.d. and evaluated as stationary medium for the separation of organic acids, nucleosides and peptides using microbore columns in hydrophilic interaction chromatography with the plate numbers up to 30,000 plates m⁻¹.

     

The use of multidimensional chromatography for the isolation of synthetic oligodeoxyribonucleotides on a preparative scale

Summary A critical step in the chemical preparation of oligonucleotides is the chromatographic purification of the deprotected oligomers. In case of large quantities of reaction products, the oligonucleotides are first enriched on a QAE-Sephadex column at low pressure. The obtained fractions are then purified by multidimensional chromatography making use of three independent physical properties of the solutes: molecular size, ionic net charge and hydrophobicity. In the first dimension size exclusion chromatography (Sephadex G-15) is used. In the second dimension the high molecular weight fraction from the size exclusion chromatography is applied to a HPLC ion-exchange column (Partisil-10 SAX). Usually the last peak is collected and transferred to a HPLC reversed phase column (Nucleosil C18) where the components are separated according to their hydrophobicity in the third dimension. The efficiency of this multi-dimensional chromatographic procedure is demonstrated by the unequivocal fingerprints after radioactive labelling of the isolated oligonucleotides. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Size-Exclusion Effect and Protein Repellency of Concentrated Polymer Brushes Prepared by Surface-Initiated Living Radical Polymerization

The adsorption of proteins on poly(2-hydroxyethyl methacrylate) (PHEMA) brushes was systematically investigated from the viewpoint of the size-exclusion effect of the concentrated brushes. By use of surface-initiated atom transfer radical polymerization, well-defined, concentrated PHEMA brushes were successfully grafted on the inner surface of the silica monolithic column with meso pores of ca. 80 nm as well as a silicon wafer and a quartz crystal microbalance (QCM) chip. By eluting low-polydispersity pullulans with different molecular weight through the modified monolithic column, the concentrated PHEMA brush was characterized and demonstrated to sharply exclude solute molecules with the critical molecular size (size-exclusion limit) comparable to the distance between the nearest-neighboring graft points d. The elution behaviors of proteins with different sizes were studied with this PHEMA-grafted column: the protein sufficiently larger than the critical size was perfectly excluded from the brush layer and separated only in the size-exclusion mode by the meso pores without affinity interaction with the brush surface. Then, the irreversible adsorption of proteins on PHEMA brushes was investigated using QCM by varying graft densities (σ = 0.007, 0.06, and 0.7 chains/nm²) and protein sizes (effective diameter = 2–13 nm). A good correlation between the protein size and the graft density was observed: proteins larger than d caused no significant irreversible adsorption on the PHEMA brushes. Thus, we experimentally substantiated the postulated size-exclusion effect of the concentrated brushes and confirmed that this effect plays an important role for suppressing protein adsorption.     

Mesoscale simulations of the behavior of charged polymer brushes under normal compression and lateral shear forces

Dissipative particle dynamics (DPD) was used to investigate the behavior of two opposing end-grafted charged polymer brushes in aqueous media under normal compression and lateral shear. The effect of polymer molecular weight, degree of ionization, grafting density, ionic strength, and compression on the polymer conformation and the resulting shear force between the opposing polymer layers were investigated. The simulations were carried out for the poly(tert-butyl methacrylate)-block-poly(sodium sulfonate glycidyl methacrylate) copolymer, referred as PtBMA-b-PGMAS, end-attached to a hydrophobic surface for comparison with previous experimental data. Mutual interpenetration of the opposing end-grafted chains upon compression is negligible for highly charged polymer brushes for compression ratios ranging from 2.5 to 0.25. Under electrostatic screening effects or for weakly charged polymer brushes, a significant mutual interpenetration was measured. The variation of interpenetration thickness with separation distance, grafting density, and polymer size follows the same scaling law as the one observed for two opposing grafted neutral brushes in good solvent. However, compression between two opposing charged brushes results in less interpenetration relative to neutral brushes when considering equivalent grafting density and molecular weight. The friction coefficient between two opposing polymer-coated surfaces sliding past each other is shown to be directly correlated with the interpenetration thickness and more specifically to the number of polymer segments within the interpenetration layer.     

原子转移自由基聚合法制备新型亲水开管毛细管柱及在毛细管电色谱上的应用

为了增加开管毛细管柱(OTCC)的相比,提高分离效率,发展了表面引发原子转移自由基聚合法(SI-ATRP)制备葡萄糖聚合物修饰的开管毛细管柱。通过扫描电镜观察,该开管柱内壁上修饰了三维波浪状聚合物,明显增加了内壁比表面积和相比。在pH 3~11范围内,对含糖聚合物修饰的开管柱和空柱的电渗流进行了比较。修饰后开管柱的电渗流仅为空柱的1/2~1/3,且在pH 6~11范围内保持平稳。稳定的电渗流保证了分离的重复性和稳定性。用该开管毛细管柱成功实现了小分子混合物(苯丙氨酸、胸腺嘧啶、腺苷、鸟苷、5-溴尿嘧啶、水杨酸)以及蛋白质大分子(核糖核酸酶B、转铁蛋白和牛血清白蛋白)的有效分离,结果表明葡萄糖聚合物修饰的开管毛细管柱具有良好的重复性和稳定性。     

Synthesis and characterization of graft copolymers from lignin and 2-propenamide

Free radical polymerization of 2-propenamide in the presence of lignin, anhydrous calcium chloride, and cericion in photolyzed dioxane solvent produces a reaction product containing lignin-(1-amidoethylene) graft copolymer. Up to 49 weight percent of the product is poly(1-amidoethylene) homopolymer. Photolysis products of dioxane and the presence of anhydrous calcium chloride are critical to the reaction. A maximum yield of polymer is obtained when the dioxane solvent is irradiated for 3 h in a Pyrex vessel and the reaction mixture contains 2.0 weight percent calcium chloride. Aqueous size exclusion chromatography of reaction products shows that the molecular size of the lignin-(2-propenamide) reaction product is significantly greater than that of unreacted lignin, that the side chain and the lignin backbone migrate as one unit through the column, and that mixtures of lignin and poly(1-amidoethylene) are easily separated by the chromatographic column.     

Self-assembly of catecholic macroinitiator on various substrates and surface-initiated polymerization

A catechol-containing macroinitiator has been designed for the surface-initiated atom transfer radical polymerization (SI-ATRP) from various substrates at ambient temperature. Temperature-sensitive poly(N-isopropyl acrylamide) (PNIPAM) brushes were successfully grafted from a range of substrates surfaces, including metals and polyimides, via SI-ATRP using the resulting macroinitiator, which were characterized by X-ray photoelectron spectroscopy (XPS), water contact angle measurements, and atomic force microscopy (AFM). Effects of the temperature response behavior of PNIPAM brushes on the water contact angles and the impedance of the modified surfaces were also exhibited. The self-assembled film of macroinitiator and the resulting polymer brushes were both stable to soaking of basic solvents, and the brushes did not show any exfoliation or delamination even after 2 h of ultrasonic test. The advantages of the macroinitiator in strong interactions with surfaces and high stability and convenience make it possible to modify the native materials with polymer brushes in a convenient and nondestructive way. Importantly, the macroinitiator is compatible with microcontact printing, and patterned polymer brushes on Ti plate were demonstrated by microcontact printing of BrDOPAMA and the following SI-ATRP.     

Size Exclusion Chromatography with Low Angle Laser Light Scattering Detection

Abstract

A review is given of the use of low angle laser light scattering (LALLS) detection in conjunction with size exclusion chromatography (SEC) to measure polymer molecular weight distributions without conventional SEC column calibration methods. A summary of light scattering theory is presented, and instrument configurations and principles of operation are described for two LALLS photometers. Also discussed are the overall performance of the SEC/LALLS technique and data from selected applications.     

Direct coupling of size exclusion chromatography and mass spectrometry for the characterization of complex monoclonal antibody products

The present study describes the possibilities offered by an innovative bioinert size exclusion chromatography column for size variant characterization of complex monoclonal antibody products. This size exclusion chromatography column includes a novel column hardware surface. The column was prepared from metallic hardware components that were treated to have prototype hydrophilically modified hybrid organic–inorganic silica surfaces called hybrid surface technology. This provides a significant reduction in nondesired hydrophobic and electrostatic interactions that can occur between column and analyte when performing size exclusion chromatography analysis with volatile mobile phase. Compared to a reference stainless-steel column packed with the same batch of packing material, peak tailing, band broadening, and above all recovery of high molecular weight species were distinctly improved for all types of monoclonal antibody products. Based on our observations, we found that 50 mM ammonium acetate in water was a suitable mobile phase offering good compromise in terms of liquid chromatography performance and mass spectrometry sensitivity. In addition, method repeatability (intra- and interday relative standard deviations) on elution times and high molecular weight species peak areas were found to be excellent. By using this innovative size exclusion chromatography material, the low and high molecular weight species contained in various stressed and nonstressed monoclonal antibody products were successfully characterized with mass spectrometry detection.     

Comments on exclusion of polymer chains from small pores and its relation to gel permeation chromatography.

Some criticisms of our theoretical treatment of the partial exclusion of flexible-chain polymers in solution from cavities of macromolecular size and its application to gel permeation chromatography are examined. In other discussion, it is confirmed by simple reasoning that the identification, explicit or implicit in various studies, of the mean projection of a polymer molecule onto a line as a characteristic dimension governing the extent of permeation of simple pores does not depend on specific molecular models. Our previous calculation of permeation by certain random-flight branched-chain species is shown to lead, incidentally, to the mean projection for these structures. From relations between the mean projection and the hydrodynamic volume of a molecule, it appears that the product of intrinsic viscosity and molecular weight is not a common calibration factor for elution of all molecular species from a gel chromatographic column, but theory and experience do support the validity of this correlation among solutes with similar molecular architecture.     

Enthalpic Partition-Assisted Size Exclusion Chromatography: 1. Principle of Method

A novel high performance liquid chromatographic (HPLC) method viz. “enthalpic partition assisted size exclusion chromatography” deliberately combines entropic and enthalpic partition mechanisms. It enables separation of homopolymers according to their molar mass with increased selectivity, as well as discrimination of polymer species differing in their nature/composition. Enthalpic partition of macromolecules takes place between the mobile phase and the stationary “liquid” of a different chemical nature, which is immobilized within pores of an appropriate carrier (a bonded phase). The extent of enthalpic partition depends on the accessibility of bonded phase for macromolecules and on the difference of polymer solubility in the mobile phase and in the solvated bonded phase. The enthalpic partition in favor of column packing arises from better solubility of polymer solutes in the solvated stationary phase compared to the mobile phase. Macromolecules are “pushed” into the solvated stationary phase and their retention volumes (V_R) increase. In the area of high molar masses, the extent of enthalpic partition as rule raises with the increasing size of macromolecules. However, under properly chosen experimental conditions the enthalpic partition may rapidly diminish with the sample molar mass (M), likely due to the solubility changes and/or due to partial exclusion of macromolecules from the pores. As result, the corresponding retention volumes sharply drop within a narrow range of M with the increasing size of macromolecules. This results in the log M vs. V_R dependences, which resemble in their form that for size exclusion chromatography but are much more flat indicating highly selective separations of homopolymers according to their molar masses. In this way, enthalpic partition “assists” entropic partition (size exclusion). Polymer species, which do not undergo enthalpic partition, elute from the HPLC column in the conventional size exclusion mode and can be discriminated from the partitioning species. Enthalpic partition assisted size exclusion chromatography can be utilized in separation and characterization of various homopolymers, and polymer blends.     

Determination of Morphological Properties of Swellable Solids by Size Exclusion Chromatography

Many technically interesting porous solids, e.g. ion exchangers or adsorbents for catalysis, are swellable polymers, i.e. the pore structure depends on the solvent medium. A method based on exclusion chromatography, permits determination of the pore size and pore size distribution in the swollen state.—Size exclusion chromatography, also referred to as gel permeation, gel filtration, or molecular sieve chromatography, is a widely employed method for the separation of dissolved substances—mostly polymer mixtures—according to their molecular size. Porous solids are used as stationary phase. Conversely, pore sizes and other structural data can be determined by exclusion chromatography. This application requires a series of standards (polymer samples) of known molecular weight. As a simple and rapid method, it has already proven valuable for such determinations in the case of rigid solids; in the case of swellable solids, this constitutes the sole method by which the pore structure can be characterized: classical methods require dry samples.     

Short metal capillary columns packed with polymer-coated fibrous materials in high-temperature gas chromatography

The high-temperature gas chromatographic (GC) separation of several semivolatile compounds is studied with a short metal capillary column packed with fibrous material, having a polydimethylsiloxane coating thereon. Taking advantage of the excellent heat-resistance of the fiber and also the combination of the surface-deactivated metal capillary, a temperature-programmed separation up to 450 degrees C is successfully demonstrated for the separation of polymer standard samples. The average molecular weight of the commercially-available polymer standard samples for size exclusion chromatography (SEC) is estimated by high-temperature GC analysis and compared with the nominal value determined by a conventional SEC method. Although a slight deviation for the number-average molecular weight is observed between the GC and SEC analysis, the data for the weight-average molecular weight shows a good agreement in these methods. The results also suggest the future possibility of the fiber-packed metal capillary as a miniaturized GC column with an increased sample loading capacity.     

Liquid chromatography at the critical condition: Thermodynamic significance and influence of pore size

Liquid chromatography at the critical condition (LCCC) is a high performance liquid chromatography (HPLC) technique that lies between size exclusion chromatography and adsorption-based interaction chromatography, where the elution of polymers becomes independent of polymer molecular weight. At LCCC, the balance between the entropic exclusion and the enthalpic adsorption interactions between polymers and stationary phases results in the simultaneous HPLC elution of polymers regardless of molecular weight. Using C18-bonded silica chromatographic columns with 5 μm particle size and different average pore size (diameter = 300 Å, 120 Å, 100 Å, and 50 Å), we report (1) the thermodynamic significance of LCCC conditions and (2) the influence of column pore size on the determination of critical conditions for linear polymer chains. Specifically, we used mixtures of monodisperse polystyrene samples ranging in molecular weight from 162 to 371,100 g/mol and controlled the temperature of the HPLC columns at a fixed composition of a mobile phase consisting of 57(v/v)% methylene chloride and 43(v/v)% acetonitrile. It was found that, at the fixed mobile phase composition, the temperature of LCCC (T_LCCC) is higher for C18-bonded chromatographic columns with larger average pore size. © 2009 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 47: 2533–2540, 2009     

Effect of poly(ethylene oxide)-silane graft molecular weight on the colloidal properties of iron oxide nanoparticles for biomedical applications

The size, charge, and stability of colloidal suspensions of magnetic nanoparticles with narrow size distribution and grafted with poly(ethylene glycol)-silane of different molecular weights were studied in water, biological buffers, and cell culture media. X-ray photoelectron spectroscopy provided information on the chemical nature of the nanoparticle surface, indicating the particle surfaces consisted of a mixture of amine groups and grafted polymer. The results indicate that the exposure of the amine groups on the surface decreased as the molecular weight of the polymer increased. The hydrodynamic diameters correlated with PEG graft molecular weight and were in agreement with a distributed density model for the thickness of a polymer shell end-grafted to a particle core. This indicates that the particles obtained consist of single iron oxide cores coated with a polymer brush. Particle surface charge and hydrodynamic diameter were measured as a function of pH, ionic strength, and in biological buffers and cell culture media. DLVO theory was used to analyze the particle stability considering electrostatic, magnetic, steric, and van der Waals interactions. Experimental results and colloidal stability theory indicated that stability changes from electrostatically mediated for a graft molecular weight of 750 g/mol to sterically mediated at molecular weights of 1000 g/mol and above. These results indicate that a graft molecular weight above 1000 g/mol is needed to produce particles that are stable in a wide range of pH and ionic strength, and in cell culture media.