共查询到20条相似文献,搜索用时 31 毫秒
1.
Gilar M Fountain KJ Budman Y Neue UD Yardley KR Rainville PD Russell RJ Gebler JC 《Journal of chromatography. A》2002,958(1-2):167-182
An ion-pair reversed-phase HPLC method was evaluated for the separation of synthetic oligonucleotides. Mass transfer in the stationary phase was found to be a major factor contributing to peak broadening on porous C18 stationary phases. A small sorbent particle size (2.5 microm), elevated temperature and a relatively slow flow-rate were utilized to enhance mass transfer. A short 50 mm column allows for an efficient separation up to 30mer oligonucleotides. The separation strategy consists of a shallow linear gradient of organic modifier, optimal initial gradient strength, and the use of an ion-pairing buffer. The triethylammonium acetate ion-pairing mobile phases have been traditionally used for oligonucleotide separations with good result. However, the oligonucleotide retention is affected by its nucleotide composition. We developed a mathematical model for the prediction of oligonucleotide retention from sequence and length. We used the model successfully to select the optimal initial gradient strength for fast HPLC purification of synthetic oligonucleotides. We also utilized ion-pairing mobile phases comprised of triethylamine (TEA) buffered by hexafluoroisopropanol (HFIP). The TEA-HFIP aqueous buffers are useful for a highly efficient and less sequence-dependent separation of heterooligonucleotides. 相似文献
2.
A DNA-silica, (dT)18-silica, was prepared and used in a study of the chromatography of the oligonucleotide, (dA)18, based upon base pairing. It was shown that hybridization efficiency did not depend upon flow-rates up to 2 ml/min for the small columns (22 × 2 mm) used. As increasing amounts of (dA)18 were loaded onto the columns, the columns were found to saturate at a well defined capacity that was always less than the amount that theoretically could have been bound. Maximum capacity was achieved whenever the loading temperature was at least 20–25°C below the temperature at which the loaded oligonucleotide would elute. The effects of porosity on both coupling efficiency and capacity were measured and suggest that pore sizes in the 300–500 Å range are most appropriate for this form of chromatography. 相似文献
3.
A novel continuous bed with high dynamic adsorption capacity for protein has been developed. It is a macroporous poly(glycidyl methacrylate-divinylbenzene-triallylisocyanurate) rod prepared by in situ copolymerization in a chromatographic tube. The bed matrix contained epoxy groups, so diethylaminohydroxypropyl groups were coupled to the matrix, leading to an anion-exchange continuous bed. The component, specific surface area, and the pore structure of the bed matrix were characterized by Fourier transform infrared spectroscopy, BET method and scanning and transmission electron microscopies, respectively. The flow properties, column efficiency and the dynamic adsorption behavior of the bed were studied. The results show that the continuous bed, a ternary copolymer of glycidyl methacrylate (GMA), divinylbenzene (DVB) and triallylisocyanurate (TAIC) with a specific surface area of 56.4 m2/g, consisted of a three-dimensional structure made up of continuous clusters of microspheres (300 nm) and interconnected irregular pores. The rate of mass transfer is enhanced by the convection of the mobile phase through the pores. The dynamic adsorption isotherm of the anion-exchange column for bovine serum albumin was expressed by the Langmuir equation with a dynamic capacity as high as 76.0 mg/g. Moreover, the separation of proteins, i.e. lysozyme, hemoglobin and bovine serum albumin, is little affected by mobile-phase velocity up to 902.5 cm/h; it was completed within 5 min at 902.5 cm/h. 相似文献
4.
David S. Hart Chithkala Harinarayan Gunnar Malmquist Andreas Axén Mandakini Sharma Robert van Reis 《Journal of chromatography. A》2009,1216(20):4372-4376
Increased recombinant protein expression yields and a large installed base of manufacturing facilities designed for smaller bulk sizes has led to the need for high capacity chromatographic resins. This work explores the impact of three pore sizes (with dextran distribution coefficients of 0.4, 0.53, and 0.64), dextran surface extender concentration (11–20 mg/mL), and ligand density (77–138 μmol H+/mL resin) of cation exchange resins on the dynamic binding capacity of a therapeutic antibody. An intermediate optimal pore size was identified from three pore sizes examined. Increasing ligand density was shown to increase the critical ionic strength, while increasing dextran content increased dynamic binding capacity mainly at the optimal pore size and lower conductivities. Dynamic binding capacity as high as 200 mg/mL was obtained at the optimum pore size and dextran content. 相似文献
5.
A high-performance liquid chromatographic (HPLC) procedure has been evaluated to establish a routine test in the clinical laboratory for measuring the profiles of estrogen and progestin receptor isoforms in human breast and endometrial tumors. This procedure will be used to determine if there is a relationship between particular isoform profiles and response to various endocrine therapies. Evaluation of various HPLC modes has shown that high-performance ion-exchange chromatography (HPIEC) with silica-based anion exchangers offers a promising approach. In this paper, we have compared HPIEC columns of different lengths (10 and 25 cm) and pore sizes (300, 500 and 1,000 A) in order to obtain an optimal separation procedure. Because of receptor lability, all investigations were performed at 4 degrees C. The mobile phase consisted of 10-500 mM phosphate buffer, supplemented with the stabilizing agent, sodium molybdate at pH 7.4. Recoveries from each of the columns were between 70-100%. The length of the column did not influence significantly the retention time and salt concentration required for elution of receptor proteins. However, pore sizes appeared to alter these parameters. With a larger pore size (1,000 A), the retention of proteins was lower (elution with 50 mM phosphate) than that observed with the 500-A pore size column (elution with 100 mM phosphate) or of the 300-A pore size column (elution with 150 mM phosphate). Based solely on recovery patterns and peak shape, we conclude that separation of receptor isoforms on a 1,000-A, 25-cm column is best suited for clinical analysis. 相似文献
6.
Hu X Skadtchenko BO Trudeau M Antonelli DM 《Journal of the American Chemical Society》2006,128(36):11740-11741
Chemically reducible micro- and mesoporous Ti oxides with controlled pore sizes from 12 to 26 A were synthesized. The hydrogen storage and adsorption capacity at 77 K was tested as a function of surface area, pore size, and reducing agent. Surprisingly, the oxidation state of the surface Ti species had an even greater effect on the storage densities than surface area or pore size. For example, the 12 A material reduced with bis(toluene) Ti possesses a surface area of less than 300 m2/g, but absorbs up to 4.94 wt % and 40.46 kg/m3 of H2 reversibly at 77 K and 100 atm. This volumetric storage capacity is higher than that of AX-21, which has a much higher surface area. The H2 binding enthalpies increased from 4.21 kJ/mol to 8.08 kJ/mol as the surface oxidation state of the Ti decreased. These results suggest that a Kubas-type sigma H2 complex may be involved and that further tuning of the H2 binding enthalpies through use of appropriate organometallic reagents may achieve even higher storage levels at more moderate temperature. 相似文献
7.
Chen B Miller ME Gross RA 《Langmuir : the ACS journal of surfaces and colloids》2007,23(11):6467-6474
Polystyrene resins with varied particle sizes (35 to 350-600 microm) and pore diameters (300-1000 A) were employed to study the effects of immobilization resin particle size and pore diameter on Candida antarctica Lipase B (CALB) loading, distribution within resins, fraction of active sites, and catalytic properties for polyester synthesis. CALB adsorbed rapidly (saturation time = 4 min) for particle sizes = 120 microm (pore size = 300 A). Infrared microspectroscopy showed that CALB forms protein loading fronts regardless of resin particle size at similar enzyme loadings ( approximately 8%). From the IR images, the fractions of total surface area available to the enzyme are 21, 33, 35, 37, and 88% for particle sizes 350-600, 120, 75, 35 microm (pore size 300 A), and 35 microm (pore size 1000 A), respectively. Titration with methyl p-nitrophenyl n-hexylphosphate (MNPHP) showed that the fraction of active CALB molecules adsorbed onto resins was approximately 60%. The fraction of active CALB molecules was invariable as a function of resin particle and pore size. At approximately 8% (w/w) CALB loading, by increasing the immobilization support pore diameter from 300 to 1000 A, the turnover frequency (TOF) of epsilon-caprolactone (epsilon-CL) to polyester increased from 12.4 to 28.2 s-1. However, the epsilon-CL conversion rate was not influenced by changes in resin particle size. Similar trends were observed for condensation polymerizations between 1,8-octanediol and adipic acid. The results herein are compared to those obtained with a similar series of methyl methacrylate resins, where variations in particle size largely affected CALB distribution within resins and catalyst activity for polyester synthesis. 相似文献
8.
The influence of the pore size of a chromatographic reversed phase material on the adsorption equilibria and diffusion of two industrially relevant peptides (i.e. a small synthetic peptide and insulin) has been studied using seven different reversed phase HPLC materials having pore sizes ranging from 90 Å to 300 Å. The stationary phase pore size distribution was obtained by inverse size exclusion measurement (iSEC). The effect of the pore size on the mass transfer properties of the materials was evaluated from Van Deemter experiments. It has been shown that the lumped mass transfer coefficient increases linearly with the average pore size. The Henry coefficient and the impurity selectivity were determined in diluted conditions. The saturation capacity of the main peptides was determined in overloaded conditions using the inverse method (i.e. peak fitting). It was shown that the adsorption equilibria of the peptides on the seven materials is well described by a surface-specific adsorption isotherm. Based on this a lumped kinetic model has been developed to model the elution profile of the two peptides in overloaded conditions and to simulate the purification of the peptide from its crude mixture. It has been found that the separation of insulin from its main impurity (i.e. desamido-insulin) was not affected by the pore size. On the other hand, in the case of the synthetic peptide, it was found that the adsorption of the most significant impurity decreases with the pore size. This decrease is probably due to an increase in silanol activity with decreasing pore size. 相似文献
9.
In process-scale antibody purification, protein-A affinity chromatography is commonly used as the initial purification step. In this paper, two different protein-A media were evaluated. These adsorbents have a porous glass backbone with different pore sizes: 700 A and 1000 A. Adsorption equilibrium data of human immunoglobulins on these media were measured via a batch technique and correlated using the Langmuir isotherm model. A larger static capacity was found for the smaller pore size material, which is probably a result of the larger specific surface area and associated higher ligand concentration. The protein uptake kinetics were also obtained via a stirred tank experiment using different initial protein concentrations. A surface layer model was used to represent the protein uptake by the media and to estimate values of a concentration-independent effective diffusivity within the particle. Experimental breakthrough curves were also obtained from packed beds operated under different conditions. Calculated breakthrough profiles were found to be in good agreement with the experimental results. Experimental breakthrough data were used to determine the dependence of the dynamic capacity of the media as a function of the fluid residence time. A larger dynamic capacity was also found for the smaller pore size media. The permeability of large scale packed beds was also reported and used in conjunction with the dynamic capacity to calculate the process production rate. 相似文献
10.
《液相色谱法及相关技术杂志》2012,35(5):839-851
Abstract The relationship between the textural properties (pore size, pore volume and surface area) of reversed-phase silica gel packings for HPLC and the dynamic loading capacity of large biomolecules was studied by using silica gels manufactured by similar processes. Several silica gels whose unbonded pore diameters range from 100 to 250 A and whose pore volumes range from 1.0 to 1.4 ml/g have been prepared and characterized. The bonded phase is monomeric C18. The textural properties of the bonded silica gels are also presented and related to the properties of the unbonded silica gels. Chromatographic evaluation with typical proteins in an underload-to-overload condition was performed in order to relate the influence of textural properties of silica gel to loading capacity and resolution. The packings with larger pore size and pore volume produced better column performance and higher loading of proteins. 相似文献
11.
Biologically important arsenic species such as arsenobetaine, arsenocholine iodide, tetramethylarsonium iodide, methylarsonic acid, and dimethylarsinic acid can be separated and quantitated by HPLC. The pH-sensitive separations on a weak anion-exchange column are described, as well as separations on a reverse-phase column with the aid of tetrabutylammonium nitrate or heptanesulfonic acid as ion-pairing agents. The thermospray mass spectra of these arsenicals in addition to those of sodium arsenate and an arsenosugar derivative are described. This technique is suitable for HPLC MS studies. 相似文献
12.
Rigid macroporous copolymers of styrene and divinylbenzene have been designed for large-scale preparative and process-scale purification of synthetic peptides and recombinant proteins. The polymeric particles are mechanically stable and hence able to operate at the required high linear velocities. The pore size and pore morphology has been optimised to enable unhindered solute diffusion whilst providing maximum available surface area to enhance loading capacity. A 100 A pore size has been developed for synthetic peptides and a 300 A pore size for recombinant proteins. Precise control of particle size, within the range 10 to 20 microm, is possible which together with the very narrow particle size distribution enables maximum resolution/loading to be obtained within the pressure limits of the instrumentation being used. The chemical stability of the polymer enables cleaning in place with 1 M sodium hydroxide without particle dissolution or a deterioration in selectivity. These materials can be packed into compression hardware and are manufactured as single lots up to 100 kg (300 l) batch size. 相似文献
13.
We report the successful size-based separations of large, neutral polysaccharides using capillary electrochromatography (CEC). As the polysaccharides possessed little chromophore for photometric detection, two separate approaches were taken. In the first approach, indirect detection was combined with size-exclusion chromatography using a sulfonated polystyrene/divinylbenzene stationary phase. The separations were performed using a 300 A pore size stationary phase under aqueous conditions. Non-size based interactions were minimal using this material, resulting in an effective calibration range of molecular masses 180 to 112 000 g.mol(-1) for pullulans. In the second approach, the polysaccharides were derivatized with phenylisocyanate and were subsequently separated on columns made using a combination of high capacity ion-exchanger and a neutral polystyrene/divinylbenzene material of various pore sizes. The sulfonated ion-exchange phase provided the electroosmotic flow, while the mixed pore size material provided the extended calibration range. The linear range for this primarily nonaqueous system using tetrahydrofuran was determined to be from molecular masses 738 to 404 000 g.mol(-1) of the original, untagged pullulan. This approach overcame the limited solubility issue associated with analysis of some polysaccharides. Analysis of pullulan and amylose samples by CEC correlated well with results obtained by conventional high-performance liquid chromatography (HPLC). The size-exclusion electrochromatographic separations provide an alternative mode for determining the relative molecular weights of polysaccharides with reduced sample and solvent consumption, as well as analysis times. 相似文献
14.
A rapid method for isolation of human hemoglobin benzo[a]pyrene diol epoxide derived adducts using high performance liquid chromatography 总被引:1,自引:0,他引:1
S Naylor L S Gan B W Day P L Skipper S R Tannenbaum 《Biomedical chromatography : BMC》1989,3(6):266-268
A method is described to isolate rapidly human hemoglobin-benzo[a]pyrene diol epoxide adducts. A combination of 300 A pore size C4 reversed phase HPLC to effect separation of adducted protein from native protein, and mu-bore C18 reversed phase HPLC to isolate and partially characterize proteolytic peptide adducts (by UV), was used. 相似文献
15.
16.
Bakry R Stöggl WM Hochleitner EO Stecher G Huck CW Bonn GK 《Journal of chromatography. A》2006,1132(1-2):183-189
In the paper we demonstrate a new approach for the preparation and application of continuous silica bed columns that involve encapsulation (entrapment) of functionalized silica microparticles, which can be used as packing material in micro high performance liquid chromatography (micro-HPLC) and capillary electrochromatography (CEC). Like traditional packed columns, these capillaries possess characterized silica particles that offer high phase ratio and narrow pore size distribution leading to high retention and separation efficiency, respectively. More importantly, immobilization of the microparticles stabilizes the separation bed and eliminates the need for retaining frits. The developed capillary columns were fabricated in exactly the same way as a packed capillary column (slurry packing) but with an additional entrapment step. This immobilization of the packed bed was achieved by in situ polymerization of styrene and divinylbenzene in presence of decanol as a porogen and azobisisobutyronitrile as thermal initiator. Silica particles with different particle sizes and pore sizes ranging from 60 to 4000 A were studied. In addition different modified silica was used, including C-18 reversed phase, anion exchange and chiral stationary phases. Efficient separation of polyphenolic compounds, peptides, proteins and even DNA mutation were achieved using the developed technique depending on the properties of the silica particles used (particles pore size). For example, using 3 microm ProntoSIL C-18 particles with 300 A pore size, separation efficiencies in the range of 120,000-200,000 plates/m were obtained for protein separation, in a 6 cm x 200 microm i.d. capillary column. Using encapsulated silica C-18 with 1000 A pore size, separation of DNA homo and hetero duplexes were achieved under denaturing HPLC conditions for mutation detection. In addition, nucleotides were separated using anion exchange material encapsulated with poly(styrene-divinylbenzene) (PS/DVB), which indicated that the chromatographic properties of the silica packing material were still active after polymerization. The prepared capillary columns were found to be stable and could easily be operated continuously up to a pressure of 350 bar without column damage and capillary can be cut to any desired length. 相似文献
17.
Romila Manchanda SunilK. Agarwal Pradeep Kumar AshwaniK. Sharma KailashC. Gupta Ramesh Chandra 《Helvetica chimica acta》2002,85(9):2754-2762
A simple protocol based on polymerization reactions has been developed for the preparation of high‐loading polymer supports, useful for large‐scale synthesis of oligonucleotides. Polymer supports of different pore sizes have been employed in the present investigation to improve the functional‐group density on them. A ten‐ to twelvefold increase in the loading of the functional groups, after the polymerization reaction, has been observed. The support was then used in the subsequent reaction to attach the leader nucleoside to obtain fully functionalized supports 6a – 6c by oligonucleotide synthesis in an automated DNA synthesizer. The aminoalkylated‐supports 5a – 5c were directly employed for the synthesis of oligonucleotide 3′‐phosphates. The oligonucleotides and oligonucleotide 3′‐phosphates synthesized on these supports were compared with the corresponding standard oligomers with respect to their retention time on HPLC. These were further characterized on MALDI‐TOF mass spectrometry. 相似文献
18.
Poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate) monolithic supports were prepared by radical polymerisation of the continuous phase of water in oil high internal phase emulsions. Morphology of monolithic materials was studied by scanning electron microscopy and mercury intrusion porosimetry. The ratio of phase volume and the degree of crosslinking influenced the void size and pore size distribution of resulting polymers. Void sizes between 1 and 10 microm were observed and average pore sizes around 100 nm. Polymers with 60, 75, 80 and 90% pore volume were prepared and even samples with highest pore volume showed good mechanical stability. They were modified to bear weak-anion exchange groups and tested on the separation of standard protein mixture containing myoglobin, conalbumine and trypsin inhibitor. Good separation was obtained in a very short time similar to the separation obtained by commercial methacrylate monoliths. However, higher dispersion was observed. Bovine serum albumin dynamic binding capacity for monolith with 90% porosity was close to 9 mg/ml. 相似文献
19.
P. Morin M. B. Amran S. Favier R. Heimburger M. Leroy 《Fresenius' Journal of Analytical Chemistry》1991,339(7):504-509
Summary Speciation and quantitative analysis of arsenical compounds are performed by using high-performance liquid chromatography (HPLC) with direct UV detection. Ion chromatography has been used to separate mixtures of arsenical compounds (arsenite, MMA, DMA, arsenate) on an anion-exchange column using phosphate buffer (1 mmol/l, pH=5.3) as eluent. Ion -pair reversed-phase chromatography has been investigated to resolve mixtures of arsenite, arsenate, MMA, DMA, arsenobetaine and arsenocholine on an octadecyl-bonded silica column using water as mobile phase (pH=7.3) and tetrabutylammonium cation as ion-pairing reagent. The influence of several parameters (pH, the ion-pairing reagent concentration or the amount of methanol in the mobile phase) has been studied to determine the best chromatographic conditions. 相似文献
20.
This work investigates the influence of temperature on the binding capacity of bovine serum albumin (BSA), soybean trypsin inhibitor and L-glutamic acid to a CIM (DEAE) weak anion-exchange disk monolithic column. The binding capacity was determined experimentally under dynamic conditions using frontal analysis. The effect on the dynamic binding capacity of dimers present in the BSA solution has been evaluated and a closed-loop frontal analysis was used to determine the equilibrium binding capacities. The binding capacity for both BSA and soybean trypsin inhibitor increased with increasing temperature. In the case of L-glutamic acid, an increase in the binding capacity was observed with temperature up to 20 degrees C. A further increase in temperature caused a decrease of the dynamic binding capacity. 相似文献