The influence of particle size distribution and operating conditions on the adsorption performance in fluidized beds

The influence of matrix properties and operating conditions on the performance in fluidized-bed adsorption has been studied using Streamline diethyl-aminoethyl (DEAE), an ion exchange matrix based on quartz-weighted agarose, and bovine serum albumin (BSA) as a model protein. Three different particle size fractions (120-160 mum, 120-300 mum, and 250-300 mum) were investigated. Dispersion in the liquid phase was reduced when particles with a wide size distribution were fluidized compared to narrow particle size distributions. When the mean particle diameter was reduced, the breakthrough capacities during frontal adsorption were enlarged due to a shorter diffusion path length within the matrix. At small particle diameters the effect of film mass transfer became more relevant to the adsorption performance in comparison to larger particles. Therefore matrices designed for fluidized-bed adsorption should have small particle diameter and increased mean particle density to ensure small diffusion path length in the particle and a high interstitial velocity to improve film mass transfer. Studies on the influence of sedimented matrix height on axial mixing showed an increased Bodenstein number with increasing bed length. Higher breakthrough capacities were also found for longer adsorbent beds due to reduced dispersion and improved fluid and particle side mass transfer. With increasing bed height the influence of flow rate on breakthrough capacity was reduced. For a settled bed height of 50 cm breakthrough capacities of 80% of the equilibrium capacity for flow rates varying from 3 to 9 cm/min could be achieved. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 54-64, 1997.     

干湿交替对红河干旱河谷区土壤优先流形成特征的影响

为明确干旱河谷气候区干湿交替作用对土壤优先流形成的影响,本研究以红河干旱河谷区的荒草地为对象,通过模拟干湿交替的方法,基于染色示踪和水分穿透曲线试验并利用图像处理技术,对比分析模拟前后土壤优先流特征的差异性规律。结果表明: 模拟干湿交替条件下基质流发生区在0~10 cm土层,染色深度高达35 cm,其优先路径的水平宽度仅为3～10 cm,且染色面积曲线波动小。模拟干湿交替条件导致土壤稳定出流速率、大孔隙数量和大孔隙率明显增加,在0~20 cm土层,实施干湿交替后的土壤稳定出流速率较非干湿交替条件高约0.27 cm³·s^-1,染色区的大孔隙数量增加约1.4倍,大孔隙率则高13.4%。大孔隙数量与稳定出流速率呈极显著正相关,模拟干湿交替后大孔隙数量从大到小依次为: 0.6～0.8 mm＞0.8～1.0 mm＞1.0～1.5 mm＞1.5～2.0 mm＞2.0～3.7 mm,非干湿交替条件下为: 0.8～1.0 mm＞0.6～0.8 mm＞1.0～1.5 mm＞2.0～3.7 mm＞1.5～2.0 mm。各孔径范围的大孔隙数量与染色面积比呈极显著相关关系,经过模拟干湿交替处理后,其相关性增大,且影响优先流发生的主导因素由孔径1.5～2.0 mm的大孔隙数量变为孔径0.8～1.0 mm的大孔隙数量。干湿交替作用会通过影响大孔隙特征进而导致土壤更易发生优先流且程度增强。     

Dye-ligand poly(GMA–TAIC–DVB) affinity adsorbent for protein adsorption

Macroporous poly(glycidyl methacrylate-triallyl isocyanurate-divinylbenzene) was prepared by a radical suspension copolymerization. Reaction of the copolymer with 2-hydroxyethyl amine was employed to obtain a hydrophilic matrix. An affinity dye, Cibacron blue 3GA, was then coupled covalently to prepare a novel macroporous affinity adsorbent. The surface and pore structure of the affinity adsorbent were examined by scanning electron micrography (SEM). SEM observations showed that the affinity adsorbent abounded in macropores. Bovine serum albumin (BSA) and lysozyme (Lys) were used as samples to examine the adsorption properties of the adsorbent. Under appropriate conditions, the affinity adsorbent had a capacity of 15.5 mg BSA/g and 22.3 mg Lys/g (wet adsorbent weight). The adsorbed proteins could be desorbed by increasing liquid phase ionic strength or by using a NaOH solution, and the adsorbent could be recycled for protein adsorption.     

HPLC resolution of diacylglycerol moieties of natural triacylglycerols on a chiral phase consisting of bonded (R)-(+)-1-(1-naphthyl)ethylamine

Chiral phase high performance liquid chromatographic resolution of sn-1,2(2,3)- and X-1,3-diacylglycerols generated by partial Grignard degradation from natural triacylglycerols was carried out using a chiral column (25 cm x 4.6 mm i.d.) containing (R)-(+)-1-(1-napthyl)ethylamine polymer chemically bonded to 300A wide pore spherical silica (5 microns particles). The diacylglycerols were chromatographed as 3,5-dinitrophenyl-urethanes and detected at 226 or 254 nm UV. By an isocratic elution with n-hexane- 1,2-dichloroethane-ethanol 40:10:1 (v/v/v) as the mobile phase, the sn-1,2(2,3)-diacylglycerols from corn, linseed, and menhaden oils were resolved into two clearly distinguishable enantiomer groups, although some peak overlappings between the enantiomers were observed in the linseed and menhaden oil diacylglycerols. In addition to the excellent enantiomer resolution, each enantiomer and the X-1,3-isomers were partially resolved into several peaks, which could be tentatively identified on the basis of equivalent carbon number. It is concluded that chiral phase high performance liquid chromatography can be utilized for effective resolution, identification, and quantitation of enantiomeric diacylglycerols from complex natural mixtures.     

芦荟苷A、B以及异芦荟色苷D的同时分离纯化

首次采用反相中压制备色谱从芦荟中一次分离制备得到芦荟苷A、B以及异芦荟色苷D。以库拉索芦荟丙酮粗提物为原料,采用中压制备色谱系统:SCO色谱柱(40 cm×26 cm,30～50μm),流动相甲醇-0.5%乙酸水(33∶67,V/V),流速20 mL/min,等度洗脱方式,柱温室温,检测波长254 nm,收集波长356 nm对芦荟样品进行分离制备,得到三种化合物单体。经高效液相色谱、紫外、红外、质谱及核磁共振等方法分析表明所得到的三种化合物分别是异芦荟色苷D、芦荟苷A和芦荟苷B,其纯度分别达到了98.0%,96.0%和98.9%。该方法简便,产品质量高,一次制备可以得到多种单体,为芦荟成分的测定与药理活性的研究提供了条件。     

A novel matrix derivatized from hydrophilic gigaporous polystyrene-based microspheres for high-speed immobilized-metal affinity chromatography

Agarose coated gigaporous polystyrene microspheres were evaluated as a novel matrix for immobilized-metal affinity chromatography (IMAC). With four steps, nickel ions were successfully immobilized on the microspheres. The gigaporous structure and chromatographic properties of IMAC medium were characterized. A column packed with the matrix showed low column backpressure and high column efficiency at high flow velocity. Furthermore, this matrix was used for purifying superoxide dismutase (SOD), which was expressed in Escherichia coli (E. coli) in submerged fermentation, on an Äkta purifier 100 system under different flow velocities. The purity of the SOD from this one-step purification was 79% and the recovery yield was about 89.6% under the superficial flow velocity of 3251 cm/h. In conclusion, all the results suggested that the gigaporous matrix has considerable advantages for high-speed immobilized-metal affinity chromatography.     

Preparation and characterization of long alkyl chain methacrylate-based monolithic column for capillary chromatography

This paper describes the fabrication of long alkyl chain methacrylate monolithic materials for using as stationary phases in capillary liquid chromatography. Following deactivation of the capillary surface with 3-(trimethoxysilyl)propyl methacrylate (gamma-MAPS), monoliths were formed by co-polymerisation of stearyl methacrylate (SMA) with ethylene glycol dimethacrylate (EDMA) in the presence of the initiator AIBN and a mixture of porogens including iso-amyl alcohol and 1,4-butanediol. The monoliths were prepared in 100 microm i.d. capillaries and the composition of the polymerisation mixtures were optimised in terms of the ratio of SMA/EDMA, the porogen composition and ratio of porogen to monomers. As the porogen weight fraction decreased, the microglobules became smaller and as expected, the total porosity decreased. In order to determine the usability of such materials, the column permeability K was measured by pumping water through the columns at different linear flow rates. Good results were obtained when these capillaries were used to separate mixtures of weak acids, neutral and basic compounds.     

Preparation and characterization of long alkyl chain methacrylate-based monolithic column for capillary chromatography

This paper describes the fabrication of long alkyl chain methacrylate monolithic materials for using as stationary phases in capillary liquid chromatography. Following deactivation of the capillary surface with 3-(trimethoxysilyl)propyl methacrylate (γ-MAPS), monoliths were formed by co-polymerisation of stearyl methacrylate (SMA) with ethylene glycol dimethacrylate (EDMA) in the presence of the initiator AIBN and a mixture of porogens including iso-amyl alcohol and 1,4-butanediol. The monoliths were prepared in 100 μm i.d. capillaries and the composition of the polymerisation mixtures were optimised in terms of the ratio of SMA/EDMA, the porogen composition and ratio of porogen to monomers. As the porogen weight fraction decreased, the microglobules became smaller and as expected, the total porosity decreased. In order to determine the usability of such materials, the column permeability K was measured by pumping water through the columns at different linear flow rates. Good results were obtained when these capillaries were used to separate mixtures of weak acids, neutral and basic compounds.     

拟南芥中4种细胞分裂素的高效液相色谱法测定

应用高效液相色谱法同时测定拟南芥中4种细胞分裂素组分玉米素(Z)、玉米素核苷(ZR)、6-r,r-二甲基烯丙基氨基嘌呤(2ip)和6-r,r-二甲基烯丙基氨基嘌呤核苷(2ipr)含量。结果表明,采用反相色谱柱Waters C₁₈柱(4.6×250 mm,5 μm),在35℃以乙腈和三乙胺缓冲液为流动相梯度洗脱,流速1 ml/min,在270 nm处能准确检测出拟南芥中4种细胞分裂素组分的含量,检测限达0.001 μg/ml。     

Quantitation of itopride in human serum by high-performance liquid chromatography with fluorescence detection and its application to a bioequivalence study

A new method was developed for determination of itopride in human serum by reversed phase high-performance liquid chromatography (HPLC) with fluorescence detection (excitation at 291 nm and emission at 342 nm). The method employed one-step extraction of itopride from serum matrix with a mixture of tert-butyl methyl ether and dichloromethane (70:30, v/v) using etoricoxib as an internal standard. Chromatographic separation was obtained within 12.0 min using a reverse phase YMC-Pack AM ODS column (250 mm x 4.6 mm, 5 microm) and an isocratic mobile phase constituting of a mixture of 0.05% tri-fluoro acetic acid in water and acetonitrile (75:25, v/v) flowing at a flow rate of 1.0 ml/min. The method was linear in the range of 14.0 ng/ml to 1000.0 ng/ml. The lower limit of quantitation (LLOQ) was 14.0 ng/ml. Average recovery of itopride and the internal standard from the biological matrix was more than 66.04 and 64.57%, respectively. The inter-day accuracy of the drug containing serum samples was more than 97.81% with a precision of 2.31-3.68%. The intra-day accuracy was 96.91% or more with a precision of 5.17-9.50%. Serum samples containing itopride were stable for 180.0 days at -70+/-5 degrees C and for 24.0 h at ambient temperature (25+/-5 degrees C). The method was successfully applied to the bioequivalence study of itopride in healthy, male human subjects.     

Determination of tianeptine in human plasma using high-performance liquid chromatography with fluorescence detection

A new, selective and sensitive high-performance liquid chromatography (HPLC) method with fluorimetric detection was developed for the determination of tianeptine (TIA) in human plasma using solid phase extraction (SPE) procedures. The method is based on the derivatization of TIA with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in borate buffer of pH 8.5 to yield a yellow, fluorescent product. The HPLC separation was achieved on a Phenomenex C(18) column (250 mm x 4.6 mm) using a mobile phase of acetonitrile-10mM orthophosphoric acid (pH 2.5) (77:23, v/v) solvent system at 1 mL/min flow rate. Gabapentin (GA) was used as the internal standard. The fluorometric detector was operated at 458 nm (excitation) and 520 nm (emission). The assay was linear over the concentration range of 5-300 ng/mL. The detection limit (LOD) was found to be 2 ng/mL. The mean recovery was determined to be 88.6%. The proposed method was applied for pharmacokinetic study of 12.5mg TIA in a healthy volunteer.     

Expanded bed protein A affinity chromatography of a recombinant humanized monoclonal antibody: process development, operation, and comparison with a packed bed method.

We show that expanded bed protein A affinity chromatography using Streamline rProtein A media is an efficient method for purifying a recombinant humanized monoclonal antibody from unclarified Chinese hamster ovary cell culture fluid and that it provides purification performance comparable to using a packed bed. We determined that the dynamic capacity of the expanded bed media is related to flow rate (measured in column volumes per hour) by a power function, which allows a high capacity at a low flow rate. At 250 cm h-1 with a 25 cm bed height (10 column volumes h-1), the dynamic capacity is 30 g l-1. The yield and purity (measured by the amount of host cell proteins, DNA, SDS-PAGE, and turbidity) of the antibody purified by expanded bed is comparable to the yield and purity obtained on a standard packed bed method using Prosep A media.     

Molecularly imprinted solid-phase extraction for the selective determination of valnemulin in feeds with high performance liquid chromatography

A simple, sensitive and reproducible high performance liquid chromatographic method was developed for determining valnemulin in feeds. Feed samples were extracted with ethyl acetate under alkaline condition, cleaned up by molecularly imprinted solid-phase extraction, and analyzed by high performance liquid chromatography with ultraviolet detection. The characteristics of the synthesized polymer were evaluated and the loading capacity of the polymer was about 1000 μg analyte/g imprinted polymer. The new procedure for the feed sample cleanup using the prepared polymer cartridge gave higher recoveries and fewer matrix interferences. The assay exhibited a linear dynamic range of 5.0-200 mg kg(-1) with the correlation coefficient above 0.9993. Recoveries of valnemulin from feed samples spiked at 5.0, 20 and 50 mg kg(-1) ranged between 76.0% and 94.4% with relative standard deviations of less than 9%. The limit of detection for valnemulin in feeds was 1 mg kg(-1).     

Incorporation of tandem mass spectrometric detection to the analysis of peptide mixtures by continuous flow fast atom bombardment mass spectrometry

The ability to acquire structurally informative daughter ion spectra for individual peptides undergoing separation and analysis by continuous flow fast atom bombardment (CF FAB) is demonstrated. To illustrate the potential of this methodology, tryptic and chymotryptic digests of the 29-residue peptide glucagon were analyzed by CF FAB using mass spectrometric and tandem mass spectrometric detection in consecutive analyses. Daughter ion spectra were recorded using B/E linked scans for the major hydrolysis products observed by liquid chromatography/mass spectrometry. The peptide mixtures were separated by gradient capillary high-performance liquid chromatography with the FAB matrix being added post-column using a coaxial flow interface between the column and flow probe. The entire effluent (3 microl min(-1)) was sampled by the mass spectrometer. Results obtained using less than 300 pmol of digested glucagon indicated several advantages to tandem mass spectrometric detection including the ability to confirm identities for products of enzymatic digestion and the potential use of this method for tandem sequence analysis of peptide mixtures.     

Optimization and validation of a high-performance liquid chromatographic method with UV detection for the determination of pyrrole-imidazole polyamides in rat plasma

A simple and sensitive high-performance liquid chromatography (HPLC) method utilizing UV detection was developed for the determination of plasma pyrrole (Py)-imidazole (Im) polyamides in rats and applied to the pharmacokinetic study of compounds. After deproteinization of plasma with methanol, Py-Im polyamides were analyzed with a reversed-phase TSK-GEL ODS-80TM (4.6 mmx15.0 cm TOSOH Co., Japan) column maintained at 40 degrees C. The mobile phase solvent A was 0.1% acetic acid and the solvent B was HPLC-grade acetonitrile (0-10 min, A: 100-20%, B: 0-80% linear gradient; 10-15 min, A: 40%, B: 60%). The flow rate was 1.0 ml/min. The detection wavelength was set at 310 nm. The method was used to determine the plasma concentration time profiles of Py-Im polyamides after intravenous injection.     

Chiral analysis of butaclamol enantiomers in human plasma by HPLC using a macrocyclic antibiotic (vancomycin) chiral stationary phase and solid phase extraction

An enantioseparation of the antipsychotic drug butaclamol in human plasma by high-performance liquid chromatography (HPLC) with solid phase extraction is presented. The separation was achieved on the vancomycin macrocyclic antibiotic chiral stationary phase (CSP) Chirobiotic V with a polar ionic mobile phase (PIM) consisting of methanol : glacial acetic acid : triethylamine (100:0.2:0.05, v/v/v) at a flow rate of 0.5 ml/min. The detection wavelength was 262 nm. Bond Elut C18 solid phase extraction cartridges were used in the sample preparation of butaclamol samples from plasma. The method was validated over the range of 100-3,000 ng/ml for each enantiomer concentration (R(2) > 0.999). Recoveries for (+)- and (-)-butaclamol were in the range of 94-104% at the 300-2,500 ng/ml level. The method proved to be precise (within-run precision ranged from 1.1-2.6% and between-run precision ranged from 1.9-3.2%) and accurate (within-run accuracies ranged from 1.5-5.8% and between-run accuracies ranged from 2.7-7.7%). The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 100 ng/ml and 50 ng/ml, respectively.     

New method for the chiral evaluation of mirtazapine in human plasma by liquid chromatography

A simple, rapid and sensitive high-performance liquid chromatography (HPLC) method was developed for the enantioselective analysis of the new antidepressant drug mirtazapine in human plasma. The procedure involved liquid-liquid extraction using toluene, followed by liquid chromatography coupled to UV detection at 292 nm. The chromatographic separation of the (+)-(S)- and (-)-(R)-enantiomers of mirtazapine was achieved on a Chiralpak AD column (250 mm x 4.6 mm, 10 microm particle size) protected with a CN guard column, using hexane-ethanol (98:2, v/v) plus 0.1% diethylamine as the isocratic mobile phase, at a flow rate of 1.2 ml/min. The total analysis time was less than 12 min per sample. The recoveries of (+)-(S)- and (-)-(R)-mirtazapine were in the 88-111% range with a linear response over the 6.25-625 ng/ml concentration range for both enantiomers. The quantification limit (LOQ) was 5 ng/ml. Within-day and between-day assay precision and accuracy were studied at three concentration levels (10, 50 and 250 ng/ml). For both mirtazapine enantiomers, the coefficients of variation (CV) and deviation from the theoretical value were lower than 15% at all concentration levels. The method proved to be suitable for pharmacokinetic studies.     

A HPLC method for determination of nicousamide in dog plasma and its application to pharmacokinetic studies

A sensitive and reproducible high performance liquid chromatography (HPLC)-UV method for determination of nicousamide, an inhibitor of rennin and transforming growth factor-beta1 (TGF-beta1) type II receptors, has been developed and validated. Following acetonitrile deproteiniation, samples were separated by isocratic reversed-phase HPLC on an Aichrom Bond-AQ C(18) column and quantified using UV detection at 320 nm. The mobile phase was acetonitrile/water (ratio 62:38 containing 0.1% H(3)PO(4)), with a flow-rate of 1.0 ml/min. A linear curve over the concentration range 5-200 ng/ml (r(2)=0.9978) was obtained. The coefficients of the variation for the intra- and inter-day precisions ranged from 1.4-10.7% and 1.8-7.1%, respectively. The percentage of relative recovery was 91.56-105.45%. The method was used to determine the plasma concentration-time profiles for nicousamide after oral doses of 30, 100 and 300 mg/kg in dogs. A nonlinear pharmacokinetics was found in dogs at doses from 30 to 300 mg/kg. Following 30 mg/kg oral dose, the C(max) and AUC in females were lower than that in male. There is a potential for accumulation in dogs following multiple doses.     

The UVB-induced synthesis of vitamin D3 and 1alpha,25-dihydroxyvitamin D3 (calcitriol) in organotypic cultures of keratinocytes: effectiveness of the narrowband Philips TL-01 lamp (311 nm)

Both calcitriol and UVB radiation exert potent antipsoriatic effects. We hypothesize that the therapeutical effect of UVB radiation may be attributed at least in part to UVB-triggered cutaneous synthesis of calcitriol. The optimum wavelength for initiation of the vitamin D(3) pathway was found to be in the range of 300+/-5 nm in vitro and in vivo. The narrowband Philips TL-01 lamp which is commonly used as UVB source for phototherapy of psoriasis has maximum spectral irradiance at around 311 nm which is presumed to be, however, of lesser importance in photochemical activation of the vitamin D(3) pathway. The aim of this study was to compare the vitamin D(3) and calcitriol-inducing potential of UVB from the TL-01 lamp with that of monochromatic UVB at 300+/-2.5 nm and 310+/-2.5 nm in organotypic cultures of keratinocytes supplemented with 25 microM 7-DHC. We found that maximum calcitriol-generating capacity of the TL-01 lamp at 500 mJ/cm(2) and 16 h after irradiation still amounts up to 44% of that found after monochromatic irradiation at 300+/-2.5 nm and 30 mJ/cm(2). Thus, the antipsoriatic effect of UVB emitted from the TL-01 lamp may, at least in part, based on the antiproliferative and prodifferentiative action of newly synthesized calcitriol on epidermal keratinocytes.     

Nanostructured synthetic carbons obtained by pyrolysis of spherical acrylonitrile/divinylbenzene copolymers

Novel carbon materials have been prepared by the carbonization of acrylonitrile (AN)/divinylbenzene (DVB) suspension porous copolymers having nominal crosslinking degrees in the range of 30-70% and obtained in the presence of various amounts of porogens. The carbons were obtained by pre-oxidation of AN/DVB copolymers at 250-350°C in air followed by pyrolysis at 850°C in an N(2) atmosphere. Both processes were carried out in one furnace and the resulting material needed no further activation. Resulting materials were characterized by XPS and low temperature nitrogen adsorption/desorption. It was found that maximum pyrolysis yield was ca. 50% depending on the oxidation conditions but almost independent of the crosslinking degree of the polymers. Porous structure of the carbons was characterized for the presence of micropores and macropores, when obtained from highly crosslinked polymers or polymers oxidized at 350°C and meso- and macropores in all other cases. The latter pores are prevailing in the structure of carbons obtained from less porous AN/DVB resins. Specific surface area (BET) of polymer derived carbons can vary between 440 m(2)/g and 250 m(2)/g depending on the amount of porogen used in the synthesis of the AN/DVB polymeric precursors.