Large‐scale isolation and purification of C‐phycocyanin from the cyanobacteria Anabaena marina using expanded bed adsorption chromatography

BACKGROUND: Phycobiliproteins are water soluble proteins useful as fluorescent markers of cells and macromolecules, and as natural colorants, and are anticarcinogenic. Although phycobiliproteins have many applications, their use is limited by the high cost of the purified macromolecules, mainly related with the cost of extraction and purification. In this study a fast and scalable method for preparative extraction and purification of C‐phycocyanin (C‐PC) from Anabaena marina is developed. RESULTS: The method developed consists in the extraction of phycobiliproteins using repeated single contact strategy, separation being performed by expanded bed adsorption (EBA) chromatography using Streamline‐DEAE. Optimal conditions for EBA were obtained at small scale, using a 15 mm internal diameter column, these being a sample load of 0.9 mg C‐PC mL^?1 adsorbent, an expanded bed volume twice the settled bed volume and a sample viscosity of 1.109 mP. The process was then scaled up 36 times, the success of the scale‐up process being verified. Finally, to obtain pure C‐PC conventional ion‐exchange chromatography was utilized. CONCLUSION: Small diameter columns was shown to be useful to simulate the behavior of larger diameter columns for use in scaled up systems. Expanded bed adsorption was demonstrated to be a scalable technology allowing large quantities of C‐PC to be obtained, maintaining high protein recovery while reducing both processing cost and time. The proposed methodology allows recovery of more than 62% of the C‐PC contained in the biomass in the form of pure C‐PC concentrates. Copyright © 2010 Society of Chemical Industry     

Simultaneous recombinant 503 antigen recovery and endotoxin removal from E. coli M15 homogenate using expanded bed adsorption chromatography

This study evaluated the simultaneous recovery of recombinant 503 antigen and endotoxin removal from E. coli homogenate by immobilized metal affinity- expanded bed adsorption chromatography (EBAC), using the non-ionic surfactant Triton X-114 during the washing step. The strategy employed was able to remove more than 99% of endotoxin in all conditions tested, and the lowest residual concentration obtained was 10.22 EU/mL, with a recovery of 503 antigen ranging from 41.4% to 61.1%. Therefore, the procedure of integrating the clarification, recovery of protein, and endotoxin removal into a unit operation exploiting the EBAC was successful.     

膨胀床离子交换吸附分离纳豆激酶

对膨胀床分离纳豆激酶过程的各个阶段进行了考察。在填充床上的探索性实验表明,吸附时最佳的pH为6.0,电导率应低于6.2mS/cm。在膨胀床上样吸附阶段,考察了保持流速不变和保持床层膨胀率不变2种操作方式,结果表明采用控制膨胀率不变的方法更适合纳豆激酶这种料液的粘度和平衡缓冲液的粘度相差不大的情况;在冲洗阶段,通过考察颗粒冲洗的效率确定采用平衡缓冲液冲洗;洗脱阶段采用填充床模式洗脱。与传统方法相比,用膨胀床对纳豆激酶进行分离和纯化,操作步骤从6步减少为2步,操作时间缩短了8—10h,回收率提高了约50%。     

Comparison of the adsorption characteristics of expanded bed adsorbent with conventional chromatographic adsorbent

The static and kinetic adsorption characteristics of Streamline DEAE and DEAE-Sepharose FF were studied under various operating conditions. The adsorption isotherms for the two types of adsorbents were obtained and found to fit well to a Langmuir-type expression. The adsorption kinetics of Streamline DEAE at different concentrations, temperatures, and viscosities were studied and a mathematical model including particle size distribution was developed to describe the adsorption performance of Streamline DEAE. Comparing the uptake curves of Streamline DEAE with DEAE-Sepharose FF, it could be concluded that Streamline DEAE achieves equilibrium faster to get equilibrium than DEAE-Sepharose FF, indicating that Streamline DEAE could be used in higher flow rate systems.     

Compositional heterogeneity in partially hydrolysed poly(vinyl alcohol) by reversed phase liquid chromatography

Reversed phase high performance liquid chromatography (HPLC) was employed to elucidate the composition distribution of partially hydrolysed samples of poly(vinyl alcohol) (PVOH), demonstrating that elution across a chromatogram proceeded from higher to lower degree of hydrolysis (DoH). Fractions isolated by preparative HPLC fractionation and characterised by ¹H nuclear magnetic resonance spectroscopy (NMR) were used as HPLC standards to construct a calibration curve of retention time versus DoH, allowing for DoH determination of any PVOH sample once its chromatogram was available. Plots of cumulative and differential distributions as a function of DoH were determined, allowing for comparisons of samples having average DoH in the range 70–90 mol%. A second set of fractions originating from a parent polymer having different molar mass was also isolated to confirm that calibration was not influenced by molar mass or size exclusion effects.     

Measurement of intraparticle diffusion in reversed phase liquid chromatography

The intraparticle diffusion coefficient was measured using a method based on the fitting of a set of experimental chromatographic profiles to the lumped pore diffusion model. For this purpose, both the analytical solution of the model in the Laplace domain and a numerical method were used. There was an excellent agreement between the results given by the two methods. These results are compared to those obtained by moment analysis of the same set of chromatographic profiles and by the determination of the intraparticle diffusion coefficient from the second central moment of these bands. Nearly identical results were obtained with these two independent methods. The values of the intraparticle diffusion coefficient, D_e, for rubrene in pure methanol was found to be by the modeling method and by the moment analysis method. These values increase with increasing water concentration, to 1.10×10⁻⁶ and , respectively, in a methanol/water solution and to 1.63×10⁻⁶ and , respectively, in a solution.These results confirm the validity and the consistency of the lumped pore model and the moment analysis theory. They show that both approaches describe correctly the mass transfer kinetics in the particles of packing material during the chromatographic process. Systematic determinations of the intraparticle diffusion coefficient can now be undertaken and the influence of various experimental parameters on this important property of packing materials can be investigated.     

从甘草中提取甘草酸和黄酮类化合物的研究

用超滤与大孔树脂吸附整合工艺路线提取甘草中有效成分－甘草酸和黄酮类化合物。实验结果表明,该工艺不但使操作过程简化,而且提高了甘草酸的提取率。此外还优化了压力、温度、循环液流量、浓度等操作参数。     

Hydrolysis of raw hide using proteolytic enzyme extracted from papaya latex

Crude proteolytic enzyme was extracted from papaya latex using two solvents, water and phosphate buffer pH 6. The yield of extracted enzyme using water as a solvent was similar to that using phosphate buffer. Following the solvent extraction, the extracted enzyme was precipitated in 45 wt% saturated ammonium sulfate solution. The yield and activity of precipitated enzyme considerably decreased. Crude proteolytic enzyme extracted using water as an extracting liquid was, therefore, selected to use in gelatin production from raw hide hydrolysis, comparing to the use of commercial papain. The effects of hydrolysis conditions on gelatin recovery and properties of obtained gelatin were investigated. The optimum conditions for the activities of both crude extracted enzyme and commercial papain were at 75 °C and pH 7. At this condition, the highest percentages of gelatin recovery were obtained from raw hide hydrolysis reactions. The gelatin recovery and gel strength of gelatin obtained from crude extracted enzyme and commercial papain hydrolysis were similar. This proved that crude extracted enzyme from papaya latex could be effectively used in gelatin production, instead of the use of commercial papain, with a comparatively low cost.     

Modeling and simulation of breakthrough curves during purification of two chitosanases from Metarhizium anisopliae using ion-exchange with expanded bed adsorption chromatography

A mathematical model was developed to predict breakthrough curves during purification of the two chitosanases from Metarhizium anisopliae by expanded bed adsorption, taking into account the axial dispersion of liquid and using Streamline DEAE and SP XL adsorbents, anion and cation exchange resins, respectively. All the experiments were performed without clarification (with cells) aiming at the reduction of unit operations in future projects of separation processes, thereby reducing capital and operating costs. Chitosanases are enzymes that hydrolyze the carbohydrate chitosan, resulting in oligosaccharides that have many remarkable biological activities, such as anti-cancer, anti-HIV and antioxidant activities. The two adsorbents had similar performance in relation to hydrodynamics and mass transfer. The results of the parametric sensitivity analysis agree with the literature, and the model was validated with an average high degree of fit (94.68%) between simulated and experimental data obtained in this work.     

疏水性电荷诱导扩张床吸附分离免疫球蛋白IgY

结合疏水性电荷诱导色谱和扩张床吸附两项新型生物分离技术,开发了一个高效分离禽类血液免疫球蛋白IgY的新过程。以2-巯基-1-甲基咪唑（MMI）为疏水性电荷诱导配基,偶联于琼脂糖扩张床吸附基质上,制备出疏水性电荷诱导扩张床吸附剂S-MMI。结果表明,S-MMI具有较高的配基密度,约105 mmol·g^-1,pH7时IgY饱和吸附容量达71.26 mg·（ml介质）^-1,扩张床内可形成稳定的分级分布,床层稳定。选用经辛酸沉淀预处理后鸡血浆为料液,通过固定床色谱确定了上样和洗脱条件,比较了扩张床中不同膨胀率的影响,优化了分离条件,实现了扩张床吸附分离鸡血IgY,纯度达到98.9%,收率为80.5%。结果表明,疏水性电荷诱导色谱结合扩张床吸附,可以实现免疫球蛋白的高效分离,为禽类血液蛋白综合利用提供了新方法     

Separation of squalene from palm fatty acid distillate using adsorption chromatography

A method to separate squalene from palm fatty acid distillate (PFAD) using neutralization‐hydrolysis‐neutralization before employing adsorption column chromatography was developed. Extraneous matters, especially free fatty acids (83.8%) and acylglycerols (12.7%), were first neutralized and removed before being subjected to hydrolysis by using commercially available, immobilized Candida antartica lipase, at 65 °C for 8 h. Neutralization followed by hydrolysis and repeated neutralization successfully concentrated squalene from an initial amount of 3.76% to 27.5%. Oil extracted from neutralized‐hydrolyzed‐neutralized PFAD was then passed through a Diaion HP‐20 column using reverse‐phase adsorption chromatography. Squalene was desorbed by hexane, with a recovery of 93%.     

反相高效液相色谱法测定化妆品中的3-O-乙基维生素

采用反相高效液相色谱法测定化妆品中的3-O-乙基维生素C,色谱条件:SH IMADZU Sh im-pack VP-C18ODS(150 mm×4.6 mm,5μm)色谱柱;C18ODS保护柱芯;流动相V(甲醇)∶V(0.025 mol/L KH2PO4)=20∶80;流速:1.0 mL/m in;柱温:30℃;紫外检测器检测,检测池温度:40℃,波长:254 nm。结果表明,在此条件下,3-O-乙基维生素C在0~100 mg/L与相应的峰面积具有良好的线性关系(r=0.999 2),线性回归方程为A=31.170 3,ρ回收率在97.75%~100.60%,方法精密度RSD约为2.6%(n=5)。     

Numerical analysis of fixed bed adsorption kinetics using orthogonal collocation

Fixed bed adsorption kinetics is analyzed to test the validity of the simplified model based on the linear driving force approximation by comparison with the exact model by using the orthogonal collocation method. The axial dispersion, the external film diffusion, and the intraparticle diffusion are considered to be the major mass transfer phenomena involved with the fixed bed adsorption kinetics in this study. It is assumed that a local equilibrium is attained at the fluid-solid interface and the equilibrium can be described by the Langmuir isotherm. A homogeneous particle diffusion model is employed to describe the intraparticle diffusion.     

反相高效液相色谱法测定化妆品中防腐剂

采用反相高效液相色谱法测定化妆品中山梨酸、苯甲酸、对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、对羟基苯甲酸丙酯和对羟基苯甲酸丁酯等6种防腐剂,用ZorbaxODS(5μm,4 6mmi d ×150mm)柱,V(磷酸二氢钾)∶V(乙腈)为90∶10作流动相,加入磷酸调pH值为2 0,检测波长为254nm。样品用乙醇-水-冰乙酸(体积比为70∶29 5∶0 5)提取,回收率为90 8%～105 7%,相对标准偏差为3 1%～8 5%。     

Thermodynamics and mass transfer kinetics of phenol in reversed phase liquid chromatography

The thermodynamics and the mass transfer kinetics of the chromatographic system made of phenol, in a water-acetonitrile mobile phase, on a C18 RPLC column, were studied in the temperature range from 21 to and the interstitial velocity range of 0.021 to 1.27 cm/s. The equilibrium isotherm was accurately approximated by a multilayer model assuming lateral interactions between adsorbed molecules. The parameters of the kinetics of the phenol mass transfer in this column were measured by the method of moments. These data were analyzed using the available models and correlations. It was proven that the parameters of the mass transfer kinetics measured under linear conditions could be successfully used for the prediction of the concentration profiles obtained under overloaded conditions.     

Separation of palm carotene from crude palm oil by adsorption chromatography with a synthetic polymer adsorbent

Palm carotene was successfully concentrated from crude palm oil by a single-stage chromatographic process on a synthetic porous polymer. Carotene was concentrated to about 10⁵ ppm solution, which is about 160 times the original concentration in crude palm oil. Carotene recovery varied from 40 to 65% depending upon chromatographic conditions. The fatty acid composition of the palm oil did not change during the carotene recovery process, and the carotene composition was also almost the same as that in palm oil. Adsorption isotherms of the adsorbent differed from other adsorbents. This new recovery method for palm carotene may be suitable as an edible palm oil pretreatment process due to its efficient mass recovery of a valuable bioresource.     

Arsenic adsorption using copper (II) oxide nanoparticles

Arsenic poisoning is a major problem in today's life. To reduce its concentration in drinking water, different metal based compounds were explored as arsenic adsorbents. In the present study, copper (II) oxide nanoparticles were prepared by thermal refluxing technique and used as an adsorbent for arsenic removal from water. Characterization of the adsorbent using TEM, BET, XRD and FTIR implied that the prepared adsorbent was in nano size and had excellent adsorption behavior with surface area of 52.11 m²/g. Systematic adsorption experiments were carried out with different process parameters such as contact time, adsorbent mass, pH, temperature and stirring speed and found that copper (II) oxide had very good efficiency towards arsenic adsorption. Thermodynamic parameters and adsorption kinetics were studied in detailed to know the nature and mechanism of adsorption. Results showed that the adsorption process followed pseudo second order kinetic and endothermic behavior. Adsorption equilibrium was studied with Langmuir and Freundlich isotherm models. The adsorption process followed Langmuir isotherm with an adsorption capacity of 1086.2 μg/g. A regeneration study was proposed in order to reuse the adsorbent for better economy of the process. Finally, a process design calculation is reported to know the amount of adsorbent required for efficient removal of arsenic from aqueous medium.     

Continuous size fractionation of magnetic nanoparticles by using simulated moving bed chromatography

The size fractionation of magnetic nanoparticles is a technical problem, which until today can only be solved with great effort. Nevertheless, there is an important demand for nanoparticles with sharp size distributions, for example for medical technology or sensor technology. Using magnetic chromatography, we show a promising method for fractionation of magnetic nanoparticles with respect to their size and/or magnetic properties. This was achieved by passing magnetic nanoparticles through a packed bed of fine steel spheres with which they interact magnetically because single domain ferro-/ferrimagnetic nanoparticles show a spontaneous magnetization. Since the strength of this interaction is related to particle size, the principle is suitable for size fractionation. This concept was transferred into a continuous process in this work using a so-called simulated moving bed chromatography. Applying a suspension of magnetic nanoparticles within a size range from 20 to 120 nm, the process showed a separation sharpness of up to 0.52 with recovery rates of 100%. The continuous feed stream of magnetic nanoparticles could be fractionated with a space-time-yield of up to 5 mg/(L∙min). Due to the easy scalability of continuous chromatography, the process is a promising approach for the efficient fractionation of industrially relevant amounts of magnetic nanoparticles.     

化妆品中抗坏血酸磷酸镁的色谱分析

建立了化妆品中抗坏血酸磷酸镁(Vc—PMG)的反相高效液相色谱法(RP—HPIC)。以四氢呋喃-磷酸盐缓冲液(30：70，体积比)溶解并稀释样品，采用乙腈-0．3mol／L磷酸盐缓冲溶液(pH4)(35：65，体积比)为流动相，选择检测波长255nm，Vc—PMG在氨丙基柱上获得了良好分离。该方法的检出限为0．4mg／L，回收率为96．97％～102．78％，相对标准偏差为1．28％。     

3种不同Protein A亲和层析填料纯化抗CD52单克隆抗体的比较

目的 比较3种Protein A亲和层析填料MabSelect SuRe、Protein A Diamond及UniMab 50纯化抗CD52单克隆抗体的载量及纯化效果.方法 将浓度为1.24 mg/mL的抗CD52单克隆抗体分别流经3种Protein A亲和层析填料,确保保留时间一致,检测流穿液的抗体浓度,以流穿液中...