Residue Analysis of Fluroxypyr-meptyl in Wheat and Soil by GC�CECD

C-Phycocyanin is a natural blue pigment with many commercial applications in foods, cosmetics, and medicines. In this paper we describe the extraction and purification of C-phycocyanin from the cyanobacterium Spirulina platensis. The procedure is based on adsorption of impurities with chitosan and activated charcoal then one-step ion-exchange chromatography. The dry algal powder was soaked in potassium phosphate buffer for 2?h to furnish crude phycocyanin extract of purity 0.93. The crude extract was then treated with chitosan and activated charcoal for 5?min, which increased the purity to 2.78. After chromatography on DEAE Sephadex A-25, the purity of phycocyanin was improved to 4.3. The identity of the purified phycocyanin was confirmed by sodium dodecyl sulfate?Cpolyacrylamide gel electrophoretic analysis and by spectroscopic measurements (UV?Cvisible spectrophotometry and spectrofluorimetry). Compared with conventional methods, this method was simple, inexpensive, and time-saving.     

Purification of secoisolariciresinol diglucoside with column chromatography on a sephadex LH-20

A simplified and efficient method is developed for the large-scale purification of the secoisolariciresinol diglucoside (SDG) from defatted flaxseed after aqueous ethanol extraction. Extractant from defatted flaxseed with aqueous ethanol is hydrolyzed with basic solution, concentrated under vacuum, subjected to Sephadex LH-20 column chromatography, and eluted with aqueous ethanol of different concentrations. Elution is monitored by a UV detector at 280 nm, and fractions containing SDG are pooled, concentrated, and applied to a second column chromatography under the same conditions. Elution with water results in a better resolution of SDG [94.5% by high-performance liquid chromatography (HPLC)] than that with pure ethanol or 50% (v/v) aqueous ethanol. HPLC-photodiode array detection-mass spectrometry and NMR are applied to identify SDG and to determine the purity of the eluted fraction. This simplified purification scheme avoids toxic organic solvent used in the common silica gel separation process and, thus, increases the safety of the process.     

微囊藻毒素-RR的纯化及鉴定

将固相萃取和凝胶色谱技术相结合,建立了以野外采集的水华蓝藻为原料提取和纯化微囊藻毒素-RR(MC-RR)的有效方法。用70%的甲醇溶液溶解35 g藻浆,经离心等系列处理,得粗提液,旋转蒸发去除甲醇;粗提液经HLB柱固相萃取后,得到7.5 mL洗脱液,然后将其浓缩至2 mL,再用Sephadex LH-20凝胶色谱柱分离纯化,洗脱液分管收集,用紫外分光光度计测定每管洗脱液在238 nm的吸收值,并绘制洗脱曲线。使用高效液相色谱对峰值组分进行鉴定,同时利用紫外分光光度计对毒素的光谱特征进行鉴定。最终获得3.65 mg纯度超过90%的MC-RR样品,产品得率为74.1%。其紫外吸收光谱在238 nm处有特征吸收,证明所纯化的样品为MC-RR。     

Expanded Bed Adsorption Chromatography for Recovery of Phycocyanins from the Microalga Spirulina Platensis

We carried out the purification of C-phycocyanin and allophycocyanin from Spirulina platensis taking advantage of the adsorption properties of the expanded beds. Initially, phycobiliproteins were released from the microalga cells by osmotic shock. Next, phycocyanins were recovered by applying the centrifuged cell suspension directly to the anion exchanger Streamline-DEAE using expanded bed columns, equilibrated with 50 mM sodium phosphate buffer, pH 7.0. After adsorption, washing was carried out in the expanded-bed mode. Having removed unbound proteins and cellular debris, the bed was allowed to sediment and phycocyanins rich solution was eluted with a downward flow of 500 mM sodium phosphate buffer, pH 7.0. Finally, we utilized conventional gel filtration and ion exchange chromatography methods for separation and purification of C-phycocyanin and allophycocyanin. The purification steps were monitored using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the purity of recovered phycocyanins was confirmed by absorption and emission spectroscopy. The main advantage of this new method is the high yield achieved in the steps of product extraction and adsorption by expanded bed adsorption, so reducing both processing times and costs.     

Purification of flavonoids from licorice using an off‐line preparative two‐dimensional normal‐phase liquid chromatography/reversed‐phase liquid chromatography method

An orthogonal (71.9%) off‐line preparative two‐dimensional normal‐phase liquid chromatography/reversed‐phase liquid chromatography method coupled with effective sample pretreatment was developed for separation and purification of flavonoids from licorice. Most of the nonflavonoids were firstly removed using a self‐made Click TE‐Cys (60 μm) solid‐phase extraction. In the first dimension, an industrial grade preparative chromatography was employed to purify the crude flavonoids. Click TE‐Cys (10 μm) was selected as the stationary phase that provided an excellent separation with high reproducibility. Ethyl acetate/ethanol was selected as the mobile phase owing to their excellent solubility for flavonoids. Flavonoids co‐eluted in the first dimension were selected for further purification using reversed‐phase liquid chromatography. Multiple compounds could be isolated from one normal‐phase fraction and some compounds with bad resolution in one‐dimensional liquid chromatography could be prepared in this two‐dimensional system owing to the orthogonal separation. Moreover, this two‐dimensional liquid chromatography method was beneficial for the preparation of relatively trace flavonoid compounds, which were enriched in the first dimension and further purified in the second dimension. Totally, 24 flavonoid compounds with high purity were obtained. The results demonstrated that the off‐line two‐dimensional liquid chromatography method was effective for the preparative separation and purification of flavonoids from licorice.     

HPLC Purification of Solid-Phase Generated Synthetic Bombesin

Abstract

A scheme based on ion-exchange and reverse-phase high pressure liquid chromatography has been utilized for the semi-preparative and preparative purification of the solid phase generated model peptide bombesin. The final product showed a purity ≥ 99% in analytical reverse-phase high pressure liquid chromatography and was identical to authentic bombesin as demonstrated by different physico-chemical and biological criteria. The results are discussed and compared to those obtained using countercurrent chromatography.     

Extraction and isolation of lithospermic acid B from Salvia miltiorrhiza Bunge using aqueous two‐phase extraction followed by high‐performance liquid chromatography

A rapid and effective method integrating separation and purification of lithospermic acid B from Salvia miltiorrhiza Bunge was developed by combining an aqueous two‐phase system extraction with preparative chromatography. An aqueous two‐phase system of n‐butyl alcohol/KH₂PO₄ was chosen from seven systems. The influence of parameters including concentration of KH₂PO₄, n‐butyl alcohol concentration, pH, and the ratio of an aqueous two‐phase system to crude extract were investigated using a single factor design. Response surface methodology was subsequently used to find the optimal compositions of an aqueous two‐phase system. Keeping a solvent‐to‐solid ratio of 10, the final optimized composition of an aqueous two‐phase system was 39.1% w/w n‐butyl alcohol and 22.6% w/w KH₂PO₄. Under these conditions a recovery yield of 99.8% and a high partition coefficient of 310.4 were obtained. In a pilot‐scale experiment using optimized conditions, 18.79 g of lithospermic acid B with a purity of 70.5% and in a yield of 99.8% was separated from 0.5 kg of crude extract. Subsequently, 9.94 g lithospermic acid B with a purity of 99.3% and recovery yield of 70.3% was obtained with a preparative chromatographic process, and the two‐step total recovery was 70.1%.     

Characterization of chemically synthesized human relaxin by high-performance liquid chromatography

Highly purified human relaxin, produced by combining chemically synthesized A- and B-chains, was analyzed by reversed-phase high-performance liquid chromatography, ion-exchange chromatography and tryptic mapping in order to ascertain purity of the material, presence of uncleaved protecting groups, correctness of disulfide linkages and presence of deamidated or oxidized variants. It was shown by a variety of analytical methods that the product was of high purity; a minimum purity level as judged by the most discriminating assay was greater than 98%. Components of the relaxin preparation removed during the purification were identified to be variants containing deletions arising from incomplete coupling reactions in the solid phase peptide synthesis and/or oxidized methionine residues.     

Efficient large-scale purification of non-histone chromosomal proteins HMG1 and HMG2 by using Polybuffer-exchanger PBE94

A method for the efficient and practical large-scale purification of high-mobility group (HMG) non-histone chromosomal proteins, HMG1 and HMG2, from porcine thymus applying Polybuffer-exchanger PBE94 gel as anion-exchanger has been developed. This method affords higher resolution, purity and yield, than the conventional procedure of CM-Sephadex C-25 ion-exchange column chromatography. Furthermore, use of Polybuffer-exchanger PBE94 column chromatography led to direct preparation of HMG1 and HMG2 from loosely bound non-histone chromosomal protein fraction of chromatin without prefractional precipitation with trichloroacetic acid or prior extraction with perchloric acid. Thus, the application of PBE94 gel as an anion-exchanger to the subfractionation of other kinds of homologous protein is possible.     

Evaluation of a PEG/hydroxypropyl starch aqueous two‐phase system for the separation of monoclonal antibodies from cell culture supernatant

In this study, an aqueous two‐phase system (ATPS) with PEG and hydroxypropyl starch (HPS) was used to separate monoclonal antibody (mAb) from Chinese hamster ovary cell culture supernatant. The phase diagram of the PEG/HPS ATPS was determined, and the effects of NaCl addition were investigated. The results showed that NaCl addition could lead to a shift of the binodal curve and that phase separation would occur at higher PEG and HPS concentrations. The effects of NaCl addition, pH, and the load of cell supernatant on the partitioning of mAb in a PEG/HPS ATPS were investigated. It was found that with 6% cell supernatant and 15% NaCl addition at pH 6.0, the yield of mAb in the upper phase was 96.7% with a purity of 96.0%. The back‐extraction of mAb with a PEG/phosphate ATPS were also studied, and the results showed that after the two‐step extraction with ATPSs the purity of mAb could reach 97.6 ± 0.5% with a yield of 86.8 ± 1.0%, which was comparable to the purification with Protein A chromatography. These results indicate that the two‐step extraction with PEG/HPS and PEG/phosphate ATPSs might be a promising alternative for the separation of mAb from cell culture supernatant.     

Integrated process for the purification of antibodies combining aqueous two-phase extraction, hydrophobic interaction chromatography and size-exclusion chromatography

We have evaluated a process incorporating aqueous two-phase extraction, hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) for the purification of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cell supernatant. These unit operations were chosen not only for allowing the removal of target impurities but also for facilitating the integration of different process units without the need for any conditioning step. Extraction in aqueous two-phase systems (ATPSs), composed of polyethylene glycol (PEG) and sodium citrate, allowed the concentration of the antibodies in the citrate-rich phase and the removal of the most hydrophobic compounds in the PEG-rich phase. An ATPS composed of 10% (w/w) PEG 3350 and 12% (w/w) citrate, at pH 6, allowed the recovery of IgG with a 97% yield, 41% HPLC purity and 72% protein purity. This bottom phase was then directly loaded on a phenyl-Sepharose HIC column. This intermediate purification step allowed the capture of the antibodies using a citrate mobile phase with 99% of the antibody recovered in the elution fractions, with 86% HPLC purity and 91% protein purity. Finally, SEC allowed the final polishing by removing IgG aggregates. HIC-eluted fractions were directly injected in a Superose 6 size-exclusion column affording a 100% pure IgG solution with 90% yield.     

Rapid purification of detergent-solubilized bovine hormone-sensitive lipase by high performance hydrophobic interaction chromatography

Hormone-sensitive lipase (HSL), the enzyme controlling the rate of adipose tissue lipolysis and also possibly involved in the regulation of steroidogenesis, has been purified from bovine omental adipose tissue. Partially detergent-solubilized, delipidated and purified HSL was obtained through step-elution at conventional DEAE ion-exchange chromatography, followed by concentration on hydroxylapatite. High performance hydrophobic interaction chromatography (HPHIC) on phenylsilica then resulted in an increase of HSL protein purity from 2% to more than 70%. Final purification of the enzyme to apparent homogeneity (greater than 95% protein purity), concentration and removal of most of the detergent was obtained by high performance cation exchange chromatography on Mono S. At least 0.5 mg of highly stable HSL was obtained from 5 kg of bovine omental fat within four working days. The purified lipase had a lower specific activity than previously reported for the corresponding rat enzyme but the preparations have proved very useful for enzyme structure studies and as an antigen.     

离子交换色谱法分离纯化鸡卵黄免疫球蛋白

建立了高效、经济、大规模获得鸡卵黄免疫球蛋白(IgY)的生产方法。在对传统的水稀释法改良的基础上,结合聚乙二醇沉淀与离子交换色谱进行IgY的分离纯化。结果显示,用8倍无菌水稀释蛋黄液,用0.1 mol/L HCl调节pH为5.2,在4 ℃下静置8 h,于5000×g力离心可得上清粗IgY液,经测定回收率可达93.47%。然后用6%聚乙二醇沉淀后经DEAE-Toyopearl 650 M离子交换纯化,最佳的纯化条件: 0.05 mol/L磷酸盐缓冲液(PBS, pH 7)平衡上样,0.075 mol/L PBS(pH 7)洗脱。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析结果显示所得的IgY的纯度为95.02%,活性保持率高达73.77%。本研究弥补了传统分离方法不能同时达到高纯度和高回收率的缺点,且可用于大规模生产。     

Purification of human immunoglobulin G by thermoseparating aqueous two-phase systems

The purification of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cells supernatant was studied using an aqueous two-phase system (ATPS) composed of ethylene oxide/propylene oxide (UCON) and dextran. In UCON/dextran systems IgG partitions preferentially to the less hydrophobic dextran-rich phase (Kp<1). The addition of triethylene glycol-diglutaric acid (TEG-COOH) shifted the IgG partition into the upper phase showing significant improvements in both the recovery yields and purity. The purification of IgG from a CHO cell supernatant with UCON 2000/dextran/TEG-COOH system was optimised using a central composite design. Using an ATPS composed of 8% UCON, 6% dextran and 20% TEG-COOH, IgG was purified, in two steps, with a global yield of 85% and 88% purity. Statistical valid models were obtained to predict the effect of the experimental conditions on the IgG yield and purity, for both extraction and back-extraction steps. A system composed of 10% UCON, 5.5% dextran and 20% TEG-COOH was identified as the best compromise between final purity and yield.     

Separation and purification of neohesperidin from the albedo of Citrus reticulata cv. Suavissima by combination of macroporous resin and high-speed counter-current chromatography

In this article, a simple and efficient protocol for rapid preparation and separation of neohesperidin from the albedo of Citrus reticulata cv. Suavissima was established by the combination of macroporous resin column chromatography and high-speed counter-current chromatography (HSCCC). Six types of resin were investigated by adsorption and desorption tests, and D101 macroporous resin was selected for the first cleaning-up procedure, in which 55% aqueous ethanol was used to elute neohesperidin. After treatment with D101 resin, the neohesperidin purity increased 11.83-fold from 4.92% in the crude extract to 58.22% in the resin-refined sample, with a recovery of 68.97%. The resin-refined sample was directly subjected to HSCCC purification with a two-phase solvent system composed of ethyl acetate-n-butanol-water (4:1:5, v/v), and 23.6 mg neohesperidin with 97.47% purity was obtained from 60 mg sample in only one run. The recovery of neohesperidin in HSCCC separation procedure was 65.85%. The chemical structure of the purified neohesperidin was identified by both HPLC and LC-MS. The established purification process will be helpful for further characterization and utilization of Citrus neohesperidin.     

Isolation and characterization of proteoglycans from different tissues

Two chromatographic procedures for the isolation and purification of proteoglycans (PG) and their related glycosaminoglycan (GAG) peptides are described. PG from human aorta were isolated from tissue extract by sequential ion-exchange, size-exclusion and hydroxyapatite chromatography. Final purification of samples was achieved by chromatography on Mono Q. Homogeneity of samples was demonstrated by Western blot analysis of biotin-labelled compounds prior to and after enzymatic digestion and dual-wavelength detection in size-exclusion chromatography. The purity of samples obtained by the procedure described was sufficient for protein sequence analysis. GAG preparations of bovine trachea cartilage were purified by the sequential use of strong anion-exchange supports. Molecular weight distribution and sensitivity to treatment with glycan-specific enzymes was shown by size-exclusion chromatography.     

Purification of domoic acid from toxic blue mussels (Mytilus edulis) and phytoplankton.

Domoic acid was the primary neurotoxin in blue mussel (Mytilus edulis) that caused poisoning in humans. Further research showed that the algae, Nitzschia pungens, was the source of this toxin. In this study, a method for the extraction and purification of domoic acid from contaminated mussels and phytoplankton was developed. Domoic acid was extracted from these sources by treatment with a mixture of chloroform and methanol (1:2, v/v). The resulting extract was subjected to ultrafiltration through a PM1 Millipore filter, followed by repeated high-performance liquid chromatography on a reversed-phase column. The purity and yield of domoic acid prepared by this method are compared with two previously described methods of extraction. The current method is relatively simple, rapid, and results in improved recovery with comparable purity of domoic acid.     

Preliminary Studies for Cephamycin C Purification Technique

A study was made for purification of cephamycin C from fermentation of Streptomyces clavuligerus. Initially, the culture broth was clarified by microfiltration and ultrafiltration, after which the resulting permeates were subjected to nonspecific adsorption and ion-exchange chromatography on resin columns. The antibiotic activity was measured by the biological method at each stage by assaying its activity against the Escherichia coli ESS, super sensitive to β-lactam antibiotic. The purification processes were assessed in relation to the variables affecting each step. The purification efficiency by nonspecific adsorption was monitored by UV spectrophotometry, while the ion-exchange adsorption fractions were assessed by NMR spectroscopy. Some of the fractions obtained during purification were also analyzed by mass spectrometry (LC/MS and LC/MS/MS) to identify the cephamycin C molecule. These preliminary results proved the process feasibility.     

Ni(II)-based immobilized metal ion affinity chromatography of recombinant human prolactin from periplasmic Escherichia coli extracts

A novel, two-step preparative technique is described for the purification of authentic recombinant human prolactin (rhPRL) secreted into the periplasm of transformed Escherichia coli cells. The first step is based on immobilized metal ion affinity chromatography of periplasmic extract, using Ni(II) as a relatively specific ligand for hPRL in this system. It gives superior resolution and yield than established ion-exchange chromatography. Size-exclusion chromatography is used for further purification to >99.5% purity. The methodology is reproducible, leading to 77% recovery. Identity and purity of the rhPRL were demonstrated using sodium dodecylsulphate-polyacrylamide electrophoresis, isoelectric focusing, mass spectrometry (matrix-assisted laser desorption ionization time-of-flight), radioimmunoassay, RP-HPLC and high-performance size-exclusion chromatography. In the Nb2 bioassay, the hormone showed a bioactivity of 40.9 IU/mg.     

重组人干扰素-γ的制备与鉴定

用聚乙二醇200疏水相互作用色谱固定相(PEG200-STHIC)分别在色谱柱和色谱饼上完成了一步复性并同时纯化来源于大肠杆菌(E.coli)表达的重组人干扰素-γ(rhIFN-γ)。为了能使色谱分离方法用于不同来源的rhIFN-γ的纯化,对rhIFN-γ在反相色谱、离子交换色谱、固定化镍离子亲和色谱上的保留行为也进行了研究。色谱柱纯化的rhIFN-γ收集液经排阻色谱除盐和冷冻干燥得到rhIFN-γ干粉。用基质辅助激光解吸电离飞行时间质谱对rhIFN-γ干粉进行了测定,rhIFN-γ单体的相对分子质量为17184.0,二聚体的相对分子质量为34204.4。用细胞病变抑制法(CPEI)测定rhIFN-γ干粉的比活性为9.5×108 IU/mg。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定rhIFN-γ干粉的纯度高于95%。用色谱柱复性并同时纯化rhIFN-γ的质量回收率达到93.7%,纯度高于95%,比活性为4.3×107 IU/mg。结果表明,采用PEG200-STHIC色谱柱复性并同时纯化rhIFN-γ是一种十分高效的方法。