Adsorption behaviors of avian immunoglobulins and purification of immunoglobulin Y from chicken serum with mixed-mode resins

The importance of immunoglobulin Y(IgY) as a specific antibody equivalent to mammalian immunoglobulin G(IgG) is well recognized. However, production of highly purified IgY is still difficult due to the lack of specific purification methods. In this study, adsorption behaviors of Ig Y on four mixed-mode resins with functional ligands of 4-mercatoethyl-pyridine(MEP), 2-mercapto-1-methyli-midazole(MMI), 5-aminobenzi-midazole(ABI) and tryptophan-5-aminobenzi-midazole(W-ABI) were evaluated. The results showed that high adsorption ratio were found at p H 6.0–7.0 with little adsorption under acidic conditions. The resin with ABI ligand was then used to separate IgY from immunized chicken serum. An efficient process with Ig Y purity of 95% and recovery of 90% was developed after optimization of loading and elution p H and injection volume. The biological activity of the purified Ig Y was fully maintained. These results indicated that mixed-mode chromatography with specially-designed ligands has great potential for the separation of Ig Y from crude feedstock.

Preparation and evaluation of mixed-mode resins with tryptophan analogues as functional ligands for human serum albumin separation

Five tryptophan analogues with a hydrophobic indole ring and an amino group on each molecule were used as functional ligands of mixed-mode resins for human serum albumin(HSA) purification. Their adsorption performance was evaluated and the effects of p H and salt addition on HSA adsorption were studied. The resins prepared showed typical p H-dependent adsorption and the highest adsorption capacity and affinity were found at pH 5.0for all the resins tested. The saturated adsorption capacity was 138.02 mg·g^(-1)with the tryptaminefunctionalized resin, which significantly decreased at p H below 4.0 due to electrostatic repulsion between ligands and HSA. Moreover, the addition of Na Cl or(NH_4)_2SO_4in media reduced HSA adsorption capacity, although the two salts showed different affecting profiles. The tryptamine-functionalized resin showed the best salt-tolerant performance, and its high adsorption capacity was maintained under high salt concentrations. In addition, the five resins prepared showed good adsorption selectivity for recombinant HSA from Pichia pastoris broth. Molecular docking results between tryptamine and HSA indicated that tryptamine was favorable to bind on Site II(indole-binding site) of HSA.

Isolation and purification of carbazole contained in anthracene slag by extraction combined with medium pressure liquid chromatography

Ultrasonic-assisted extraction(UAE)combined with medium pressure liquid chromatography(MPLC)was designed for carbazole separation from anthracene slag(AS).The effects of liquid/solid ratio,temperature,and extraction times on carbazole separation were investigated.When using CC14 and ethyl acetate as extraction solvents and combining with MPLC,carbazole recovery and purity are 75.1%and 95.4%,respectively.The mechanism for carbazole separation were presumed by examining intermolecular interactions such as N-H…π,π-π,and C-Cl…πinteractions.These results demonstrate that UAE/MPLC has a considerable potential as a green and promising strategy for separating and purifying carbazole and other chemicals from AS.

High Performance Liquid Chromatography Separation and Simultaneous Fluorometric Detection of Trace Amount of Polyamines in Cell Culture Media

A highly sensitive and precise method for the determination of the polyamines (including putrescine, spermidine and spermine) in cell culture media is described. The samples were concentrated by C18 column and the polyamines were converted to fluorometric derivatives with DNS-Cl. The polyamine derivatives were then completely separated by HPLC and determined by simultaneous fluorometric detection. The CV of intragroup and intergroup were 2. 49 %-4. 26% and 4. 29 %-5. 16 %, respectively. The rate of recovery was 103%- 99%. There was trace amount of polyamines detected by this method in the media of F12, 8900, RPMI1640, DMEM and M199 even without incubation with cells. So this method can be used for detecting the changes of polyamines in a medium before and after incubation with cells. It is helpful for the researches on the regulation of cell proliferation by polyamines.

荔枝草中乙酸乙酯层化学成分分析

目的:对荔枝草的乙酸乙酯层进行化学成分研究。方法:采用硅胶柱色谱、中压快速色谱和Sephadex-LH凝胶色谱等方法进行分离纯化,运用波谱手段、理化性质和文献对照进行各单体化合物的结构鉴定。结果:从荔枝草的乙酸乙酯层分离出2个单体化合物,分别鉴定为泽兰黄酮和高车前素。结论:采用中压色谱能从荔枝草乙酸乙酯萃取层中快速大量提取泽兰黄酮和高车前素,该方法省时、简便、高效,值得推广应用。

Combination of reversed phase liquid chromatography and zwitterion exchange-reversed phase-hydrophilic interaction mixed-mode liquid chromatography coupled with mass spectrometry for the analysis of antibiotics and their impurities

In this study, a system involving two-dimensional, column-switching high-performance liquid chromatography （HPLC） coupled with tandem mass spectrometry （LC-MS/MS） was developed and optimized for the analysis of antibiotics and their related substances. In the first-dimensional chromatography, the analytes were separated on a zwitterion exchange-reversed phase-hydrophitic interaction （ZIC-RP-HILIC） mixed-mode column coupled with tandem mass spectrometry （LC-MS/MS）. A commonly used reversed phase LC column was employed in the second-dimensional chromatography. The mobile phase for the ZIC-RP-HILIC mixed-mode chromatography consisted of a volatile buffer that was compatible with LC-MS/MS, which enhanced the efficiency of ionization for structure elucidation. The antibiotic impurities eluted in the ion-pairing reversed phase chromatography were directly characterized by the ZIC-RP-HILIC-MS system, and the orthogonal separation of ZIC-RP-HILIC mixed-mode chromatography and reversed phase LC provided extra confidence that no impurity was missed. The efficiency of this method was demonstrated in the analysis of penicillin V potassium, oxacillin sodium, ceftriaxone sodium, and their impurities. In addition, this method is convenient for impurity profiling of antibiotics, and may be used for the analysis of other pharmaceutical ingredients.

“Grafting from”法制备高载量大孔弱阳离子交换层析介质

针对大孔聚合物层析介质孔径大、比表面积低而导致的蛋白结合容量低的问题,采用“graftingfrom”策略,以大孔聚丙烯酸酯微球为基质,通过氧化还原引发甲基丙烯酸在微球表面接枝聚合,制备了高载量弱阳离子交换层析介质.研究了单体浓度、过硫酸钾浓度及反应温度等因素对蛋白结合容量的影响.所得介质的蛋白静态结合容量和动态结合容量分别达到252.21和157.25mg/mL;同时发现具有一定离子交换容量的该类介质能够在0.2 mol/L NaCl缓冲液中保持100 mg/mL的蛋白结合容量.将该层析介质用于鸡卵清中溶菌酶的纯化,获得了较高的纯化效率.

利用中压制备液相色谱从蓝莓叶多酚中分离纯化绿原酸

该研究以蓝莓叶为试验材料,采用超声辅助纤维素酶的方法获得蓝莓叶多酚粗提物,然后利用中压制备液相色谱技术对粗提物进行分离纯化,分别考察不同流动相、进样量、流动相流速对绿原酸得率的影响。结果表明,最佳制备条件为乙腈-水溶液为流动相、进样量6 mL、流动相流速10 mL/min,在此条件下,绿原酸得率为21.39%。经高效液相色谱分析,得到的绿原酸纯度可达94.98%。该方法能够快速、高效地分离蓝莓叶中的绿原酸。该方法为研究和应用蓝莓叶中活性成分提供了一种有效的手段。

A reversed phase/hydrophilic interaction/ion exchange mixed-mode stationary phase for liquid chromatography

A reversed phase (RP)/hydrophilic interaction (HILIC)/ion exchange (IEX) mixed tri-mode stationary phase (TMSP) has been prepared via a divergent synthesis scheme starting from propylamine on silica then by amine-epoxy reactions with 1,4-butanedioldiglycidyl ether and tertiary amines (N,Ndimethyldecylamine, DMDA). Its retention mechanism was found to follow RP/HILIC/IEX mixed-mode.The stop-flow test revealed that TMSP had good compatibility with 100% aqueous mobile phase. It demonstrated effective separation towards several kinds of compounds or drug molecules and their counterions within a single run.

基于负化学电离技术的中链氯化石蜡检测研究

建立了测定塑料中的中链氯化石蜡含量的气相色谱-负化学电离质谱法。以乙酸乙酯为溶剂提取样品,提取温度为60℃,提取时间为45min,提取液滤膜过滤后,采用气相色谱-负化学电离质谱法进行定性和定量分析。结果表明,中链氯化石蜡在2~20mg·L^(-1)质量浓度范围内呈良好的线性关系,方法检出限为20.1mg·kg^(-1),在3个不同浓度加标水平下的平均回收率为102.3~115.2%相对标准偏差(RSD,n=6)为1.9~8.2%。该方法操作简捷、准确度高、精密度好,可用于塑料样品中MCCPs的检测。

Two-dimensional MOF Cu-BDC nanosheets/ILs@silica core-shell composites as mixed-mode stationary phase for high performance liquid chromatography

Here,silica microspheres were decorated with two-dimensional metal-organic frameworks(2 D MOFs)nanosheets and ionic liquids,and evaluated as the mixed-mode stationary phase for chromatographic separation.The ionic liquids were used to assist the synthesis of 2 D MOFs nanosheets,and also acted as adhesives among the nanosheets and silica.In contrast with the 2 D MOFs-based column without ionic liquids and commercial columns,the prepared column exhibited enhanced chromatographic separation performance for partially hydrophilic compounds such as alkaloids,sulfonamides and antibiotics,etc.In addition to excellent chromatographic repeatability and stability,it has also been verified that the composites could be easily and repeatedly prepared.The relative standard deviation of the retention time of the same type of analyte between the three batches of materials was ranging from 0.21%to 1.7%.In short,these results indicated that the synthesized composites were promising separation material for liquid chromatography,which made it possible to broaden the application of 2 D MOFs in the field of chromatography.

Facile Fabrication of Polymeric Ionic Liquid Grafted Porous Polymer Monolith for Mixed-Mode High Performance Liquid Chromatography

A novel polymeric ionic liquid grafted porous polymer monolith has been facilely fabricated for mixed-mode chromatography.The column is prepared from poly(glycidyl methacrylate-co-ethylene dimethacrylate)monolith through hydrolyzation of the epoxy moieties into hydroxyl groups,followed by“grafting from”polymerization of ionic liquid of 1-vinyl-3-butylimidazolium chloride.Successful modification is characterized by scanning electron microscope,infrared spectroscopy,elemental analysis and mercury intrusion porosimetry.The HPLC performance of developed column is evaluated by separating acidic vitamin B analytes,neutral steroids and basic aromatic amines in mixed-mode chromatography on a single column,respectively.The ionic liquid affords the monolith with both enhanced separation ability and improved column efficiency.

气相色谱-负化学离子源质谱法测定中链氯化石蜡3种定量方法比较研究

为阐明中链氯化石蜡现行3种定量方法的测定结果差异,配制不同浓度、不同氯含量的标准样品及含有短链氯化石蜡的模拟样品和基质干扰样品,比较研究3种定量方法对不同试样的测定数据。结果表明:校准曲线法和多元线性回归法适用于待测试样氯含量与曲线氯含量相近的样品测定,总体响应因子校正法适用于浓度与曲线浓度相近的待测试样测定。实际检测时,总体响应因子校正法的偏差相对可控,应尽量建立与待测试样氯含量接近的标准曲线使定量结果更加可靠。

超高效液相色谱法测定细胞培养液中游离氨基酸浓度

目的探讨基于超高效液相色谱(ultra performance liquid chromatography,UPLC)法,对细胞培养液中游离氨基酸浓度的测定方法。方法采用UPLC技术,结合优化的样品处理方法,对细胞培养液中游离氨基酸进行高效、精准的测定。结果该UPLC法能够快速、可靠地测定细胞培养液中的游离氨基酸浓度,为生物制药工业中细胞培养监控与优化提供了有效手段。结论对氨基酸浓度的准确测定,不仅有助于更好地了解细胞培养液中关键营养成分的动态变化,还为生产过程中的工艺优化提供了重要数据支持。

卷烟爆珠溶剂中链甘油三酯的分离与鉴定

为明确卷烟爆珠常用溶剂中链甘油三酯(MCT)的化学组成,利用GC/MS法对3种市售MCT和7种爆珠卷烟的爆珠溶剂进行了定性分析,利用中压制备液相色谱-紫外检测技术分离制备了MCT中极性相近的4个组分,并通过对各个组分的化学表征确定了其成分信息。结果表明:(1)卷烟爆珠溶剂与市售MCT的主要化学成分一致。(2)收集的4个组分的质量总和占MCT产品的92%。(3)通过核磁共振与质谱比对,4个组分及其占MCT产品的质量分数分别为三辛酸甘油酯22.8%、二辛酸一癸酸甘油酯37.9%、一辛酸二癸酸甘油酯25.5%和三癸酸甘油酯5.8%。

中压柱层析法分离白藜芦醇的研究

对虎杖(PolygonumcuspidatumSieb.etZucc.)中的白藜芦醇进行了提取分离和含量测定。考察了浸提温度、浸提液浓度、浸提时间和次数、料液比等5个因素对白藜芦醇提取的影响,确立了白藜芦醇最佳提取条件为:浸提液体积分数为95%,浸提温度60℃,料液比11∶1,回流提取3次,每次60min。采用中压硅胶柱层析法分离白藜芦醇,产物经重结晶后用高效液相色谱测定含量达99.28%。结果表明所建立的制备及分离纯化工艺可用于白藜芦醇的生产。

金属螯合亲和色谱介质的合成及其色谱特性研究

详细研究了亚氨基二乙酸（ＩＤＡ）在硅胶基质上键合的适宜条件，利用国产小颗粒大孔径硅胶（７μｍ，３０ｎｍ）制备了固定有Ｃｕ２＋，Ｎｉ２＋，Ｃｏ２＋，Ｚｎ２＋的金属螯合吸附剂，比较了蛋白质在金属螯合柱和裸柱上的保留特性，利用合成的Ｃｕ－ＩＤＡ－硅胶柱分离了来自牛血红细胞的超氧化物歧化酶（ＳＯＤ）。

连续中压硅胶柱层析纯化法夫酵母虾青素

采用连续中压硅胶柱层析纯化虾青素,原料为含量高于20%的法夫酵母虾青素。连续中压柱层析分离工艺条件为:不锈钢中压层析柱装填120～160μm层析硅胶,以石油醚∶1,2-二氯乙烷∶丙酮(体积比)为5∶2.5∶1作洗脱剂,负载量为1∶7,可以得到纯度大于97%的虾青素产品,平均回收率大于60%。回收的洗脱液经校正后可重复使用。使用后的层析柱以丙酮为再生液再生,石油醚为平衡液平衡,平衡后的层析柱可重复使用多次。利用该工艺方法纯化虾青素,与传统方法相比溶剂用量少、层析填料价格低、生产周期短、分离效果好,易于实现连续工业化清洁生产。

中压硅胶柱层析连续纯化茶叶中EGCG及ECG的研究

采用一种连续中压硅胶柱层析分离高纯度表没食子儿茶素没食子酸酯(EGCG)及表儿茶素没食子酸酯(ECG),原料为含量高于98%的茶多酚。连续中压柱层析分离工艺条件为:160～280μm硅胶为填充料,1200mm×80mm的自制不锈钢中压层析柱,洗脱液为乙酸乙酯-石油醚-甲酸(体积比6∶4∶1),洗脱流速为30mL/min,负载量为35g/kg(以硅胶计,下同),可以得到纯度大于98%的EGCG产品(平均回收率为85.5%)和98%的ECG产品(平均回收率均为80.3%)。回收的洗脱剂先校正pH值,再经薄层层析校正后可重复使用。使用后的层析柱用乙酸乙酯再生,石油醚为平衡剂平衡,平衡后的层析柱可重复使用。

北虫草培养残基中虫草素的提取纯化及抗肿瘤活性

以北虫草固体培养残基为实验材料,比较闪式提取、超声波提取和超高压提取培养残基中虫草素的效果,用陶瓷膜过滤提取液,获得虫草素粗提液,利用高速逆流色谱分离制备虫草素,通过LC-MS/MS对产物结构进行鉴定,并验证了虫草素对小鼠S180肉瘤的抑制作用。结果显示,超高压提取培养基中虫草素较好,高速逆流色谱分离纯化条件是以V(乙酸乙酯)∶V(正丁醇)∶V(水)=2∶3∶5的两相溶剂系统,上相为固定相,下相为流动相,流速3.0 m L/min,主机转速900 r/min,分离温度28℃,以此分离条件经一步洗脱,高速逆流色谱收集馏分,其中峰Ⅲ产物经HPLC检测纯度达97.6%,结构经LC-MS/MS鉴定为虫草素。从100 g北虫草培养残基中可制备得虫草素181.90mg,并对其活性验证发现剂量为100 mg/kg时小鼠S180肉瘤瘤重抑制率为64.48%。