Refolding of Denatured/Reduced Lysozyme Using Weak-Cation Exchange Chromatography

Oxidative refolding of the denatured/reduced lysozyme was investigated by using weak-cation exchange chromatography (WCX). The stationary phase of WCX binds to the reduced lysozyme and prevented it from forming intermolecular aggregates. At the same time urea and ammonium sulfate were added to the mobile phase to increase the elution strength for lysozyme. Ammonium sulfate can more stabilize the native protein than a common eluting agent, sodium chloride. Refolding of lysozyme by using this WCX is successfully. It was simply carried out to obtain a completely and correctly refolding of the denatured lysozyme at high concentration of 20.0 mg/mL.

Denatured Thermodynamics of Proteins in Weak Cation-exchange Chromatography

The thermostability of some proteins in weak cation-exchange chromatography was investigated at 20-80 ℃. The results show that there is a fixed thermal denaturation transition temperature for each protein. The appearance of the thermal transition temperature indicates that the conformations of the proteins are destroyed seriously. The thermal behavior of the proteins in weak cation-exchange and hydrophobic interaction chromatographies were compared in a wide temperature range. It was found that the proteins have a higher thermostability in a weak cation-exchange chromatography system. The thermodynamic parameters(Δ H 0, Δ S 0) of those proteins were determined by means of Vant Hoff relationship(ln k -1/ T ). According to standard entropy change(Δ S 0), the conformational change of the proteins was judged in the chromatographic process. The linear relationships between Δ H 0 and Δ S 0 can be used to evaluate 'compensation temperature'( β ) at the protein denaturation and identify the identity of the protein retention mechanism in weak cation-exchange chromatography.

Poly [ (chloromethyl) styrene-co-divinylbenzene] Continuous Rod Column of Weak Cation Exchange Chromatography and its Applications in the Separation of Biopolymers

Macroporous poly [(chloromethyl) styrene-co-divinylbenzene] continuous rod was prepared by direct polymerization of the monomers in the presence of a porogenic diluent inside an empty chromatographic column. A new 'in-situ' technique was used to modify the synthesized polymer rod for a weak cation exchanger and it has been used successfully for the separation of biopolymers. It was found that the back pressure of the continuous rod column was much lower and its surface was proved to be modified well.

Determination of ephedrine and codeine in human urine by cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography

A sensitive method for the determination of ephedrine and codeine in human urine by capillary electrophoresis （CE） was described. In order to improve the sensitivity, two online concentration techniques including cation-selective exhaustive injection （CSEI） and sweeping micellar electrokinetic chromatography （sweeping-MEKC） were used. Under the optimum conditions, the detection limits （S/N = 3） were 0.10 μg/L for ephedrine and 0.80 μg/L for codeine. This method was successfully applied to real urine sample analysis.

Impact of Hydrogen Ion Concentration on Amino Acids Composition of Macadamia Protein: Approached Using Cation-Exchange Chromatography

In the present context, the objective of this study was to synthesize and analyze the content of AA of macadamia protein and the impact of hydrogen ion concentration (pH) on AA composition. The determination of AA mainly by cation-exchange chromatography was also investigated. Reproducible and reliable techniques for quantification and identification of AA usually require derivatization. However, techniques such as AA analyzer are composed of cation-exchange chromatography and other components can sideline the derivatization with significant accuracy. The present analysis revealed a higher concentration of essential amino acids especially acidic AA, Glu and Asp and basic AA, Arg than other AA in macadamia protein. The study constitutes first report of use of bubble chart for evaluation of AA and explaination of AAS. The results may elaborate that the degradation of AA of macadamia protein for extraction of pH 11 is caused by the impact of pH. Moreover, the nutritional values of AA present in macadamia protein could change for the better by adjusting pH of extraction.

离子色谱法同时检测大气颗粒物PM_(2.5)中低级脂肪胺和常规阳离子

大气中存在大量的有机胺污染,其中低级脂肪胺是促进颗粒形成和生长成为PM_(2.5)的诱因,会对人体肾脏、心肺功能健康造成损害。而PM_(2.5)是大气中常见的颗粒污染物,是雾霾天气产生的主要原因,其成分非常复杂,测定其中的阳离子和低级脂肪胺,能直接监测环境大气质量,保护人体健康。本研究建立了抑制电导离子色谱法同时测定大气细颗粒物PM_(2.5)中4种低级脂肪胺(甲胺、二甲胺、三甲胺、乙胺)和5种常见阳离子(Na^(+)、NH_(4)^(+)、K^(+)、Mg^(2+)、Ca^(2+)),通过优化色谱条件,实现了K^(+)和甲胺、二甲胺和乙胺等难分离物质的有效分离,分析结果可用于评估空气中的颗粒物污染情况。本研究采用负载石英滤膜的中流量采样器采集大气中的PM_(2.5)颗粒物,裁剪1/2滤膜并剪碎于10 mL超纯水中超声提取2次共60 min,提取液过0.22μm滤膜后用离子色谱检测。比较了3种阳离子色谱柱IonPac^(TM) CS17、IonPac^(TM) CS16和SH-CC-9后,最终选用SH-CC-9阳离子分析柱(200 mm×4.6 mm)进行分离:柱温30℃,检测器温度35℃,进样量25μL,流动相为甲基磺酸(MSA)水溶液,流速为1.1 mL/min。在此色谱条件下,大气中可能存在的其他胺(N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、丙胺、二乙胺、三乙胺、三乙醇胺、异丙胺)对目标离子没有影响。待测的4种低级脂肪胺和5种常见阳离子在各自相应的浓度范围内呈现良好的线性关系,线性相关系数(r)均不小于0.997,检出限(LOD)为0.02~1.90μg/L,定量限(LOQ)为0.07~6.32μg/L, 6个平行样品测定的相对标准偏差(RSD)小于2%,样品加标回收率为93.2%~104%。采用建立的方法对189个大气颗粒物PM_(2.5)样品进行检测,9种离子均有检出,其中Na^(+)、NH_(4)^(+)、Ca^(2+)等离子含量较多,4种低级脂肪胺含量少,但部分样品中乙胺含量偏高。结果表明,本研究建立的离子色谱方法前处理简单,灵敏度高,准确性好,可满足大气颗粒物PM_(2.5)中低级脂肪胺和常规阳离子同时定量检测的需求,能够快速处理大量样品,准确评价空气中颗粒物的污染程度,追溯污染来源,保护人类健康。

“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.

基于离子色谱法快速测定血清中钠、钾、钙、镁4种阳离子水平

目的建立并优化一种基于微波消解结合离子色谱法同时测定血清中钠、钾、钙、镁4种阳离子的方法,并对其性能进行评价。方法采用微波消解技术对血清样品进行前处理,使用离子色谱定量检测4种阳离子。参照美国临床和实验室标准协会(CLSI)发布的EP6-A和EP15-A3文件对建立的方法进行线性、精密度、加标回收率和正确度等性能验证。结果钠、钾、钙、镁4种离子的检测不精密度以变异系数(CV)表示,分别为0.10%~0.36%、0.15%~0.48%、0.22%~0.87%、0.21%~0.73%。4种阳离子的加标回收率均在97.3%~103.0%之间。参加RELA2020和RELA2021计划的分析结果偏差分别为-0.57%~-0.14%、-0.84%~-0.16%、-1.82%~-0.37%、-0.60%~0.34%,比对均通过。4种阳离子的检出限/定量限分别为0.0175/0.0583mmol/L、0.0005/0.0017mmol/L、0.0099/0.0329mmol/L、0.0011/0.0035mmol/L,表明经前处理后,其他杂质对测定无明显干扰。结论成功建立并优化了一种基于微波消解结合离子色谱法同时测定血清中钠、钾、钙、镁4种阳离子的方法。该方法精密度和正确度良好,操作简单,分析时间短,成本低,可用于临床血清电解质检测的质量改进。

万通离子色谱仪测定雨水中阴阳离子的方法验证分析

检出限是测试仪器性能及评价一个分析方法的重要指标,依据《环境监测分析方法标准制修订技术导则》(HJ 168-2010)中有关方法验证相关的规定,利用万通离子色谱仪验证《大气降水中氟、氯、亚硝酸盐、硝酸盐、硫酸盐的测定离子色谱法》(GB 13580.5-1992)及《酸沉降监测技术规范(附录B离子色谱法对阳离子的测定)》(HJ/T 165-2004)中Na^(+)、NH_(4)^(+)、K^(+)、Mg^(2+)、Ga^(2+)五种阳离子和F^(-)、Cl^(-)、NO_(3)^(-)、SO_(4)^(2-)四种阴离子的研究方法,验证过程涉及线性关系、检出限、精密度、准确度及加标回收率5个项目,并对验证结果进行分析。

离子色谱法中降低可溶性阳离子空白值的对策

离子色谱法具有选择性好、灵敏度高、方便快捷的特点,常用于环境监测中阴阳离子的测定。自然界的化合物多为阴阳离子相互组合而成,因此阴阳离子伴随着颗粒物及水分会残留在实验室环境中,使用离子色谱法测定阴阳离子时常出现空白值偏高的现象,特别是可溶性阳离子。文章以美国戴安公司(DIONEX)ICS-900型离子色谱仪测定水中可溶性阳离子Na^(+)、NH_(4)^(+)、K^(+)、Mg^(2+)和Ca^(2+)为例,提出降低可溶性阳离子空白值的对策,供检测实验室参考。

阴阳离子色谱联用技术在氯化钾检测中的应用

随着氯化钾在医药、食品及无机等工业领域的运用越来越广,如何有效选择氯化钾检测技术对于其产品质量的提高起着至关重要的作用。目前工业氯化钾中各种化学物质的检测方法通常选择质量法、容量法及光度法等,但是这类技术手段灵敏度不高,操作复杂,不能同时进行多组分检测,不能满足工业领域对于质量的要求。为了克服上述问题,通过技术研究,采用阴阳离子色谱联用技术检测工业氯化钾样品中各种化学物质含量,该方法操作简单,准确度高,而且可以检测容量法不能检测的钠离子含量,通过一次进样就能完成氯化钾溶液中所有需要检测的离子浓度。

氯化苯副产盐酸的精制工艺方法研究

利用树脂吸附和脱吸附原理,对氯化苯生产过程中产生的盐酸进行脱色和去除有机物,从吸附和脱附工艺流程、树脂配比、盐酸吸附定量等方面展开分析,设计相应模拟实验进行过程探索,形成了一套比较完善的氯化苯副产盐酸精制的工艺流程。

Molecular weight determination of a newly synthesized guanidinylated disulfide-containing poly(amido amine) by gel permeation chromatography

A cationic gene delivery vector, guanidinylated disulfide-containing poly(amido amine)(CARCBA), was synthesized by Michael addition reaction between N,N′-cystaminebisacrylamide(CBA) and guanidine hydrochloride(CAR). Gel permeation chromatography(GPC) was used to evaluate the molecular weight of synthesized CAR-CBA. Polyethyleneimine(PEI) with molecular weight of 25 kDa was adopted as a reference, and polyethylene glycols(PEG) with different molecular weights were used to establish a standard curve for determining the molecular weight of CAR-CBA. The effects of two critical factors, namely columns and eluents,on the molecular weight measurement of CAR-CBA were investigated to optimize the GPC quantitative method. The results showed that Ultrahydrogel columns(120, 250) and HAc–NaAc(0.5 M, pH 4.5) buffer solution were the optimal column and GPC eluent, respectively.The molecular weight of the synthesized CAR-CBA was analyzed by the optimized GPC method and determined to be 24.66 kDa.

Acarbose Isolation with Gel Type Strong Acid Cation Exchange Resin： Equilibrium, Kinetic and Thermodynamic Studies

Acarbose, a potent α-glucosidase inhibitor, is widely used as an oral anti-diabetic drug for the treatment of the type 2, non-insulin-dependent diabetes. In this work, a gel type strong acid cation exchange resin 001×4 was applied to isolate acarbose from fermentation broth. It was demonstrated that cation exchanger 001×4 displayed a large adsorption capacity and quick exchange rate for acarbose. The static adsorption equilibrium data were well fitted to the Langmuir equation. Column adsorption experiments demonstrated that high dynamic adsorption capacity was reached at bed height of 104.4 mm, feed flow rate of 1.0 ml·min 1and acarbose concentration of 4.0 mg·ml 1.Under the optimized conditions, the column chromatography packed with cation exchanger 001×4 recovered 74.3%(by mass) of acarbose from Actinoplanes utahensis ZJB-08196 fermentation broth with purity of 80.1%(by mass),demonstrating great potential in the practical applications in acarbose separation.

Synthesis of Monodisperse Poly(chloromethylstyrene divinylbenzene)Particle and Its Applications

Monodisperse poly (chloromethylstyrene divinylbenzene) particles were prepared by a two-step swelling method. It was demonstrated that the packings have comparative advantages for biopolymer separation with high column efficiency, low interstitial volume and low back pressure to the packings prepared with suspension polymerization method. Using size exclusionchromatography and weak ion exchange chromatography as examples, it was shown that these packings could be modified chemically more easily than poly (styrene divinylbenzene) packings.

ISOLATION AND PURIFICATION OF ALKALOIDS FROM PLUMULA NELUMBINIS BY DOUBLE-COLUMN ADSORPTION CHROMOTOGRAPHY

The performance of adsorption and separation for liensinine, isoliensinine and neferine was studied by double-column adsorption chromatography using macroporous adsorption and cation exchange resins. The alkaloid extract with 49.2% total contents by mass representing 10.6% liensinine, 10.6% isoliensinine and 28.0% neferine respectively was prepared by D72 cation exchange resins, in which most of the impurities were water-soluble alkaloids. Furthermore,the alkaloid extract with 82.6% total contents by mass containing 33.1%, 15.0% and 34.5% of the three adsorbates respectively was prepared by double-column adsorption chromatography using AKS-W macroporous adsorption and D72 cation exchange resins. As a result, the content of single and total alkaloids has been greatly increased by the double-column adsorption chromatography.

Elution Behaviour of Monocarboxylic Acids on a Cation-exchange Resin Column

The retention mechanism of monocarboxylic acids on a cation-exchange resin column was investigated. It was assumed that both Donnan membrane equilibrium and adsorption equilibrium were involved in the chromatographic process. On the basis of the proposed mechanism, an equation was derived for correlating distribution coefficient, Kd, dissociation constant, Aa, and adsorption equilibrium constant, K, of the analyzed acid. By this approach, retention data for some aliphatic acids under different operating conditions were predicted. Results are reasonably in agreement with experiment.

离子色谱法同时测定北京市不同区域水体6种阳离子

建立离子色谱法测定不同区域水体Li^(+)、Na^(+)、NH_(4)^(+)、K^(+)、Ca^(2+)、Mg^(2+)等6种阳离子的方法,以及验证方法在实际应用的可行性.以ICS1000型离子色谱仪配有DionexIonPac CG12A阳离子保护柱(4 mm×50 mm)和CS 12A阳离子分离柱(4 mm×250 mm),CSRS 300型抑制器(4 mm),甲基磺酸为淋洗液,流量为1.0 mL·min^(−1),6种阳离子的质量浓度在0.01—0.50 mg·L^(-1)到5.0—100.0mg·L^(-1)范围内线性较好,相关系数>0.9995,方法检出限为0.002—0.020 mg·L^(-1),与有证标准溶液相对误差−2.50%—4.38%(n=6),不同环境水样加标回收率90.7%—107.5%.利用以上色谱条件分析北京市不同区域地表水阳离子分布特征,结果显示城乡结合部、城区和郊区水体pH稳定,呈弱碱性;城乡结合部水体6种阳离子总浓度为150.05 mg·L^(-1),明显高于城区和郊区25.35%和63.54%,阳离子浓度均值大小为:Ca^(2+)>Na^(+)>Mg^(2+)>K^(+)>NH_(4)^(+)>Li^(+),其中Ca^(2+)和Na^(+)是3个区域水体的优势离子,NH_(4)^(+)和Li^(+)质量浓度最低;城乡结合部和城区水体阳离子浓度在夏季和秋季低于春季和冬季.实验结果表明,离子色谱法样品处理简便,分析速度快,准确可靠,灵敏度高,重复性好,适用于地表水中6种水溶性阳离子的快速批量测定.

离子色谱法测定不同生长期烟叶中的5种阳离子

【目的】研究不同生长期烟叶中阳离子含量的变化规律。【方法】新鲜烟叶样品以0.05 mol/L盐酸水溶液(HCl)为提取溶剂,经RP 1.0cc固相萃取柱净化,采用梯度淋洗程序,基于离子色谱法对不同生长期烟叶中的NH_(4)^(+)、K^(+)、Na^(+)、Ca^(2+)、Mg^(2+)进行定量分析。【结果】(1)该方法中5种阳离子在1~100 mg/mL范围内具有较好的线性相关性,其检出限在0.01~0.07 mg/mL之间,平均加标回收率在96.9%~101.2%之间,相对标准偏差均在6%以内。(2)5种阳离子的含量在不同生长时期有明显差异:K+与NH4+在苗期时含量最高,成熟期时含量最低;Na^(+)在苗期时含量相对较高,现蕾期与成熟期的含量无显著性差异;Mg2+在苗期时含量较低,现蕾期与成熟期时含量升高,且无显著性差异;Ca^(2+)在苗期时含量较低,现蕾期与成熟期含量显著增加。【结论】不同生长期的烟叶中,各阳离子的变化规律差异较大,研究结果可为后续烟叶品质与内在阳离子含量的关联分析提供技术支撑和数据积累。

离子色谱法快速测定秦陵土遗址中无机阴、阳离子含量

研究发现,可溶盐在土遗址内反复溶解重结晶产生的体积膨胀,是导致其产生酥粉、脱落、盐析病害的主要原因,严重影响着遗址类文物的安全与稳定。因此,对土遗址可溶盐组成、分布的系统性研究是判断文物病害成因、病害发展趋势的基础性工作。建立离子色谱技术快速测定秦陵土遗址中无机阴、阳离子的方法,有利于对土遗址或埋藏文物面临的潜在风险进行有效识别,从而为文物预防性保护提供数据支撑。以30 mmol/L氢氧化钾溶液作为淋洗液用于5种阴离子(F^(-)、Cl^(-)、SO_(4)^(2-)、NO_(3)^(-)、PO_(4)^(3-))的分离及20 mmol/L甲烷磺酸(MSA)溶液作为淋洗液用于5种阳离子(Na^(+)、NH_(4)^(+)、K^(+)、Mg^(2+)、Ca^(2+))的分离,测定中采用抑制电导检测器。结果表明,几种离子的质量浓度均在一定范围内呈线性关系,F^(-)、Cl^(-)、SO_(4)^(2-)、NO_(3)^(-)、PO_(4)^(3-)等5种阴离子的检出限分别为0.003、0.002、0.001、0.001、0.003 mg/L,Na^(+)、NH_(4)^(+)、K^(+)、Mg^(2+)、Ca^(2+)等5种阳离子的检出限分别为0.003、0.004、0.004、0.005、0.010 mg/L,阴离子的加标回收率在95.0%~101%,阳离子的加标回收率在91.0%~99.0%。方法具有精密度高、准确度好、分析速度快等优点,可用于秦陵土遗址中常见无机阴、阳离子的快速测定。