离线pH梯度-强阳离子交换色谱法分离肽段混合物

复杂肽段混合物的有效分离是高覆盖率地鉴定蛋白质混合物的前提。Shotgun蛋白质组学研究通常采用二维液相色谱(强阳离子交换色谱-反相色谱)分离后接串联质谱检测的方法。但由于离子交换色谱体系中含有高浓度的盐,使得在线分析的难度较大;而在离线分析时,也常因需要对高盐组分进行脱盐处理而易引起样品损失。因此,该文尝试用pH梯度替代盐梯度,实现pH梯度-强阳离子交换色谱方法应用于复杂肽段混合物的分离。通过对缓冲体系pH值的计算,优化了乙酸-乙酸铵体系线性pH梯度配合盐梯度的离子交换色谱方法,以及柠檬酸-氨水体系线性pH梯度的离子交换色谱方法。将这两种方法应用于牛血清白蛋白酶切产物的分离取得了与常规强阳离子交换色谱相似的分离效果。乙酸-乙酸铵体系采用的是低浓度的可挥发性铵盐,采用真空冻干的方法可以有效除盐,基质辅助激光解吸质谱靶上自然挥发也可以达到较好的脱盐效果,简化了常规方法繁琐费时的脱盐步骤及避免了由此造成的样品损失。柠檬酸-氨水体系采用pH梯度洗脱替代盐梯度洗脱,大大降低了体系中的盐浓度。这两种方法在复杂体系蛋白质组研究的样本预分离中具有较好的应用前景。     

用阳离子交换树脂和pH梯度洗脱分离头孢菌素混合物

合成了一种聚苯乙烯结构的凝胶型1×2强酸性阳离子交换树脂,结合pH梯度或盐浓度梯度洗脱法分离头孢菌素类抗生素的混合物,并与市售的树脂比较,考察其分离性能。1×2型树脂具有较小的颗粒度(0.2～0.3mm)和较小的交换容量(在60%乙醇溶液中),用pH梯度洗脱法可以将5种头孢菌素按照等电点顺序完全分离,结构稳定的3种头孢菌素的收率均大于95%,1×2型强酸性阳离子交换树脂结合pH梯度洗脱法适用于水溶性头孢菌素类抗生素的制备分离。     

刺激响应梯度聚合物

近年来,刺激响应聚合物因其独特的性质而受到广泛关注,这类聚合物能够感受外界环境刺激(包括光、电、热、力、氧化还原、pH、化学、环境和生物信号等刺激)而产生响应,从而引起体系物理或化学性质的改变。而梯度聚合物则是伴随着可控自由基聚合技术的诞生、发展而出现和形成的一类新型聚合物,区别于无规和嵌段聚合物,梯度聚合物表现出单体组成沿分子链渐变的性质,其中,由亲水性单元和憎水性单元组成的两亲性梯度聚合物在选择性溶剂中可自组装形成多种聚集体,因此具有多方面的潜在应用价值,比如超分子组装体、智能涂料、网络或这些可能应用的组合。本文调研了有关梯度聚合物方面的研究工作,综述了几种梯度聚合物的合成方法,如活性阴离子聚合法、可控自由基聚合法(包括氮氧稳定自由基聚合、原子转移自由基聚合及可逆加成断裂链转移自由基聚合)和开环聚合法等,同时阐述了梯度聚合物的自组装及刺激响应行为,尤其是其独特的热和pH敏感性。此外,还对由两亲性嵌段聚合物和两亲性梯度聚合物自组装形成的聚集体的刺激响应行为进行了详细地论述和比较,并指出了梯度聚合物的应用前景和发展方向。     

偏最小二乘法—流动注射pH梯度技术用于同时测定铜和钴

以PAR作显色剂,用流动注射pH梯度技术测定多个不同pH下的吸光度,以偏最小二乘法建立校正模型并预测,对Cu~(2+)、Co~(2+)二元素进行了同时测定,其计算结果优于主成分回归及多元线性回归法。     

高效液相色谱法同时检测蜂蜜中的5类抗生素残留

建立了蜂蜜中氯霉素类、硝基咪唑类、喹诺酮类、磺胺类、四环素类5类共15种抗生素残留同时检测的高效液相色谱分析方法.蜂蜜经pH 2.0的磷酸水溶液溶解后直接用PCX固相萃取小柱净化富集,以乙腈-磷酸溶液(pH 2.5)作流动相,梯度洗脱,紫外检测器在274、315 nm波长下进行检测.15种抗生素在0.2 ～20.0 m...     

薄层等电聚焦技术及其在生化分析中的应用

等电聚焦是1966年建立起来的一种高分辨率的蛋白质分离分析新技术,10多年来发展很快。它的基本原理是利用蛋白质分子或其它两性分子等电点的不同,在一稳定、连续、线性的pH梯度中进行蛋白质分离分析。     

离子交换法分离固相合成胸腺五肽

采用离子交换法分离纯化,对影响分离的多种因素包括吸附动力学、pH值、缓冲液和树脂的种类进行了深入讨论.结果发现,采用SP sepharose FF树脂、pH 4.4的醋酸缓冲液分离效果较好,经过方案优化,在pH 4.4醋酸缓冲液平衡、盐梯度洗脱条件下纯化TP5,HPLC和质谱分析表明,一步分离纯度可达98%以上.该法可以满足大规模制备的要求.     

高效液相色谱-电感耦合等离子体质谱同时测定虾类中6种砷形态

建立了高效液相色谱-电感耦合等离子体质谱(HPLC-ICP-MS)同时测定虾类中6种砷形态的方法。研究了不同流动相、梯度洗脱方法对6种砷形态化合物的分离效果。在优化的质谱条件下,采用pH=8.5的50 mmol/L(NH4)2CO3-甲醇(99∶1,V/V)溶液及pH=8.5的0.5mmol/L(NH4)2CO3-甲醇(99∶1,V/V)溶液为流动相进行梯度洗脱,10 min之内6种砷形态达到基线分离。同时采用0.5 mmol/L(NH4)2CO3-甲醇(99∶1,V/V)溶液(pH=8.5)作为提取剂能较好地提取养殖虾样品中的砷形态。该联用技术重现性好,能快速地同时测定养殖虾中6种砷形态化合物。     

反相高效液相色谱双梯度洗脱分离肽混合物及质谱分析

复杂肽段混合物的有效分离是高覆盖率地鉴定蛋白质混合物的前提。“鸟枪法”(Shotgun)蛋白质组学研究策略通常采用蛋白酶切、二维液相色谱-串联质谱分析肽段混合物从而鉴定蛋白质,其中高效率地分离肽段混合物是关键步骤之一。本文通过pH梯度结合有机溶剂梯度的反相高效液相色谱(RP-HPLC)进行一维液相色谱分离,按等时间间隔收集馏分并将一个梯度的前段的一个馏分与后段一个馏分混合,然后进行纳升级液相色谱-质谱联用(nanoRPLC-MS/MS)分析。将该方法应用于酵母蛋白质的分离和鉴定,实验结果为: 与常规的强阳离子色谱-反相液相色谱-质谱分离鉴定方法相比,采用pH梯度结合有机相梯度的RP-HPLC-RPLC-MS分离鉴定方法多鉴定到567个酵母蛋白质(簇,含有3035个唯一肽段);其中鉴定到肽段的pI分布范围为3.42～12.01,相对分子质量范围为587.67～3499.79;蛋白质的pI分布范围为3.82～12.19,相对分子质量范围为3446.55～432905。该结果表明这种方法在复杂体系蛋白质组分离鉴定中具有明显的优势,在蛋白质组学研究中有较好的应用前景。     

离子色谱—积分安培检测法测定气溶胶中的氨基酸

离子色谱直接测定氨基酸方法采用的是氢氧化钠／醋酸钠梯度淋洗，金电极为工作电极，pH电极为参比电极，积分脉冲安培检测，同时测定17种α—氨基酸及部分糖类和糖胺。该方法测定氨基酸灵敏度高，但由手梯度而造成的漂移较大-且高浓度的醋酸钠，使色谱的系统压力较高，在梯度过程中的系统压力有很大的差异。因此，本实验对该方法进行了     

Spectrophotometric evaluation of acidity constants of tetra-step overlapping equilibria

The simultaneous evaluation of acidity constants of tetra-step overlapping equilibria from spectrophotometric data may be carried out if the absorbance versus pH graph of a tetra-protic acid at a given wavelength shows two maxima and a minimum of two minima and a maximum. In such cases, a tetraprotic acid may be treated like a monoprotic one by substituting the pH values with a function of the pH values which have corresponding absorbances.     

Optimization of performance and minimization of silicate interference in continuous flow phosphate analysis

Specific reaction conditions for automated continuous flow analysis of phosphate are optimized in regard to minimizing coating and silicate interference, while maintaining high sensitivity. Use of Sb in the reagent increases sensitivity and yields absorbances with little temperature dependence. Coating can be minimized by using a final solution at a pH>0.5. At final pH of 0.78 there is maximum interference from silicate in the sample. We recommend therefore as an optimal reaction condition with minimal silicate interference, the use of Sb, a final solution pH of 1.00, room temperature for the reaction and a [H(+)]/[Mo] ratio of 70. An equation is provided to correct silicate interference in high precision phosphate determination.     

Colorimetric determination of low pH with Malachite Green

A spectrophotometric method was developed for determination of concentration-based pH values from 0 to 2 with Malachite Green indicator. A quadratic model equation was based on the ratio of the absorbances of the peak at 618 nm and the isosbestic point at 518 nm. Normalization to the isosbestic point was used to stabilize the response because the color faded; the useful time interval was within 5 min after indicator addition. Model and verification sets agreed within +/-0.02 pH units between pH 0.3 and 1.8. This excellent precision makes the colorimetric method useful for acid determinations with a relative precision of > +/-5%. The presence of salts at a salt/acid equivalent ratio >0.1 caused a low pH bias.     

Homogeneous permanganate oxidation in non-aqueous organic soution. A stable organomanganese intermediate in the oxidation of endo-dicyclopentadiene

The absorption spectrum of a stable organomanganese intermediate in the homogeneously catalyzed permanganate oxidation of endo-dicyclopentadiene in a non-aqueous system was directly determined. The formation and the decomposition of the intermediate were monitored by following the changes of absorbances.     

双波长双指示剂催化光度法测定铜的研究

研究了在pH 4.0的HAc～NaAc缓冲介质中,利用痕量铜(Ⅱ)催化过氧化氢氧化罗丹明B和亚甲基蓝褪色的指示反应,通过测量540 nm和660 nm下,催化体系和非催化体系吸光度的变化,建立了双波长双指示剂催化动力学光度法测定痕量铜的新方法.方法的线性范围为0.00080～0.048μg/mL,检出限为4.0×10~(-11) g/mL.方法可用于水中铜的测定.     

Studies on fluorescein-VIII Notes on the mathematical work-up of absorbance data for systems with single and multiple dissociations, the limitations of the conventional logarithmic treatment, and the dissociation constants of fluorescein

A mathematical study has been made of a commonly used method for obtaining dissociation constants from data for absorbace as a function of pH. Once the limiting working absorbances have been selected (usually simply the asymptotes to the curve of absorbance vs. pH), the value for the dissociation constant is fixed by the average of the values chosen and no treatment of the data, by graphical or least-squares methods, will alter the result. The mathematical reason for this is explained. An alternative procedure based on measuring the point of inflection of the curve and the slope through this point has been developed. Certain limitations of the Rosenblatt method for obtaining dissociation constants from absorbance data are also pointed out.     

Studies on fluorescein-VI Absorbance of the various prototropic forms of yellow fluorescein in aqueous solution

The absorbance of yellow fluorescein in water, at ionic strength 0.10, as a function of pH at 437, 455, 464, 475 and 490 nm has been resolved into four components, the absorbances of the individual prototropic forms of fluorescein in water. The molar absorptivity of each species at each of the five wavelengths is reported. A novel type of isosbestic point is described.     

A rapid and precise computer-aided method for the spectrophotometric determination of substances in solution

The Newton—Raphson iteration method for the solution of mass-balance equations is used. Given accurate spectrophotometric data (absorbances, absorptivities) and equilibrium constants, this procedure permits rapid determination of substances in solution. The program is convenient to use and gives satisfactory results on a number of systems. The pyridoxal hydrochloride system was selected for evaluation of equilibrium constants and molar absorptivities from spectrophotometric and potentiometric data and was then used as the main test of the SPEDEAD program (SPEctrophotometric DEtermination by Absorbance Data).     

Indicator characteristics of bromothymol blue derivatives

Some Bromothymol Blue derivatives with a nitro, amino, isothiocyanato or sulfonamide group substituted on the sulfonated ring of the dibromothymolsulfonephthalein have been studied spectrometrically. All the dyes have two characteristic absorption peaks which can be used to measure pH in the physiological range. The molar absorptivities, wavelengths of maximum absorption and pK(a) values have been determined from the absorbances, and are similar for all four dyes.     

Sequential determination of iron and titanium by flow-injection analysis

A flow-injection method has been developed for the sequential spectrophotometric determination of iron and titanium using 3,4 dihydroxybenzoic acid as chromogenic reagent. The system involves the sequential measurement of the absorbances of the complexes at 380 and 570 nm. The system is designed using a simultaneous injection of sample and reagent into separate carrier streams. The proposed method is characterized by a precision of about 2%, a sampling rate of about 50 samples per hour, and a reagent consumption of 200 mul (0.50% solution) per sample. It is relatively free of interferences and was used for the sequential determination of titanium and iron in rocks.