双水相萃取结合液相色谱法分离蛋白质

建立了PEG/( NH4)2SO4双水相体系萃取富集,结合液相色谱分离分析多种蛋白质的方法.考察了无机盐种类和浓度、PEG分子量、pH值和温度等因素对双水相形成以及对细胞色素C、肌红蛋白、牛血清白蛋白、溶菌酶、胰蛋白酶分配行为的影响.结果表明,上述5种蛋白在室温、pH 3.5～9.0范围内,可在15％ PEG-4000/10％ (NH4)2SO4双水相体系中得到富集,且主要集中在下相.同样条件下,血清中的高丰度蛋白在上下相均有分配,下相分配量较大.通过双水相萃取分离蛋白质及对液相色谱一定时间段的色谱峰收集,可初步实现血清中高丰度蛋白质的分离去除.     

通过一步萃取将组蛋白分为多个亚组分的蛋白质组预分离方法的建立

将基于正丙醇、氯化钠和水的双水相体系用于蛋白质组的预分离研究, 并考察了体系酸度及在不同浓度的盐、正丙醇及蛋白质存在时该双水相体系对蛋白质的分离效果. 将经该双水相体系预分离过的蛋白质组样品在未与成相试剂分离的条件下直接用于凝胶电泳分析. 结果表明, 该双水相体系可通过一步萃取将蛋白质组样品分为3个亚组群. 该蛋白质组预分离方法简单、快速、成本低, 并具有生物相容性、可连续操作性、无需昂贵复杂仪器以及在进行电泳分析前无须(或易于)将目标蛋白与成相试剂分离等优点. 该蛋白质组预分离方法的建立在蛋白质组学和方法学方面均有着极为重要的意义.     

聚焦微波辅助双水相萃取/高效液相色谱法测定农吉利中牡荆素与异牡荆素

以乙醇-硫酸铵双水相体系为萃取溶剂,采用聚焦微波辅助萃取法萃取农吉利中的牡荆素和异牡荆素,HPLC测定,建立了微波辅助双水相萃取(FMAATPE)/HPLC方法测定牡荆素和异牡荆素含量的分析方法。利用单因素试验和正交试验设计方法优化了乙醇质量分数、微波功率、料液比、萃取时间等萃取条件以及色谱分析条件。萃取优化条件为:双水相的组成:35%乙醇-16%硫酸铵,药材颗粒度:80目,料液比:1∶50,微波功率:140 W,萃取时间:20 min。以乙腈-0.5%磷酸(14∶86)为流动相在340 nm检测可较好地分离目标组分。将该方法用于农吉利药材中牡荆素和异牡荆素的萃取测定,可获得满意结果,其回收率为96.9%~103.8%,RSD为1.9%~2.6%。     

亚硝基R盐液-固萃取体系分离Co(Ⅱ)、Ni(Ⅱ)、Pd(Ⅱ)

亚硝基Ｒ盐液┐固萃取体系分离Ｃｏ（Ｉ）、Ｎｉ（Ｉ）、Ｐｄ（ＩＩ）孙小梅彭文沈静茹（中南民族学院化学系武汉４３００７４）聚乙二醇（ＰＥＧ）２０００的无机盐水溶液在一定条件下可分成液－液两相［１］。此类双水相萃取体系多用于分离生物物质酶、蛋白质等［２］，...     

液相微萃取-高效液相色谱法快速测定唾液中尼古丁含量

建立了一种以液相微萃取为样品前处理技术,结合高效液相色谱快速、有效测定唾液中尼古丁含量的方法。确定了以磷酸三丁酯为有机溶剂、2 mL 0.05 mol/L KOH调节2 mL样品溶液为给出相,10 mmol/LKH2PO4(pH=3.0)为接收相;搅拌速率为500 r/min,萃取时间为17 min的尼古丁优化萃取条件。方法的线性范围0.1-50 mg/L,相关系数r2=0.9996;检出限为0.05 mg/L(S/N=3);相对标准偏差<5%(n=5);相对回收率为96.3%-102.2%。实验证明该法可用于唾液等生物体液中碱性物质的测定。     

十四烷基三甲基氯化铵/十二烷基苯磺酸钠混合表面活性剂双水相体系的萃取性能

研究了十四烷基三甲基氯化铵(TTAC)与十二烷基苯磺酸钠(SDBS)混合表面活性剂水溶液双水相体系的分相情况、萃取性能及两相的微观结构.结果表明,TTAC/SDBS混合表面活性剂水溶液在30℃下能够形成稳定的双水相体系;该双水相体系对亚甲基蓝、靛红都具有一定的萃取分离作用.其上、下两相的微观结构明显不同,这是其能够形成稳定双水相体系且具有萃取作用的重要原因.     

分散固相萃取-超声辅助分散液液微萃取/高效液相色谱法测定土壤中溴氰菊酯残留

建立了以分散固相萃取-超声辅助分散液液微萃取为样品前处理技术,结合高效液相色谱法(HPLC)测定土壤中溴氰菊酯。样品用甲醇∶水(1∶4,V/V)提取,经布氏漏斗减压抽滤,滤液经N-丙基乙二胺(PSA)、C18、石墨炭黑粉(GCB)净化后,用氯仿萃取,超声,离心后沉积相进行HPLC测定。对分散固相萃取吸附剂的选择及影响分散液液微萃取的因素进行了优化,在最优条件下,溴氰菊酯的富集倍数达到565倍,线性范围为0.005~2.5mg/kg,线性相关系数为0.9998,检出限为0.001mg/kg,平均加标回收率为70.3%~94.5%,相对标准偏差为2.5%~4.7%。该方法具有简便快速、准确灵敏、萃取效率高等特点,可用于土壤中溴氰菊酯残留检测。     

甲醇-磷酸氢二钾-水双水相萃取-高效液相色谱检测肉类产品中四环素类残留

以甲醇-无机盐-水双水相体系,建立了一种高效的用于液相色谱法检测肉类产品中四环素类抗生素残留的样品前处理方法。实验考察了双水相的形成条件以及对四环素类药物的提取效率,用液相色谱法与瑞利散射法研究了蛋白质对甲醇-无机盐-水双水相体系萃取四环素的影响。结果表明,只有加入适量的磷酸氢二钾才能形成甲醇水双水相体系,该双水相体系对极性较强的四环素类药物具有较高的萃取效率;体积分数为70%的甲醇水溶液作为萃取液可以使肉类产品中的蛋白质基质发生缓慢而彻底地变性,有利于释放与蛋白质结合的药物。由于甲醇-K_2HPO_4-水双水相体系的上相萃取液中水溶性蛋白等杂质较少,样品净化步骤大大简化,在上相中加入少量PSA与C_(18)固体吸附剂,离心后取上清液即可进行HPLC分析。该方法用于肉类产品牛肉、鱼肉中四环素残留的萃取测定,回收率为76.8%~98.2%,检出限为0.039~0.084μg·g~(-1)。     

离子液体在蛋白质萃取分离中的应用

离子液体具有独特的物化性质如热稳定性好、不挥发、不易燃和良好的生物兼容性,近年来作为传统有机溶剂的替代物在有机合成、电化学、催化和萃取分离等领域得到了广泛应用。本文简要综述了以离子液体为媒介的萃取体系在蛋白质分离富集和分析中的相关研究和应用进展,包括辅助萃取体系、直接萃取体系、双水相萃取体系、微乳萃取体系、结晶体系和基于离子液体的固相萃取体系等。     

乙腈-无机盐-水双水相体系萃取-气相色谱法检测鱼肉中拟除虫菊酯

以乙腈-无机盐-水双水相体系,建立了一种环保、高效的用于气相色谱法检测鱼肉样品中拟除虫菊酯的样品前处理方法.实验利用荧光猝灭法与气相色谱法研究了拟除虫菊酯药物在以蛋白质为主要基质的水产品中的存在状态,考察了双水相的形成条件,并探讨了蛋白质对双水相萃取率的影响.结果表明,拟除虫菊酯药物通过疏水作用力与蛋白质牢固结合.以体积分数为80%的乙腈水溶液作为萃取液可以引起水产品中的蛋白质基质缓慢而彻底地变性,从而充分释放与蛋白质结合的药物,实现拟除虫菊酯的均相高效萃取.80%的乙腈水溶液具有较强的化学极性,因而在双水相系统上相中与药物残留共同萃入的脂溶性杂质较少,萃取液净化步骤大大简化.在双水相上相中加入无水MgSO4,PSA去除水分与杂质后,直接进样进行气相色谱检测.该方法用于鱼肉样品中六种拟除虫菊酯的萃取检测,回收率为81.1%～96.4%,检出限为8～14ng·mL-1.     

Selective separation and enrichment of proteins in aqueous two-phase extraction system

A simple aqueous two-phase extraction system(ATPS) of PEG/phosphate was proposed for selective separation and enrichment of proteins.The combination of ATPE with HPLC was applied to identify the partition of proteins in two phases.Five proteins (bovine serum albumin,Cytochrome C,lysozyme,myoglobin,and trypsin) were used as model proteins to study the effect of phosphate concentration and pH on proteins partition.The PEG/phosphate system was firstly applied to real human saliva and plasma samples,some pro...     

Integration of carboxyl modified magnetic particles and aqueous two-phase extraction for selective separation of proteins

Both of the magnetic particle adsorption and aqueous two-phase extraction (ATPE) were simple, fast and low-cost method for protein separation. Selective proteins adsorption by carboxyl modified magnetic particles was investigated according to protein isoelectric point, solution pH and ionic strength. Aqueous two-phase system of PEG/sulphate exhibited selective separation and extraction for proteins before and after magnetic adsorption. The two combination ways, magnetic adsorption followed by ATPE and ATPE followed by magnetic adsorption, for the separation of proteins mixture of lysozyme, bovine serum albumin, trypsin, cytochrome C and myloglobin were discussed and compared. The way of magnetic adsorption followed by ATPE was also applied to human serum separation.     

高效液相色谱法分离和测定小麦中的5种内源激素

建立了高效液相色谱法(HPLC)用于分离和测定小麦中的吲哚乙酸(IAA)、脱落酸(ABA)、赤霉素(GA₃)、玉米素(ZT)和水杨酸(SA)5种植物内源激素。经过条件优化,选用甲醇作为样品提取溶剂。然后,经石油醚和乙酸乙酯萃取,经Sep-Pak C18小柱纯化。液相色谱的分离采用Eclipse XDB-C₁₈ (250 mm×4.6 mm, 5 μm)反相色谱柱;流速为1 mL/min;进样量10 μL。检测器波长设置为254 nm; 14.5 min时切换到240 nm; SA洗脱后即18 min时切换回254 nm。流动相A为甲醇,B为乙酸溶液(pH 3.6)。梯度条件为0~7 min, 20%A; 7~10 min, 20%A~28%A; 10~17 min, 28%A; 17~19 min, 28%A~40%A; 19~35 min, 40%A。结果表明,小麦中各激素的分离效果理想,加标回收率达96.9%~98%,相对标准偏差在1.54%~2.29%之间。因此,该方法的建立为快速、准确地分离和测定小麦内源激素提供了可靠的方法。     

单分散亲水两性离子交换树脂的制备及其在生物大分子分离中的应用

采用分散聚合与溶胀聚合相结合的方法及高分子溶液致孔技术, 成功地制备了粒径为5.0 μm大孔和超大孔结构的单分散亲水性交联聚甲基丙烯酸环氧丙酯树脂, 并进行了结构表征. 将该树脂经胺化后再与1,3-丙磺酸内酯反应, 得到一种新型的两性离子交换(强阳-强阴型)高效液相色谱填料. 研究了该填料对标准蛋白分离性能及流动相中有机溶剂、 流速和pH值对蛋白保留的影响. 实验结果表明, 在流速为3 mL/min时, 采用线性梯度洗脱, 在4.0 min内可同时快速基线分离3种酸性和2种碱性蛋白.     

Evaluation of capillary liquid chromatography-electrospray ionization ion mobility spectrometry with mass spectrometry detection

Due to the proteomics revolution, multi-dimensional separation and detection instruments are required to evaluate many peptides and proteins in single samples. In this study, electrospray ionization (ESI) ion mobility spectrometry (IMS) was evaluated as an additional separation after HPLC separations. Common HPLC mobile phase compositions (solvents, acid modifiers, and buffers) were assessed for the effect on ESI-IMS response. Up to 5 mM sodium phosphate, a non-volatile buffer, was able to be electrosprayed into the IMS without degradation of the instrumental performance. Due to the rapid separation times of IMS, multiple IMS spectra were obtained within a single HPLC peak. A five-peptide mixture was separated in a capillary HPLC column under isocratic conditions within 3 min. Coelution of two peaks due to non-optimal HPLC conditions occurred and these two peaks could not be distinguished by HPLC with UV detection. In contrast, the single ion mobility chromatograms provided separation of each peptide as well as providing a second degree of analyte identification (HPLC retention time and IMS mobility). Furthermore, IMS-MS analysis of the five peptides and comparison with HPLC retention times showed that each peptide had a unique retention time-ion mobility-mass to charge value. This work showed that IMS could be employed for direct separation and detection of HPLC eluents and also could be combined with HPLC-MS for three unique dimensions of separation.     

高效液相色谱法同时测定尿液中4种非蛋白氮的含量

建立了高效液相色谱(HPLC)同时测定尿液中4种常见的非蛋白氮物质--肌酸(Cr)、尿肌酐(Cn)、尿酸(Ua)及假尿嘧啶核苷(Pu)含量的方法。尿液首先经丙酮沉淀法除去大分子化合物,然后冷冻干燥,复溶后直接进行HPLC分析。色谱分离选用Waters RP18 Column (150 mm×4.60 mm,3.5 μm),以10.0 mmol/L磷酸缓冲溶液 (pH 4.78)和乙腈为流动相进行梯度洗脱,流速为0.8 mL/min,于220 nm波长下检测,可在7 min内完成4种非蛋白氮的快速分离分析。结果表明Cr、Cn、Ua、Pu在0.1~250 mg/L范围内线性关系均良好(相关系数均大于0.999),检出限分别为9.31、26.19、4.70、6.30 μg/L,加标回收率为81%~111%,峰高的相对标准偏差(RSD, n=3)为0.23%~2.78%,满足定量要求。该法简便、快速,结果准确可靠,为2型糖尿病(T2DM)患者肾功能损坏的研究提供了检测手段。     

Separation of rat pancreatic secretory proteins by cation-exchange fast protein liquid chromatography.

Rat pancreatic secretory proteins were separated by an automated liquid chromatography system utilizing a Mono S cation-exchange column. Optimal resolution was obtained with a multistep salt and pH gradient (0.01-2 M LiCl, pH 5.3-63). A total of fourteen well-separated peaks, as well as several minor peaks, were detected by UV absorption. The main pancreatic enzymes were resolved (two amylases, two chymotrypsinogens, two trypsinogens, proelastase, lipase, prophospholipase A2, procarboxypeptidase A, procarboxypeptidase B, and ribonuclease). In addition, proteins without enzymic activity, such as lithostathine and pancreatitis-associated protein, were identified. Activation of proenzymes did not occur during the separation. At a flow-rate of 0.5 ml/min, ca. 250 micrograms to 5 mg of protein could be applied with equal resolution. The reproducibility of retention volumes and peak areas was high (less than 1% or 5% variation, respectively). When radiolabeled proteins were separated, a comparable pattern of peaks was obtained. The technique described is, therefore, not only useful for analytical and preparative separation of pancreatic proteins but can additionally serve for quantitative determination of the pancreatic isoenzyme pattern.     

Separation of urine proteins on the anion-exchange resin mono Q

Proteins excreted in urine due to renal failure were separated on Mono Q, a new strong anion exchange designed for fast high-resolution protein separations. The separation procedure was divided into two steps. The first step involved removal of low-molecular-weight substances by rapid desalting on a Sephadex G-25 Superfine column. In the second step, the total protein fraction (3--6 ml) was loaded onto the Mono Q column with the aid of a superloop. The proteins were adsorbed onto the top of the ion-exchanger column and gradually displaced by a combined pH and salt gradient in 40 min. The choice of ion exchanger and initial operating conditions were based on data obtained from electrophoretic titration curve experiments. Identification of separated proteins was achieved by fused rocket electrophoresis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively.     

High-performance liquid chromatography of seized drugs at elevated pressure with 1.7 microm hybrid C18 stationary phase columns

High-performance liquid chromatography (HPLC) separation of drugs at elevated pressure with 1.7 microm hybrid C18 stationary phase columns was investigated. This technique, which uses instrumentation engineered to handle the narrow peaks and high back pressures generated by 1.7 microm particle columns, provided significantly better resolution and/or faster analysis than conventional HPLC and capillary electrophoresis (CE). The use of 2mm internal diameter (i.d.) columns of 3-10 cm length has been evaluated for the separation of basic and neutral drugs, drug profiling, and general screening (including acidic drugs). For these applications, compared to conventional HPLC and CE, it provided up to 12x and 3x faster analyses, respectively. Precision was excellent for both isocratic and gradient analyses. For retention time and peak area, RSDs of < or =0.1% were obtainable. Fifteen anabolic steroids and esters were well separated in a 2.5 min gradient. For drug profiling, compared to HPLC and CE, approximately twice as many peaks were resolved. HPLC at elevated pressure is also well suited as a general screening technique. Twenty-four solutes of varying drug classes including narcotic analgesics, stimulants, depressants, hallucinogens, and anabolic steroids were fully separated in a 13.5 min gradient.