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.     

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%。     

双水相超声萃取-分光光度法测定蒲公英中总黄酮

向50.0mg蒲公英粉中加入正丙醇2.00mL及磷酸二氢钠2.5g,于50℃进行双水相超声萃取30min。经过滤并静止分层后,黄酮进入正丙醇相中,使其与水相分离后,将正丙醇相蒸干,所得残渣用乙醇(1+1)溶液溶解定容至50mL,在510nm波长处测量其吸光度。黄酮的质量浓度在8.0mg·L~(-1)以内与其对应的吸光度呈线性关系。方法用于蒲公英中总黄酮的测定,测得萃取率达4.12%。     

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。     

双水相体系萃取分离-紫外分光光度法测定桑叶中芦丁

通过试验选择聚乙二醇(PEG 1000)与聚乙烯吡咯烷酮(PVP 30000)为组合表面活性剂,(NH4)2SO4-H2O为双水相体系。在VPEG 1000/VPVP 30000=2.00 mL/2.00 mL,(NH4)2SO4用量为1.5 g,pH 6.0的B.R缓冲溶液最佳萃取条件下,经两次萃取,桑叶中芦丁定量进入PEG-PVP相。用紫外分光光度法在375 nm波长处直接萃取测定桑叶提取液中的芦丁含量。试验结果表明,方法的相对标准偏差≤3.8%(n=7),样品加入回收率为96.5%。试验表明,方法具有良好的选择性。此外,用试样超声提取率较常规索氏提取率高。     

双水相萃取/超高效液相色谱-串联质谱法测定倒提壶中8种生物碱

建立了乙醇-磷酸氢二钾双水相萃取技术结合超高效液相色谱-串联质谱法(UHPLC-MS/MS)同时测定倒提壶中8种生物碱含量的方法。采用单因素实验考察了双水相的萃取条件对倒提壶中8种生物碱萃取率的影响,最佳萃取条件为:乙醇质量分数为30%,K_2HPO_4·3H_2O的质量分数为20%,萃取剂与样品相比为6∶1,水相的pH值为4.0。以乙腈-0.1 mol/L醋酸铵为流动相进行UHPLC-MS/MS分析,8种生物碱之间具有良好的分离度,各成分在一定质量浓度范围内具有良好的线性关系(r~20.99)。方法的精密度、重复性及加标回收率的相对标准偏差(RSD)均小于3.0%。方法简单快捷、灵敏准确,可满足倒提壶中生物碱检测的要求。     

液相微萃取-高效液相色谱法测定尿样中的利多卡因

应用液相微萃取与高效液相色谱联用技术快速分析尿样中的利多卡因。考察了萃取溶剂、体积、萃取时间及料液pH值对液相微萃取的影响,建立了液相微萃取与高效液相色谱联用技术分析尿样中利多卡因的方法。优化的实验条件为：料液pH值12．0,萃取溶剂为5μL邻苯二甲酸二丁醅,萃取时间40min,搅拌速度80r／min。方法的线性范围为0．2-5mg／L;检出限为0．1mg／L;相对标准偏差小于6．3％。通过液相微萃取后,能有效地去除检测尿样中利多卡因的干扰物质,获得了较高的选择性。该方法简便、快速、灵敏、消耗有机溶剂少,是尿样中利多卡因检测的一种有效方法。     

固相萃取-高效液相色谱法测定水产品中喹乙醇

建立了固相萃取-高效液相色谱法测定水产品中喹乙醇的方法。匀浆后的水产品试样经甲醇溶液提取、正己烷除脂、固相萃取小柱直接净化浓缩后,以体积分数15%的甲醇溶液作为流动相,流速:1.0 m L/min,采用Symmetryshield TM RP18色谱柱进行分离,紫外检测器检测,外标法定量。方法的检出限和定量限分别为0.02 mg/kg和0.05 mg/kg,浓度在0.05~1.00 mg/L范围内,线性良好(r=0.9999)。添加浓度在0.05~1.00 mg/kg时,不同品种水产品的回收率在78%~99.6%之间,相对标准偏差小于7.7%。方法适合于水产品样品的成批定量检测。     

碱性离子液体水解-双水相胶束体系提取-超高效液相色谱法测定水产品中胆固醇的含量

基于碱性离子液体水解以及双水相胶束体系(ATPMS)提取,以超高效液相色谱法(UHPLC)测定水产品中胆固醇的含量,并以分配系数为指标对ATPMS组成进行了优化.称取0.10 g肉糜状水产品,加入0.36 mol·L-1氢氧化1-丁基-3-甲基咪唑盐([C4 mim]OH)碱性离子液体1 mL,于90℃加热30 min...     

双水相萃取-液相色谱法检测人尿中的羟基多环芳烃

建立了乙腈-(NH4)2SO4双水相提取,液相色谱-荧光法同时检测人尿中10种羟基多环芳烃的分析方法。尿样中结合态的羟基多环芳烃用β-葡萄糖苷酸酶-芳基硫酸酯酶缓冲液(pH 5.0)于37℃下恒温震荡4 h后,以乙腈-硫酸铵双水相提取富集、净化,采用ZORBAX Eclipse C18色谱柱分离,流动相为乙腈-水,梯度洗脱,荧光检测器检测,外标法定量。方法用有机化合物激发光谱和发射光谱的"镜像"关系确定并优化了化合物的激发波长和发射波长,讨论了无机盐、萃取剂的选择以及pH、离子强度、乙腈析出体积、静置时间、甲醇含量等对测定的影响。10种羟基多环芳烃线性范围为5.00～200.00 ng/mL,相关系数大于0.999,平均回收率为43.6%～94.4%,相对标准偏差为4.2%～9.2%,检出限为0.06～2.32 ng/mL。方法可用于尿中10种羟基多环芳烃的测定。     

丙酮双水相萃取预富集荧光法测定痕量1-萘胺

基于丙酮水溶液在适当电解质作用下能形成双水相及1-萘胺能发射荧光的特点,建立了一种丙酮-电解质-水体系双水相萃取、蠕动泵进样荧光法测定1-萘胺的新方法.研究了电解质的种类和用量、丙酮体积、1-萘胺浓度、温度、酸度和共存物等的影响.发现在最佳萃取体系丙酮-K3PO4-H2O中,1-萘胺的一次性萃取率82.7%～89.9%,测定1-萘胺的线性范围7.2×10-8～6.0×10-1 mol/L,检出限2.6×10-9 mol/L.方法用于强化水样和豆芽样品中痕量1-萘胺的测定,回收率96%～106%,相对标准偏差2.8%～5.8%.     

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...     

Purification of phospholipase D by two-phase affinity extraction

An aqueous two-phase system of polyethylene glycol (PEG)-salt was used for purification of phospholipase D (PLD) from peanuts and carrots. Alginate, a known macroaffinity ligand for PLD, was incorporated in the PEG phase and resulted in 91 and 93% of the enzyme activity (from peanuts and carrots, respectively) getting partitioned in the PEG phase. The elution of the enzyme from alginate was facilitated by exploiting the fact that the latter can be reversibly precipitated in the presence of Ca2+. The enzyme was eluted from the polymer by using 0.5 M NaCl. Peanuts and carrots PLD could be purified 78- and 17-fold with 82 and 85% activity recovery, respectively. The purified enzyme from both sources gave a single band on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis.     

Influence of magnetic field on aqueous two-phase extraction of horse ferritin in the polyethylene glycol/hydroxyethyl starch system

The presented experiments show the model of expectation of equine spleen ferritin extraction in a new aqueous two-phase system which was formed by mixing polyethylene glycol (PEG) and hydroxyethyl starch (HES). The tendency of the protein to migrate in the analyzed systems was dependent on the concentrations of HES and PEG as well as PEG molecular weight. The highest concentration of ferritin in the top phase (rich in PEG) was recorded in the system composed of 6% PEG 3000 and 3% HES. The obtained concentration was 0.88 mg mL⁻¹. The lowest concentration was 0.42 mg mL⁻¹ in the system composed of 5% PEG 6000 and 1% HES.     

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.     

Drop formation in aqueous two-phase systems

Extraction using aqueous two-phase systems (ATPSs) is a versatile technique for the downstream processing of various proteins/enzymes. The study of drop formation deals with the fundamental understanding of the behavior of liquid drops under the influence of various external body as well as surface forces. These studies provide a basis for designing of the extractions in column contactors in which liquid drops play a major role. Most of the drop formation studies reported so far is restricted to aqueous-organic systems. ATPSs, differ from aqueous-organic systems in their physical properties. In view of this, an attempt was made to develop a model for drop formation in ATPSs adopting the information available on aqueous-organic systems. In order to validate the model, experiments were performed by using polyethylene glycol (PEG)/salt systems of different phase compositions at various flow rates. At low flow rates the single stage model and at high flow rates the two stage model are able to predict the drop volume during its formation from tip of capillary. The experimental results were found to agree reasonably well with those predicted by the model.     

Purification of plasmid DNA vectors by aqueous two-phase extraction and hydrophobic interaction chromatography

The current study explores the possibility of using a polyethyleneglycol(PEG)-ammonium sulphate aqueous two-phase system (ATPS) as an early step in a process for the purification of a model 6.1 kbp plasmid DNA (pDNA) vector. Neutralised alkaline lysates were fed directly to ATPS. Conditions were selected to direct pDNA towards the salt-rich bottom phase, so that this stream could be subsequently processed by hydrophobic interaction chromatography (HIC). Screening of the best conditions for ATPS extraction was performed using three PEG molecular weights (300, 400 and 600) and varying the tie-line length, phase volume ratio and lysate load. For a 20% (w/w) lysate load, the best results were obtained with PEG 600 using the shortest tie-line (38.16%, w/w). By further manipulating the system composition along this tie-line in order to obtain a top/bottom phase volume ratio of 9.3 (35%, w/w PEG 600, 6%, w/w NH4)2 SO4), it was possible to recover 100% of pDNA in the bottom phase with a three-fold increase in concentration. Further increase in the lysate load up to 40% (w/w) with this system resulted in a eight-fold increase in pDNA concentration, but with a yield loss of 15%. The ATPS extraction was integrated with HIC and the overall process compared with a previously defined process that uses sequential precipitations with iso-propanol and ammonium sulphate prior to HIC. Although the final yield is lower in the ATPS-based process the purity grade of the final pDNA product is higher. This shows that it is possible to substitute the time-consuming two-step precipitation procedure by a simple ATPS extraction.     

离子液体双水相萃取分离苋菜红的研究

建立了由亲水性离子液体四氟硼酸1-丁基-3-甲基咪唑([Bmim]BF4)和NaH2PO4形成的双水相体系萃取分离苋菜红的新方法.研究了盐的浓度、离子液体浓度、溶液酸度、其它共存物质对苋菜红萃取率的影响.结果表明,NaH2PO4加入量在2～2.5 g,离子液量在1.0～2.0 mL,苋菜红溶液量在1.5 mL,溶液酸度在pH 4～6范围,离子液体双水相体系对苋菜红有较高的萃取率(E%＞90).用加入无机离子、不同类型表面活性剂和吸收光谱探讨了离子液体与苋菜红之间的作用.     

Distribution behavior of some drug enantiomers in an aqueous two-phase system using counter-current extraction with protein

Summary The partial resolution of some drug enantiomers by counter-current extraction in an aqueous two-phase system using bovine serum albumin (BSA) or ovomucoid (OVM) as chiral discriminator is described. The phase system was prepared with dextran 40, polyethylene glycol and a protein. Ofloxacin (OFLX) enantiomer was well recognized by the BSA system, whereas the enantiomers of carvedilol (-blocker) and DG-5128 (a new oral hypoglycaemic drug) were hardly recognized by the OVM system. The distribution coefficients of (S)-, (R)- and rac-OFLX varied with pH. Good enantioselectivity for OFLX was obtained at relatively high pH ( pH 9). The crude optically enriched OFLX enantiomers were extracted from the aqueous fractions and finally purified by thin-layer chromatography.     

火焰原子吸收光谱法研究三乙醇胺双水相体系对铁的萃取

研究了三乙醇胺(TEA)水溶液在电解质作用下的相分离及其通过形成TEA-Fe3+配合物实现三乙醇胺相对Fe3+的萃取,萃取过程将萃取剂和萃取溶剂合二为一。同时还讨论了表面张力、溶液提升率对火焰原子吸收光谱法(FAAS)测定三乙醇胺相中铁的影响。建立了TEA-K2HPO4-H2O双水相萃取、FAAS测定铁的新方法:在试液中加入1 mL三乙醇胺(1+1)和5 g K2HPO4后以水定容为10 mL,离心分离后用FAAS测定三乙醇胺相中的铁。线性范围20～240μg/L,检出限6.1μg/L(S/N=3),已用于自来水和井水中铁的萃取。