载液组成对于卵白蛋白在非对称流场流分离通道中膜吸附和聚集行为的影响

非对称流场流分离技术对于蛋白质等生物大分子的分析具有温和、分离范围广的特点。然而,在场流分离通道中,受载液组成的影响而产生的蛋白质与通道膜的相互作用和蛋白质在通道内的聚集行为,会影响分析物的回收率和尺寸形态,这些现象一定程度上限制了场流分离仪器的进一步应用。该文研究了载液组成对于卵白蛋白在非对称流场流分离中膜吸附和聚集行为的影响。考察了不同pH （6.2、7.4、8.2）、阳离子种类（Na⁺、K⁺、Mg²⁺）及多种离子强度（0~0.1 mol/L）等条件对卵白蛋白洗脱过程的影响。结果表明a）载液的离子强度越大,卵白蛋白的吸附和聚集行为越严重;b） pH和蛋白质的等电点pI的相对大小决定了蛋白质的表面电荷,从而影响蛋白质的吸附聚集行为;c）二价阳离子Mg²⁺更易引发通道中蛋白质的吸附和聚集。这些结果有助于今后使用非对称流场流分离技术分析蛋白质样品时,改善载液组成以获得更高的回收率,降低蛋白质聚集作用,对AF4更广泛地应用于蛋白质生化分析中有较好的参考价值。     

非对称流场流分离技术在纳米材料及生物分子表征方面的应用

场流分离作为一类分离技术可分离、提纯和收集流体中的悬浮物微粒,它是将流体与外场联合作用于待分离物质,利用样品质量、体积和密度等性质的差异实现分离,然后利用分离物质的保留性质来确定样品颗粒粒径及分布、分子量等性质。其中非对称流场流分离能够提供连续的、高分辨率的分离,近年来越来越受到科研人员的欢迎。本文介绍了场流分离的分类及其原理,重点介绍了非对称流场流分离的原理及其应用,包括非对称流场流分离的影响因素及与其他分离技术的比较;最后总结了该技术的发展趋势。     

非对称流场流分离-超高效液相色谱-串联四极杆飞行时间质谱用于过敏原蛋白表位筛选

应用非对称流场流分离（AF4）技术结合超高效液相色谱-四极杆飞行时间质谱（UPLC-QTOF-MS）对过敏原蛋白表位进行筛选。将选择的过敏原蛋白（虾原肌球蛋白，TM）酶解后经UPLC-QTOF-MS分析，建立蛋白质肽谱。将TM酶解后的肽段与免疫球蛋白E混合孵育30 min，孵育过程中含有抗原表位的特异性肽段与免疫球蛋白E（IgE）结合，未结合的肽段仍留在溶液中。将孵育后的溶液进行AF4分离，已结合的肽段随IgE一起由出口流出，未结合的肽段透过分离通道膜，滤出至废液。收集出口流出的组分进行UPLC-QTOF-MS分析，与蛋白质肽谱匹配，找到特异性肽段，进而检测抗原表位。本研究扩展了非对称流场流分离技术的应用，对过敏原蛋白表位的检测进行了初步探索，为过敏原蛋白表位的研究提供了一种新的研究策略。     

基于非对称场流分离技术分离表征小米淀粉

基于非对称场流分离技术耦合多角度激光光散射检测器和示差折光检测器,建立了分离表征小米淀粉的方法。研究了进样量、交叉流流速、半衰期（t_1/2）、载液离子强度和pH值对小米淀粉分离效果的影响;考察了该方法的重现性;探究了小米淀粉分子结构。结果表明,在进样体积为50 μL、进样质量浓度为0.50 g/L、交叉流流速为1.2 mL/min、t_1/2=3 min、载液为10 mmol/L pH 7.00 NaNO₃（含3 mmol/L NaN₃）的条件下,小米淀粉分离效果最佳。该方法具有良好的重现性,得到的小米淀粉的回转半径相对标准偏差为3.4%、摩尔质量相对标准偏差为7.0%。     

芯片自由流梯度电场电泳及其应用

尽管在垂直的电场和流体场作用下,采用芯片自由流电泳（μ-FFE）可实现样品的连续微分离和制备,但是由于在运行过程中,存在分析物的区带展宽问题,会直接影响样品的分离效果．在本文中,在施加固定电压的情况下,通过向和分离缓冲液相同的电极缓冲液中添加硫酸钠的方法,在分离腔内形成了梯度电场．通过对罗丹明B和甲基绿混合物的分离发现,在均一电场下,施加400V分离电压,混合物需2min才能完全分离：甲基绿的区带宽度为3．8mm,与罗丹明B的分辨率是3．2．在向电极缓冲液中添加5mmol／L硫酸钠形成的电场梯度下,施加300V的分离电压,两种染料可在10S内完成分离;在20S时,甲基绿的区带宽度被压缩到015mm,检测灵敏度提高了7倍以上;与罗丹明B的分辨率可达到16．2．此外,该方法还被用于牛血清白蛋白的富集．与施加均一电场相比,蛋白质的检测灵敏度得到了显著提高．上述结果表明,通过在μ-FFE中引入梯度电场,可有效提高样品的分辨率、检测灵敏度和分析速度．     

重力场流分离系统中载液及流速条件对分离的影响

重力场流分离作为最简单的一种场流分离技术,常用于分离微米级颗粒。选择两种不同粒径(20 μ m和6 μ m)的聚苯乙烯(PS)颗粒作为样品,通过改变载液中叠氮化钠浓度、混合表面活性剂的比例及载液流速,利用自行设计生产的重力场流分离(gravitational flow field-flow fractionation, GrFFF)仪器,对颗粒混合样品进行分离,得到了相关谱图与数据,考察了这3种因素对分离效果(保留比(R)、塔板高度(H))的影响。结果表明:20 μ m PS颗粒的R值均大于6 μ m PS颗粒的R值,H值均小于6 μ m颗粒的H值;PS颗粒的R值与H值均随着载液中叠氮化钠浓度的增加而增加;但随着载液流速的增加,R值增加,H值减小。该研究为GrFFF系统的开发及应用提供了重要的参考价值。     

非对称场流分离技术用于纳米颗粒的表征

采用非对称场流分离技术(Asymmetrical flow field-flow fractionation,AF4)对标准聚苯乙烯颗粒粒径进行表征。利用非对称场流分离仪以0.1%SDS(十二烷基磺酸钠)和0.02%NaN3的水溶液为流动相,测定标准的聚苯乙烯纳米颗粒在流体流场作用下通过分离腔室的保留时间,以确定纳米颗粒的平均粒径。优化了聚焦时间、横向流速、进样量、主体流速等实验条件,建立了利用AF4准确表征纳米颗粒的方法,并与扫描电镜(Scanning electron microscope,SEM)的表征结果进行比较。结果表明,AF4的表征结果比SEM更接近于聚苯乙烯颗粒的标准粒径,具有更高的稳定性和准确度。本方法可作为纳米粒径表征的一种准确方法。     

重力场流分离系统用于聚苯乙烯颗粒的分离条件优化

重力场流分离是最简单的场流分离(gravitational flow-field fractionation,GrFFF)技术,常用于分离粒径几微米到几十微米的颗粒及生物样品。利用自组装加工的重力场流分离仪器分离3种不同粒径(3、6、20μm)的聚苯乙烯(PS)颗粒。自制了一种混合表面活性剂,并与商品化的表面活性剂FL-70进行了比较。通过均匀设计优化流速、混合表面活性剂中聚乙二醇辛基苯基醚(Triton X-100)的质量分数、载液黏度、停流时间等分离条件,以分离度(Rs)和保留比(R)为评价指标,发现FL-70的分离效能略优于自制的混合表面活性剂,可实现3种PS颗粒的完全分离(Rs1为1.771,Rs2为2.074)。结果表明该系统具有良好的分离性能。     

纳米材料在蛋白质分离中的应用

蛋白质的分离技术不仅在药物检测和制药工程中具有重要意义,而且还是生化工程和蛋白质分析的一个研究热点.纳米材料具有许多与众不同的特性,广泛应用于化工、生物、医药、航天等多个领域,被认为是21世纪最有前途的材料.本文作者从非金属氧化物纳米材料、非金属单质纳米材料、金属氧化物纳米材料、金属单质纳米材料、纳米聚合物、纳米复合材料等方面综述了纳米材料在蛋白质分离方面的应用现状,总结了其在蛋白质分离中的优缺点,并就其在蛋白质固定和分离领域的应用前景进行了展望.     

离子液与蛋白质和核酸相互作用的研究

离子液因其具有良好的生物兼容性和独特的理化性质,近年来在生物催化和生物大分子蛋白质与核酸的分离分析领域得到广泛应用。离子液与生物大分子相互作用的研究是离子液相关理论与应用研究的基础,有关离子液与蛋白质和核酸相互作用的机理研究受到关注。本文简要介绍了常用离子液的分类,离子液与蛋白质分子作用的机理,离子液与核酸分子作用的机理,以及离子液在酶催化反应、生物分子分离、生物分子电化学分析和毛细管电泳分析中的应用,并主要综述了近年的相关研究和应用进展。     

Low-capacity channel designed for particle separation with controlled electric fields and evaluation of involved forces

Electric field is one of the suitable physical fields applicable to particle separations. Although long rectangular channel is used for particle separation in usual electrical field flow fractionation (FFF), a short low-capacity channel can replace it if the field is precisely controlled. Several separation principles are proposed with this channel. The elution behavior of particles has revealed that the gravitational, diffusion, and hydrodynamic lift force (HLF) play important roles in the determination of the elution behavior of particles. The elution threshold voltage (V(th)) was defined and experimentally determined for various system configurations and particles. The electric force no longer overcomes the other forces, and particles are taken off the wall, when the applied voltage becomes lower than V(th). V(th) values have allowed us not only to estimate surface charge density of a particle but also to evaluate the hydrodynamic lift force against particle.     

Asymmetrical flow field-flow fractionation technique for separation and characterization of biopolymers and bioparticles

Field-flow fractionation (FFF) is one of the most versatile separation techniques in the field of analytical separation sciences, capable of separating macromolecules in the range 10³–10¹⁵ g mol⁻¹ and/or particles with 1 nm–100 μm in diameter. The most universal and most frequently used FFF technique, flow FFF, includes three types of techniques, namely symmetrical flow FFF, hollow fiber flow FFF, and asymmetrical flow FFF which is most established variant among them. This review provides a brief look at the theoretical background of analyte retention and separation efficiency in FFF, followed by a comprehensive overview of the current status of asymmetrical flow FFF with selected applications in the field of biopolymers and bioparticles.     

Geometric scaling effects on instrumental plate height in field flow fractionation

This paper examines geometric scaling models for field flow fractionation systems to understand how channel dimensions affect resolution and retention. Specifically, the changing contribution of the instrumental plate height during miniaturization of field flow fractionation (FFF) systems is reported. The work is directed towards determining the optimal geometrical parameters for miniaturization of field flow fractionation systems. The experimental relationship between channel height in FFF systems and instrumental plate heights is reported. FFF scaling models are modified to: (i) better clarify the dependence of plate height and resolution on channel height in FFF and (ii) include a more complete geometrical scaling analysis and model comparison in the low retention regime. Electrical field flow fractionation has been shown to benefit from miniaturization, so this paper focuses on that subtype, but surprisingly, the results also indicate the possibility of improvement in performance with miniaturization of other field flow fractionation systems including general FFF subtypes in which the applied field does not vary with channel height. This paper also discusses the potential role of more powerful microscale field flow fractionation systems as a new class of sample preparation units for micro-total-analysis systems (mu-TAS).     

Size selectivity in field-flow fractionation: lift mode of retention with near-wall lift force

A simple theoretical model for the size selectivity, S(d), in the lift mode of retention in field-flow fractionation (FFF) is developed on the basis of the near-wall lift force expression. S(d) is made up of two contributions: the flow contribution, S(d,f), arising from the variation of the flow velocity at center of particle due to a change in particle position with particle size, and a slip contribution, S(d,s), arising from the concomitant change in the extent of retardation due to the presence of a nearby channel wall. The slip contribution is minor, but not negligible, and amounts to 10-20% of the overall size selectivity. It contributes to reduce S(d) in sedimentation FFF but to enhance it in flow FFF. S(d) would steadily increase with particle size if the flow profile was linear (Couette flow). Because of the curvature of the flow profile encountered in the classical Poiseuille flow, S(d) exhibits a maximum at some specific particle size. The model predicts a significant difference in S(d) between sedimentation FFF and flow FFF, arising from the different functional dependences of the field force with particle size between these two methods. The predictions are in good agreement with the various S(d) values reported in the literature in both sedimentation and flow FFF. On the basis of the model, guidelines are given for adjusting the operating parameters (carrier flow rate and field strength) to optimize the size selectivity. Finally, it is found that S(d) generally decreases with decreasing channel thickness.     

Impact of Ionic Strength of Carrier Liquid on Recovery in Flow Field-Flow Fractionation

Asymmetrical flow field-flow fractionation (AF4) and hollow-fiber flow field-flow fractionation (HF5) are techniques widely used in analytical, industrial and biological analyses. The main problem in all AF4 and HF5 analyses is sample loss due to analyte–membrane interactions. In this work the impact of liquid carrier composition on latex nanoparticles (NPs) separation in water and two different concentrations of NH₄NO₃ was studied. In AF4, a constant trend of decreasing the size of 60 and 121.9 nm particles induced by the ionic strength of the carrier liquid has been observed. In contrast, an agglomeration effect of the biggest 356 nm particles was observed when increasing ionic strength, which induced a significant drop of recovery to 35%. H5F provides better resolution and intensified peaks of NPs, but careful optimisation of system parameters is mandatory to obtain good separation.     

Dielectrophoretic field‐flow fractionation of electroporated cells

We describe the development and testing of a setup that allows for DEP field‐flow fractionation (DEP‐FFF) of irreversibly electroporated, reversibly electroporated, and nonelectroporated cells based on their different polarizabilities. We first optimized the channel and electrode dimensions, flow rate, and electric field parameters for efficient DEP‐FFF separation of moderately heat‐treated CHO cells (50°C for 15 min) from untreated ones, with the former used as a uniform and stable model of electroporated cells. We then used CHO cells exposed to electric field pulses with amplitudes from 1200 to 2800 V/cm, yielding six groups containing various fractions of nonporated, reversibly porated, and irreversibly porated cells, testing their fractionation in the chamber. DEP‐FFF at 65 kHz resulted in distinctive flow rates for nonporated and each of the porated cell groups. At lower frequencies, the efficiency of fractionation deteriorated, while at higher frequencies the separation of individual elution profiles was further improved, but at the cost of cell flow rate slowdown in all the cell groups, implying undesired transition from negative into positive DEP, where the cells are pulled toward the electrodes. Our results demonstrate that fractionation of irreversibly electroporated, reversibly electroporated, and nonelectroporated cells is feasible at a properly selected frequency.     

Separation of carbon nanotubes by frit inlet asymmetrical flow field-flow fractionation

Flow field-flow fractionation (flow FFF), a separation technique for particles and macromolecules, has been used to separate carbon nanotubes (CNT). The carbon nanotube ropes that were purified from a raw carbon nanotube mixture by acidic reflux followed by cross-flow filtration using a hollow fiber module were cut into shorter lengths by sonication under a concentrated acid mixture. The cut carbon nanotubes were separated by using a modified flow FFF channel system, frit inlet asymmetrical flow FFF (FI AFIFFF) channel, which was useful in the continuous flow operation during injection and separation. Carbon nanotubes, before and after the cutting process, were clearly distinguished by their retention profiles. The narrow volume fractions of CNT collected during flow FFF runs were confirmed by field emission scanning electron microscopy and Raman spectroscopy. Experimentally, it was found that retention of carbon nanotubes in flow FFF was dependent on the use of surfactant for CNT dispersion and for the carrier solution in flow FFF. In this work, the use of flow FFF for the size differentiation of carbon nanotubes in the process of preparation or purification was demonstrated.     

Analysis of complex polymers by multidetector field-flow fractionation

Field-flow fractionation (FFF) is a powerful alternative to column-based polymer fractionation methods such as size-exclusion chromatography (SEC) or interaction chromatography (IC). The most common polymer fractionation method, SEC, has its limitations when polymers with very high molar masses or complex structures must be analysed. Another limitation of all column-based methods is that the samples must be filtered before analysis and shear degradation of large macromolecules may be caused by the stationary phase and/or the column frits. Finally, the separation of very polar polymers may be a challenge because such polymers interact very strongly with the stationary phase, causing irreversible adsorption or other negative effects. This article reviews the latest developments in field-flow fractionation of complex polymers. It is demonstrated that some of the limitations of column-based chromatography can be overcome by FFF. When appropriate, results from column-based fractionations are compared with those from FFF fractionations to highlight the specific merits and challenges of each method. In addition to the fractionations themselves, various detector setups are discussed to show that different polymer distributions require different experimental procedures. Examples are given of the analysis of molar mass distribution, chemical composition, and microstructure. Advanced detector combinations are discussed, most prominently the very recently developed coupling to ¹H NMR. Finally, analysis of polymer nanocomposites by asymmetric flow field-flow fractionation (AF4)–FTIR is presented.

Field-flow fractionation in bioanalysis: A review of recent trends

Field-flow fractionation (FFF) is a mature technique in bioanalysis, and the number of applications to proteins and protein complexes, viruses, derivatized nano- and micronsized beads, sub-cellular units, and whole cell separation is constantly increasing. This can be ascribed to the non-invasivity of FFF when directly applied to biosamples. FFF is carried out in an open-channel structure by a flow stream of a mobile phase of any composition, and it is solely based on the interaction of the analytes with a perpendicularly applied field. For these reasons, fractionation is developed without surface interaction of the analyte with packing or gel media and without using degrading mobile phases. The fractionation device can be also easily sterilized, and analytes can be maintained under a bio-friendly environment. This allows to maintain native conditions of the sample in solution.In this review, FFF principles are briefly described, and some pioneering developments and applications in the bioanalytical field are tabled before detailed report of most recent FFF applications obtained also with the hyphenation of FFF with highly specific, sensitive characterization methods. Special focus is finally given to the emerging use of FFF as a pre-analytical step for mass-based identification and characterization of proteins and protein complexes in proteomics.