Separation and characterization of particles in natural water and soil using flow field-flow fractionation

Flow field-flow fractionation (FlFFF) is used to characterize particles in natural water (ground and surface water) and soil. The opposed flow sample concentration (OFSC) mode of FlFFF (OFSC-FlFFF) is employed, where the colloidal sample is continuously fed into the channel so that the particles are focused into a narrow band near the inlet of the FlFFF channel before the separation is initiated. There is no need for stopping the flow for the sample relaxation, which is usually required in conventional FlFFF operations. First, the OFSC-FlFFF is tested with mixtures of polystyrene latex spheres. Then the OFSC-FlFFF procedure is optimized for the analysis of particles in natural water and soil by varying various experimental parameters including the flow rates. Ground water of up to 100 mL has been successfully loaded, concentrated, and characterized by OFSC-FlFFF. Results show that the OFSC-FlFFF provides a simplified alternative to existing off-line concentration procedures, and it shows high potential for application to analysis of dilute colloidal particles in environmental water. The composition of the samples was analyzed using atomic absorption spectrometry.     

Low-density lipoprotein analysis in microchip capillary electrophoresis systems

Due to the mounting evidence for altered lipoprotein and cholesterol-lipoprotein content in several disease states, there has been an increasing interest in analytical methods for lipoprotein profiling for diagnosis. The separation of low- and high-density lipoproteins (LDL and HDL, respectively) has been recently demonstrated using a microchip capillary electrophoresis (CE) system [1]. In contrast to this previous study, the present report demonstrates that LDL analysis can be performed in an uncoated glass microchannel. Moreover, by adding sodium dodecyl sulfate (SDS) to the sample at a concentration well below the critical micellar concentration prior to injection, the LDL peak undergoes a focusing effect and exhibits an apparent efficiency of 2.2 x 10(7) plates/m. Laser light scattering experiments demonstrate that the low concentration of SDS used does not significantly alter lipoprotein particle size distribution within the time course that the analysis is performed. It is thus hypothesized that SDS nondisruptively coats LDL particles. The peak sharpening effect, observed only when SDS is added solely to the sample, is probably due to a mobility gradient created between the sample and the running buffer. The chip-based method demonstrated here has the potential for rapid analysis and sensitive detection of different LDL forms of clinical relevance.     

微流控芯片电泳快速分离脂蛋白

描述了一种芯片电泳快速分离脂蛋白的方法. 利用自制的微流控芯片及激光诱导荧光技术电泳分离经硝基苯并噁二唑-C₆-酰基鞘胺醇预染的脂蛋白标本, 在40 mmol/L tricine缓冲液(pH 9.4)中加入40 mmol/L甲基葡胺, 在500 V电压下40 s进样, 在2000 V 电压下2 min内完成分离, 可出现低密度脂蛋白(LDL)与高密度脂蛋白(HDL)两条脂蛋白区带, 5次重复性试验其出峰时间变异系数(CV)为2.6%. 本法为高血脂患者提供了一种快速、简便、灵敏、重复性好的诊断方法.     

Application of poly(dimethylsiloxane)/glass microchip for fast electrophoretic separation of serum small,dense low-density lipoprotein

Due to the mounting evidence of altered low-density lipoprotein (LDL) size in several disease states, there has been an increasing interest in developing new analytical methods for small, dense low-density lipoprotein (sdLDL) for diagnosis. The present report demonstrates that sdLDL analysis can be performed in a poly(dimethylsiloxane) (PDMS/glass) microchannel. n-Dodecyl β-d-maltoside (DDM) was utilized to alter channel surface to make it become hydrophilic and nonionic, thus reducing the interaction between the protein and the surface. Moreover, hydroxypropylcellulose (HPC) was added into the running buffer to suppress the adsorption of analytes and also to serve as a sieving matrix. Under optimal conditions, two baseline separations of lipoproteins including high-density lipoprotein (HDL), sdLDL, and lLDL were achieved with different selectivity. LDL particles shown on the electropherogram were also identified by several procedures. This method affords high separation speed and high reproducibility. The intraassay and interassay RSDs of lipoprotein migration times were in the range of 2.01–2.45%. The variation of serum sdLDL of a patient between prior treatment and post-treatment was assessed by this method. This system has the potential for rapid and sensitive detection of different LDL forms, and thus will be applicable to clinical diagnosis.     

Application of flow field-flow fractionation for the characterization of macromolecules of biological interest: a review

An overview is given of the recent literature on (bio) analytical applications of flow field-flow fractionation (FlFFF). FlFFF is a liquid-phase separation technique that can separate macromolecules and particles according to size. The technique is increasingly used on a routine basis in a variety of application fields. In food analysis, FlFFF is applied to determine the molecular size distribution of starches and modified celluloses, or to study protein aggregation during food processing. In industrial analysis, it is applied for the characterization of polysaccharides that are used as thickeners and dispersing agents. In pharmaceutical and biomedical laboratories, FlFFF is used to monitor the refolding of recombinant proteins, to detect aggregates of antibodies, or to determine the size distribution of drug carrier particles. In environmental studies, FlFFF is used to characterize natural colloids in water streams, and especially to study trace metal distributions over colloidal particles. In this review, first a short discussion of the state of the art in instrumentation is given. Developments in the coupling of FlFFF to various detection modes are then highlighted. Finally, application studies are discussed and ordered according to the type of (bio) macromolecules or bioparticles that are fractionated.     

Miniaturized asymmetrical flow field-flow fractionation: application to biological vesicles

Asymmetrical flow field-flow fractionation (AFlFFF) has been carried out in a miniaturized channel by reducing the channel dimensions. Performance of the miniaturized AFlFFF (mAFlFFF) channel was evaluated with standard proteins and polystyrene latex spheres from nanometer to micrometer size. By reducing the channel dimension, proteins or particulate materials can be separated within a few minutes without a significant loss in resolution. The mAFlFFF channel was applied for the separation of exosomes harvested from immortalized human mesenchymal stem cell line. It shows a potential to fractionate exosome vesicles according to sizes which can be useful for proteomic studies in relation to immunotherapeutic applications.     

Field and flow programming in frit-inlet asymmetrical flow field-flow fractionation

The separation of wide molecular mass (Mr) ranges of macromolecules using frit inlet asymmetrical flow field-flow fractionation (FI-AFlFFF) has been improved by implementing a combination of field and flow programming. In this first implementation, field strength (governed by the cross flow-rate through the membrane-covered accumulation wall) is decreased with time to obtain faster elution and improved detection of the more strongly retained (high Mr) materials. The channel outlet flow-rate is optionally held constant, increased, or decreased with time. With circulation of the flow exiting the accumulation wall to the inlet frit, the dual programming of cross flow and channel outlet flow could be implemented using just two pumps. With this flow configuration, the channel outlet flow-rate is always equal to the channel inlet flow-rate, and these may be programmed independently of the cross flow-rate through the membrane. FI-AFlFFF retains its operational advantage over conventional asymmetrical flow FFF (AFlFFF). Unlike conventional AFlFFF, FI-AFlFFF does not require time consuming, and experimentally inconvenient, sample focusing and relaxation steps involving valve switching and interruption of sample migration. The advantages of employing dual programming with FI-AFlFFF are demonstrated for sets of polystyrene sulfonate standards in the molecular mass range of 4 to 1000 kDa. It is shown that programmed FI-AFlFFF successfully expands the dynamic separation range of molecular mass.     

Natural sample fractionation by FlFFF-MALLS-TEM: sample stabilization, preparation, pre-concentration and fractionation

Two flow field flow fractionation (FlFFF) systems: symmetrical (SFlFFF) and asymmetrical (ASFlFFF) were evaluated to fractionate river colloids. Samples stability during storage and colloids concentration are the main challenges limiting their fractionation and characterization by FlFFF. A pre-fractionation (<0.45 microm) and addition of a bactericide such as NaN3 into river colloidal samples allowed obtaining stable samples without inducing any modification to their size. Stirred cell ultra-filtration allowed colloidal concentration enrichment of 25-folds. Scanning electron microscope (SEM) micrographs confirmed the gentle pre-concentration of river samples using the ultra-filtration stirred cell. Additionally, larger sample injection volume in the case of SFlFFF and on channel concentration in the case of ASFlFFF were applied to minimize the required pre-concentration. Multi angle laser light scattering (MALLS), and transmission electron microscope (TEM) techniques are used to evaluate FlFFF fractionation behavior and the possible artifacts during fractionation process. This study demonstrates that, FlFFF-MALLS-TEM coupling is a valuable method to fractionate and characterize colloids. Results prove an ideal fractionation behavior in case of Brugeilles sample and steric effect influencing the elution mode in case of Cézerat and Chatillon. Furthermore, comparison of SFlFFF and ASFlFFF fractograms for the same sample shows small differences in particle size distributions.     

Hyperlayer separation in hollow fiber flow field-flow fractionation: effect of membrane materials on resolution and selectivity

Hollow fiber flow FFF (HF FlFFF) has recently shown its capability to separate and characterize the size of submicrometer particles and has demonstrated the potential to be developed into a disposable flow FFF channel. In this work, HF FlFFF was used for the hyperlayer separation of micron-sized particles and the separation capability was examined by using various hollow fiber membrane materials (Polysulfones, cPVC, and PAN). From the experiments, PAN (polyacrylonitriles) showed an outstanding performance in particle separation compared to the other membranes. By orienting the fiber module in an upright direction, the upstream flow migration reduced band broadening of eluted peaks. When the efficiency of the PAN hollow fiber system was tested by varying the ratio of outflow-rate to radial flow-rate, it was found that optimum separation in hyperlayer HF FlFFF can be obtained at the ratio of about 6–7. From the examination of retention at or around steric inversion diameter, it was observed that experiments showed a good agreement with predictions by semi-empirical calculation. In hyperlayer HF FlFFF the diameter based selectivity values were shown to be 1.2–1.7 depending on the type of membranes and the field strength (the radial flow-rate) conditions.     

Poly（dimethylsiloxane） Microchips with Two Sharpened Stretching Tips and Its Application to Protein Separation Using Dynamic Coating

An integrated poly（dirnethylsiloxane） （PDMS） microchip with two sharpened stretching tips for convenient sample injecting, running buffer refreshing and channel cleaning has been presented. The sample was directly introduced into the separation channel through the stretching inlet tip without complicated power switching supplies and injection cross channel. The operation of running buffer refreshing or channel cleaning was simplified by vacuuming one end of the tip and placing the other tip into the solution vial. Therefore, this fabrication method can be easily applied to most analytical laboratories economically without soft lithography and plasma bonding equipments. The attractive performance of the novel PDMS microchips has been demonstrated by using laser-induced fluorescence detection for separation of proteins. The addition of 0.04% Brij 35 in 0.04 mol/L phosphate buffer （pH 7.0） can reduce the adhesion of proteins in multienzyme tablet and make separation more easily. The electroosmotic flow （EOF） exhibits pH-independence in the range of 3-1 1 in dynamic modified microchannel.     

A novel method of high-purity extracellular vesicle enrichment from microliter-scale human serum for proteomic analysis

We have developed a rapid, low-cost, and simple separation strategy to separate extracellular vesicles (EVs) from a small amount of serum (i.e.,<100 μL) with minimal contamination by serum proteins and lipoprotein particles to meet the high purity requirement for EV proteome analysis. EVs were separated by a novel polyester capillary channel polymer (PET C-CP) fiber phase/hydrophobic interaction chromatography (HIC) method which is rapid and can process small size samples. The collected EV fractions were subjected to a post-column cleanup protocol using a centrifugal filter to perform buffer exchange and eliminate potential coeluting non-EV proteins while minimizing EV sample loss. Downstream characterization demonstrated that our current strategy can separate EVs with the anticipated exosome-like particle size distribution and high yield (∼1 × 10¹¹ EV particles per mL of serum) in approximately 15 min. Proteome profiling of the EVs reveals that a group of genuine EV components were identified that have significantly less high-abundance blood proteins and lipoprotein particle contamination in comparison to traditional separation methods. The use of this methodology appears to address the major challenges facing EV separation for proteomics analysis. In addition, the EV post-column cleanup protocol proposed in the current work has the potential to be combined with other separation methods, such as ultracentrifugation (UC), to further purify the separated EV samples.     

Hollow-fiber flow/hyperlayer field-flow fractionation for the size characterization of airborne particle fractions obtained by SPLITT fractionation

Hollow-fiber flow field-flow fractionation (HF FlFFF) was applied for the separation and size characterization of airborne particles which were collected in a municipal area and prefractionated into four different-diameter intervals >5.0, 2.5-5.0, 1.5-2.5, <1.5 microm) by continuous split-flow thin (SPLIIT) fractionation. Experiments demonstrated the possibility of utilizing a hollow-fiber module for the high-performance separation of supramicron-sized airborne particles at steric/hyperlayer operating mode of HF FlFFF. Eluting particles during HF FlFFF separation were collected at short time intervals (approximately 10 s) for the microscopic examination. It showed that particle size and size distributions of all SPLITT fractions of airborne particles can be readily obtained using a calibration and that HF FlFFF can be utilized for the size confirmation of the sorted particle fraction during SPLITT fractionation.     

Hydrodynamic injection with pneumatic valving for microchip electrophoresis with total analyte utilization

A novel hydrodynamic injector that is directly controlled by a pneumatic valve has been developed for reproducible microchip CE separations. The PDMS devices used for the evaluation comprise a separation channel, a side channel for sample introduction, and a pneumatic valve aligned at the intersection of the channels. A low pressure (≤ 3?psi) applied to the sample reservoir is sufficient to drive sample into the separation channel. The rapidly actuated pneumatic valve enables injection of discrete sample plugs as small as ~ 100?pL for CE separation. The injection volume can be easily controlled by adjusting the intersection geometry, the solution back pressure, and the valve actuation time. Sample injection could be reliably operated at different frequencies (< 0.1?Hz to > 2?Hz) with good reproducibility (peak height relative standard deviation ≤ 3.6%) and no sampling biases associated with the conventional electrokinetic injections. The separation channel was dynamically coated with a cationic polymer, and FITC-labeled amino acids were employed to evaluate the CE separation. Highly efficient (≥ 7.0 × 103 theoretical plates for the ~2.4-cm-long channel) and reproducible CE separations were obtained. The demonstrated method has numerous advantages compared with the conventional techniques, including repeatable and unbiased injections, little sample waste, high duty cycle, controllable injected sample volume, and fewer electrodes with no need for voltage switching. The prospects of implementing this injection method for coupling multidimensional separations for multiplexing CE separations and for sample-limited bioanalyses are discussed.     

电泳芯片结构和芯片电泳分离操作参数的模拟、优化和实验验证

选择了L-精氨酸和L-苯丙氨酸为分离样品体系,根据电泳实验提出样品基本参数,通过模拟计算考察了进样管道宽度和进样时间对进样方差的贡献;根据分离度与分离长度拟合曲线确定电泳芯片的有效分离长度;对化学发光柱后衍生管道施加的夹流电压进行了模拟优化,得出氨基酸体系分离分析的电泳芯片设计方案和操作参数为:进样管道宽度为分离管道宽度的1/2,简单进样充样时间应大于5 s,分离管道有效分离长度为30 mm,衍生夹流比1.0~1.6。根据模拟优化结果提出了电泳芯片设计方案,采用整体浇注法制作带有柱后衍生反应器的PDMS电泳芯片,按照模拟计算提出的电压操作参数实现了精氨酸和苯丙氨酸样品体系的准确进样、芯片电泳分离和柱后衍生化学发光检测。电泳过程模拟结果和实验结果相结合,考察了柱后衍生对样品谱带展宽的影响,简单进样过程样品泄露引起的谱峰拖尾现象,并讨论了夹流进样法对减小进样方差和抑制样品泄露的贡献。     

Flow field-flow fractionation for the analysis and characterization of natural colloids and manufactured nanoparticles in environmental systems: a critical review

The use of flow field flow fractionation (FlFFF) for the separation and characterization of natural colloids and nanoparticles has increased in the last few decades. More recently, it has become a popular method for the characterization of manufactured nanoparticles. Unlike conventional filtration methods, FlFFF provides a continuous and high-resolution separation of nanoparticles as a function of their diffusion coefficient, hence the interest for use in determining particle size distribution. Moreover, when coupled to other detectors such as inductively coupled plasma-mass spectroscopy, light scattering, UV-absorbance, fluorescence, transmission electron microscopy, and atomic force microscopy, FlFFF provides a wealth of information on particle properties including, size, shape, structural parameters, chemical composition and particle-contaminant association. This paper will critically review the application of FlFFF for the characterization of natural colloids and natural and manufactured nanoparticles. Emphasis will be given to the detection systems that can be used to characterize the nanoparticles eluted from the FlFFF system, the obtained information and advantages and limitation of FlFFF compared to other fractionation and particle sizing techniques. This review will help users understand (i) the theoretical principles and experimental consideration of the FlFFF, (ii) the range of analytical tools that can be used to further characterize the nanoparticles after fractionation by FlFFF, (iii) how FlFFF results are compared to other analytical techniques and (iv) the range of applications of FlFFF for natural and manufactured NPs.     

Evaluation of multiplexed hollow fiber flow field-flow fractionation for semi-preparative purposes

A multiplexed hollow fiber flow field-flow fractionation (MxHF5) is introduced to increase throughput of an HF5 channel system for semi-preparative purposes. HF5, a cylindrical version of the flow field-flow fractionation (FlFFF) operated with a porous, hollow fiber membrane by controlling the ratio of radial and axial flow rates, is capable of fractionating proteins, cells, and macromolecules by size. An advantage of HF5 is its inexpensive channel construction, allowing for disposability that can reduce run-to-run carryover problems. MxHF5 constructed in this study was made with six parallel HF5 modules connected to seven-port manifolds for the semi-preparative scale separation of proteins or biological particles. For the evaluation of MxHF5 separation efficiency, protein standards were utilized to test peak recoveries, band broadening, and throughput. The assembly showed the possibility of handling up to 50 μg of proteins without incurring overloading. The developed channel was applied to demonstrate size sorting of lipoproteins for the future study of size dependent lipidomic and proteomic analysis. The current trial offers a unique advantage of scaling up HF5 separation without using wide-bore, hollow fibers which sacrifice separation speed.     

Biocompatible channels for field-flow fractionation of biological samples: correlation between surface composition and operating performance

Biocompatible methods capable of rapid purification and fractionation of analytes from complex natural matrices are increasingly in demand, particularly at the forefront of biotechnological applications. Field-flow fractionation is a separation technique suitable for nano-sized and micro-sized analytes among which bioanalytes are an important family. The objective of this preliminary study is to start a more general approach to field-flow fractionation for bio-samples by investigation of the correlation between channel surface composition and biosample adhesion. For the first time we report on the use of X-ray photoelectron spectroscopy (XPS) to study the surface properties of channels of known performance. By XPS, a polar hydrophobic environment was found on PVC material commonly used as accumulation wall in gravitational field-flow fractionation (GrFFF), which explains the low recovery obtained when GrFFF was used to fractionate a biological sample such as Staphylococcus aureus. An increase in separation performance was obtained first by conditioning the accumulation wall with bovine serum albumin and then by using the ion-beam sputtering technique to cover the GrFFF channel surface with a controlled inert film. XPS analysis was also employed to determine the composition of membranes used in hollow-fiber flow field-flow fractionation (HF FlFFF). The results obtained revealed homogeneous composition along the HF FlFFF channel both before and after its use for fractionation of an intact protein such as ferritin.     

Numerical analysis of an electrokinetic double-focusing injection technique for microchip CE

The injection techniques in electrophoresis microchips play an important role in the sample-handling process, whose characteristics determine the separation performance achieved, and the shape of a sample plug delivered into the separation channel has a great impact on the high-quality separation performance as well. This paper describes a numerical investigation of different electrokinetic injection techniques to deliver a sample plug within electrophoresis microchips. A novel double-focusing injection system is designed and fabricated, which involves four accessory arm channels in which symmetrical focusing potentials are loaded to form a unique parallel electric field distribution in the intersection of injection channel and separation channel. The parallel electric field effectuates virtual walls to confine the spreading of a sample plug at the intersection and prevents sample leakage into separation channel during the dispensing step. The key features of this technique over other injection techniques are the abilities to generate regular and nondistorted shape of sample plugs and deliver the variable-volume sample plugs by electrokinetic focusing. The detection peak in the proposed injection system is uniform regardless of the position of the detection probe in the separation channel, and the peak resolution is greatly enhanced. Finally, the double-focusing injection technique shows the flexibility in detection position and ensures improved signal sensitivity with good peak resolution due to the delivered high-quality sample plug.     

Fractionation of human serum lipoproteins and simultaneous enzymatic determination of cholesterol and triglycerides

A method based on Asymmetric Flow Field-Flow Fractionation (AF4) was developed to separate different types of lipoproteins from human serum. The emphasis in the method optimization was on the possibilities to characterize the largest lipoprotein fractions (LDL and VLDL), which is usually not possible with the size-exclusion chromatography methods applied in routine analysis. Different channel geometries and flow programs were tested and compared. The use of a short fractionation channel was shown to give less sample dilution at the same fractionation power compared to a conventional, long channel. Different size selectivities were obtained with an exponential decay and a linear cross flow program. The ratio of the UV absorption signal to the light scattering signal was used to validate the relation between retention time and size of the fractionated particles.An experimental setup was developed for the simultaneous determination of the cholesterol and triglycerides distribution over the lipoprotein fractions, based on enzymatic reactions followed by UV detection at 500 nm. Coiled and knitted PTFE tubing reactors were compared. An improved peak sharpness and sensitivity were observed with the knitted tubing reactor. After optimization of the experimental conditions a satisfactory linearity and precision (2-3% rsd for cholesterol and 5-6% rsd for triglycerides) were obtained. Finally, serum samples, a pooled sample from healthy volunteers and samples of sepsis patients, were analyzed with the method developed. Lipoprotein fractionation and cholesterol and triglyceride distributions could be correlated with the clinical background of the samples.     

A parylene-based dual channel micro-electrophoresis system for rapid mutation detection via heteroduplex analysis

A new dual channel micro-electrophoresis system for rapid mutation detection based on heteroduplex analysis was designed and implemented. Mutation detection was successfully achieved in a total separation length of 250 microm in less than 3 min for a 590 bp DNA sample harboring a 3 bp mutation causing an amino acid change. Parylene-C was used as the structural material for fabricating the micro-channels as it provides conformal deposition, transparency, biocompatibility, and low background fluorescence without any surface treatment. A new dual channel architecture was derived from the traditional cross-channel layout by forming two identical channels with independent sample loading and waste reservoirs. The control of injected sample volume was accomplished by a new u-turn injection technique with pull-back method. The use of heteroduplex analysis as a mutation detection method on a cross-linked polyacrylamide medium provided accurate mutation detection in an extremely short length and time. The presence of two channels on the microchip offers the opportunity of comparing the sample to be tested with a desired control sample rapidly, which is very critical for the accuracy and reliability of the mutation analyses, especially for clinical and research purposes.