Isoelectric focusing field-flow fractionation : III. Investigation of the influence of different experimental parameters on focusing of cytochrome c in the trapezoidal cross-section channel

In addition to the electric field and pH gradient used in isoelectric focusing, a recently introduced technique, isoelectric focusing (or electrical hyperlayer) field-flow fractionation, employs the flow of the liquid carrier through a thin separation channel as a third factor affecting separation. Focusing of cytochrome c (CYTC) in a trapezoidal cross-section channel of 0.875 ml volume and 25 cm length was investigated as a function of the injection procedure, relaxation time, flow-rate of the carrier ampholyte solution and applied electric power. The influence of different initial conditions was also investigated by computer simulation. Both computed and experimental data showed an important contribution of the injection procedure and relaxation time on the retention and shape of the CYTC zone. It follows from these data that the sample should be injected as a narrow zone into the centre of the stream rather than homogeneously together with the carrier solution. For the described experimental set-up it could be demonstrated that the time necessary for zone formation should be at least 15 min and that relaxation times in excess to 20 min do not influence the final shape of the CYTC zone. It could further be shown experimentally that the sample must be injected under an applied electric field, that the relaxation time should be about 10 min, that the elution flow-rate should not be larger than 100 μl/min, that focusing becomes more efficient with increasing electric fields and that, for a given assembly and specified flow conditions, there is an electric power window only within which proper operation is possible.     

High-resolution polymer separations in a four-pass hairpin thermal field-flow fractionation column

A thin-channel four-pass hairpin thermal field-flow fractionation (FFF) column is described, and the advantages of its unique dimensional characteristics are explained. The problem of isolating the performance of this and other separations columns from the ubiquitous polydispersity effects are discussed and treated theoretically. The discussion is extended to size exclusion chromatography, and it is shown that the 2 to 6 times lower selectivity of the latter compared to FFF leads to the requirement for 4 to 36 times more theoretical plates to encounter polydispersity effects and thereby obtain information on polymer molecular weight distribution. A fractogram of six narrow polystyrene samples obtained from the hairpin system is shown to imitate closely the fractograms obtained from two totally different thermal FFF columns, showing that polydispersity dominates and that molecular weight information is revealed for these samples with only a few hundred theoretical plates. Various experimental and theoretical attempts are made to isolate the polydispersity and column contributions to plate height, including cut-and-recycle experiments, the observation of plate height versus velocity curves, and the direct calculation of the contributing effects. The various methods are subject to moderate errors, but are in rough agreement. The plate height plots show that the polydispersity effect contributed 53% and 68% to the measured plate height for 51,000 and 160,000 molecular weight samples, respectively. The latter polymer is shown to emerge with ca. 1300 true column plates. It is suggested that much higher column efficiency will be observed in the future if higher retention levels can be experimentally realized.     

Lowering the molecular mass limit of thermal field-flow fractionation for polymer separations

Thermal field-flow fractionation (ThFFF) is capable of separating a wide molecular mass range of polymers by their molecular mass (Mr) and chemical composition. However, retention and resolution decrease significantly for polymers with Mr<20 kDa. Various approaches for increasing the retention of low Mr (<15 kDa) polymers were investigated. Our results showed that temperature conditions and single-component solvents had a limited effect on polymer retention and that certain binary solvent mixtures caused a dramatic increase in retention. The binary solvents approach has enabled the use of a ThFFF system and temperature conditions to separate 2.6 kDa PS from 4.4 kDa PS, thereby extending the applicability of ThFFF to lower molecular masses. The effect of binary solvent mixtures on polymer retention is correlated with the mixture viscosity.     

Effect of channel width on the retention of colloidal particles in polarization, steric, and focusing micro-thermal field-flow fractionation

The effect of the channel width on the performance of separation by micro-thermal field-flow fractionation (micro-TFFF) of the carboxylated polystyrene latex particles was studied by using the particles in diameter range from 100 nm to 3800 nm. It has been shown that the retention order follows the anticipated polarization, steric, and focusing mechanism in the corresponding size range and under the specific conditions, appropriate to each channel thickness. However, the attractive interactions of the particles with the accumulation wall can complicate the separation as has been proven by the experiments carried out by using the carrier liquids of different ionic strengths. Three channel thicknesses (0.025, 0.100, and 0.250 mm) were tested thus imposing the volumes of micro-channels of roughly 9, 37, and 92 microl. Such an experimental investigation has never been performed with respect to the applicability of the TFFF within an extended range of molar masses or particle sizes. The advantages and drawbacks of different channel widths are discussed with respect to the performance of separation of micro-TFFF but also by taking into account the practical requirements of the construction of the micro-TFFF channel. The principal finding is that very thin channel (w = 0.025 mm) substantially reduces the range of particle sizes or polymer molar masses that can effectively be separated due to the mixed separation mechanism, steric exclusion being effective from smaller particle size. The found dependence of the resolution on the imposed experimental conditions including the channel width has allowed the elucidation of some peculiar results published in the literature, which were contradictory with regard to the known theoretical and experimental findings.     

Theory of field-flow fractionation with the reversible adsorption on channel walls

Summary The particle-wall interactions in field-flow fractionating channels are described phenomenologically using the distribution coefficient, k. The time dependencies of the first and second dispersion coefficients are calculated and compared with the known solutions for the case of no adsorption. The results for the retention ratio and peak broadening are compared with the known experimental work. The model predicts the increase of peak broadening with the growth of particle-wall interactions that is consistent with the experimental data. The dependence of peak asymmetry on transverse Pe number and distribution coefficient is studied.Presented at the 19th ISC, Aix-en-Provence, France, September 14–18, 1992.     

Effect of asymmetrical flow field-flow fractionation channel geometry on separation efficiency

The separation efficiencies of three different asymmetrical flow field-flow fractionation (AF4) channel designs were evaluated using polystyrene latex standards. Channel breadth was held constant for one channel (rectangular profile), and was reduced either linearly (trapezoidal profile) or exponentially (exponential profile) along the length for the other two. The effective void volumes of the three channel types were designed to be equivalent. Theoretically, under certain flow conditions, the mean channel flow velocity of the exponential channel could be arranged to remain constant along the channel length, thereby improving separation in AF4. Particle separation obtained with the exponential channel was compared with particle separation obtained with the trapezoidal and rectangular channels. We demonstrated that at a certain flow rate condition (outflow/inflow rate = 0.2), the exponential channel design indeed provided better performance with respect to the separation of polystyrene nanoparticles in terms of reducing band broadening. While the trapezoidal channel exhibited a little poorer performance than the exponential, the strongly decreasing mean flow velocity in the rectangular channel resulted in serious band broadening, a delay in retention time, and even failure of larger particles to elute.     

Mass overloading in the flow field-flow fractionation channel studied by the behaviour of the ultra-large wheat protein glutenin

Flow field-flow fractionation (FFF) has previously been used in successful fractionation and characterisation of the ultra-large wheat protein glutenin. The many parameters, which may influence the retention behaviour, especially when analysing extremely high-molecular-mass samples such as glutenin, are here reported. Size determination from the sample retention time, using FFF theory, will as a result have a very low accuracy. The need for direct molecular mass determination, such as by light scattering, in combination with FFF, in order to do accurate size measurements of glutenin is pointed out as well as the importance to minimise the overloading.     

Flow field-flow fractionation: recent trends in protein analysis

Flow field-flow fractionation (F4) is the gentlest flow-assisted separation technique for analysis of macromolecules. The use of an empty channel as separation device and of a second mobile phase flow as perpendicular field enable F4 to separate analytes under native conditions without any modification of their original structure. Because of this unique peculiarity, F4 has been shown to be ideal for "gentle" separation of biological samples, for example intact proteins and protein complexes, since its early development. Today's F4 is an appealing technique which complements most established separation techniques, for example liquid chromatography and electrophoresis. The number of applications that show the unique advantages of F4 for analysis of protein samples is constantly increasing. In particular, F4 is finding increasing application on very high-molecular-weight species such as protein oligomers, aggregates, and complexes. This review critically discusses recent literature on the application of F4 to proteins. Either stand-alone or coupled with other characterization techniques, F4 is particularly promising for quality control of protein therapeutics, characterization of amyloid proteins, lipoprotein profiling, and as a pre-MS separation step in proteomics.     

Performance of hollow-fiber flow field-flow fractionation in protein separation

Since hollow-fiber flow field-flow fractionation (HF FIFFF) utilizes a cylindrical channel made of a hollow-fiber membrane, which is inexpensive and simple in channel assembly and thus disposable, interests are increasing as a potential separation device in cells, proteins, and macromolecules. In this study, performance of HF FIFFF of proteins is described by examining the influence of flow rate conditions and length of fiber (polyacrylonitrile or PAN in this work) on sample recovery as well as experimental plate heights. The interfiber reproducibility in terms of separation time and recovery was also studied. Experiments showed that sample recovery was consistent regardless of the length of fiber when the effective field strength (equivalent to the mean flow velocity at the fiber wall) and the channel void time were adjusted to be equivalent for channels of various fiber lengths. This supported that the majority of sample loss in HF FIFFF separation of apoferritin and their aggregates may occur before the migration process. It is finally demonstrated that HF FIFFF can be applied for characterizing the reduction in Stokes' size of low density lipoproteins from blood plasma samples obtained from patients having coronary artery disease and from healthy donors.     

Different elution modes and field programming in gravitational field-flow fractionation. Effect of channel angle

Gravitational field-flow fractionation (GrFFF) has been shown to be useful for separation and characterization of various types of micrometer-sized particles. It has been recognized however that GrFFF is less versatile than other members of FFF because the external field (Earth's gravity) in GrFFF is relatively weak and is not tunable (constant), which makes the force acting on the particles constant. A few approaches have been suggested to control the force acting on particles in GrFFF. They include (1) changing the angle between the Earth's gravitational field and the longitudinal axis of the channel, and (2) the use of carrier liquid having different densities. In the hyperlayer mode of GrFFF, the hydrodynamic lift force (HLF) also act on particles. The existence of HLF allows other means of changing the force acting on the particles in GrFFF. They include (1) the flow rate programming, or (2) the use of channels having non-constant cross-section. In this study, with polystyrene latex beads used as model particles, the channel angle was varied to study its effect on elution parameters (such as selectivity, band broadening and resolution) in the steric or in the hyperlayer mode of GrFFF. In addition, the effects of the channel thickness and the flow rate on the elution parameters were also investigated. It was found that, in the steric mode, the resolution decreases as the flow rate increases due to increased zone broadening despite of the increase in the selectivity. At a constant volumetric flow rate, both the zone broadening and the selectivity increase as the channel thickness increases, resulting in the net increase in the resolution. It was also found that the retention time decreases as the channel angle increases in both up- and down-flow positions. The zone broadening tends to increase almost linearly with the channel angle, while no particular trends were found in selectivity. As a result, the resolution decreases as the channel angle increases.     

Experimental study of isopycnic focusing generated by coupled electric and gravitational field forces: Use in thin layer focusing and focusing field-flow fractionation

Various operational parameters affecting the formation of the density gradient generated by the electric field action on a binary pseudo-continuous carrier liquid composed of charged colloidal silica particles suspended in water and the isopycnic focusing of sample particles were investigated under conditions of static thin layer focusing and dynamic focusing field-flow fractionation. The properties and the behavior of the density gradient forming carrier liquid were studied. The experimental results are compared with theoretical predictions and discussed with respect to potential applications of the proposed concept not only for separation purposes but also for studies of interparticle interactions.     

New concept in focusing field-flow fractionation and thin layer isopycnic focusing: Coupling of primary electric field with secondary gravitational force

A new concept of isopycnic focusing exploiting the simultaneous action of coupled electric and gravitational fields is described. Experimental implementation of this concept is demonstrated under conditions of focusing field-flow fractionation and of newly proposed thin layer isopycnic focusing. Both methods are aimed for particle separations. Although the demonstration was performed by using model cross-linked polymer and latex particles, the most important field of potential applications is intended for analytical and preparative separations of biological particles.     

Improved accuracy in the determination of field-flow fractionation elution volumes.

Conventional operation of field-flow fractionation (FFF) systems involves carrying out the analysis at a constant flow of carrier; the flow is temporarily interrupted after injection of a sample in order to permit its equilibration under the applied field. Retention is calculated as the ratio of elution times for a non-retained species and the sample of interest, respectively. Such time-based retentions are only valid if the flow-rate is precisely known at all times during the run. The peristaltic pumps often used with FFF equipment are shown to have an output which varies unpredictably in time. Furthermore, initiation of flow after relaxation is shown to result in significant periods of transient behaviour while the system adjusts to the operating pressure. These and other variations in flow-rate can be eliminated as sources of error by basing the retention measurement on effluent weight, rather than on time. For this purpose, an electronic balance is interfaced with the system's computer, so that detector response/effluent weight data pairs are continuously monitored during the course of the FFF analysis.     

Newly designed flow field-flow fractionation channel for macromolecules and particles in the submicrometer and micrometer range

The flow field-flow fractionation (FIFFF) technique is a promising method for separating and analysing particles and large size macromolecules from a few nanometers to approximately 50 μm. A new fractionation channel is described featuring well defined flow conditions even for low channel heights with convenient assembling and operations features. The application of the new flow field-flow fractionation channel is proved by the analysis of pigments and other small particles of technical interest in the submicrometer range. The experimental results including multimodal size distributions are presented and discussed.     

Isoelectric focusing in long immobilized pH gradient gels to improve protein separation in proteomic analysis.

The number of protein spots detected on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) gels increases as the gel size increases. The largest commercially available systems resolve a few thousand spots, being only a fraction of the total proteome. We have developed an extremely long isoelectric focusing (IEF) system aimed at more complete protein profiling. The system is especially well suited to sensitive detection methods, such as radioactive detection. The major constraint preventing progress in this area has been the inability to create an even density gradient during the immobilized pH gradient (IPG) casting process. We demonstrate for the first time that this constraint can be effectively overcome, to enable greatly increased IEF separating power with all the advantages of IPG technology,     

Osmolarity effects on red blood cell elution in sedimentation field-flow fractionation.

Field-flow fractionation (FFF) is an analytical technique particularly suitable for the separation, isolation, and characterization of macromolecules and micrometer- or submicrometer-sized particles. This chromatographic-like methodology can modulate the retention of micron-sized species according to an elution mode described to date as "steric hyperlayer". In such a model, differences in sample species size, density, or other physical parameters make particle selective elution possible depending on the configuration and the operating conditions of the FFF system. Elution characteristics of micron-sized particles of biological origin, such as cells, can be modified using media and carrier phases of different osmolarities. In these media, a cells average size, density, and shape are modified. Therefore, systematic studies of a single reference cell population, red blood cells (RBCs), are performed with 2 sedimentation FFF systems using either gravity (GrFFF) or a centrifugational field (SdFFF). However, in all cases, normal erythrocyte in isotonic suspension elutes as a single peak when fractionated in these systems. With carrier phases of different osmolarities, FFF elution characteristics of RBCs are modified. Retention modifications are qualitatively consistent with the "steric-hyperlayer" model. Such systematic studies confirm the key role of size, density, and shape in the elution mode of RBCs in sedimentation FFF for living, micronsized biological species. Using polymers as an analogy, the RBC population is described as highly "polydisperse". However, this definition must be reconsidered depending on the parameters under concern, leading to a matricial concept: multipolydispersity. It is observed that multipolydispersity modifications of a given RBC population are qualitatively correlated to the eluted sample band width.