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.     

Nonequilibrium and Polydispersity Peak Broadening in Thermal Field-Flow Fractionation

Abstract

New theoretical procedures and an expanded data base have been combined to yield improved calculations for nonequilibrium plate height and polydispersity contributions in field-flow fractionation (FFF). Five thermal FFF column systems having a fourfold range in column width and length were operated with four solutes to yield eleven distinct channel-solute systems. After data were corrected for relaxation effects, comparison of experimental plate height measurements with theory showed good overall agreement for nonequilibrium parameter X. Calculated values of polydispersity agreed reasonably well with supplier's values. Data reliability has been discussed in relationship to column dimensions and experimental parameters.     

Two‐dimensional chromatography as a tool for studying band broadening in size‐exclusion chromatography

Comprehensive 2‐D size‐exclusion chromatography (SEC×SEC) has been realized. SEC×SEC is not a useful technique for characterizing complex polymers. However, it is potentially an elegant tool to study band‐broadening phenomena. If narrow fractions can be collected from the first dimension, the band broadening in the second dimension is only due to chromatographic dispersion. This would allow a clear distinction to be made between chromatographic band broadening (column and extra‐column) and SEC selectivity (band broadening due to sample polydispersity). In comparison with MALDI‐MS, SEC×SEC allows the study of polymers across a much broader molar‐mass range.     

Band broadening in size-exclusion chromatography of polydisperse samples

Understanding and controlling the band broadening is essential to obtain accurate molar-mass distributions by size-exclusion chromatography (SEC). In this paper, band broadening in SEC is reviewed from a contemporary perspective. The observed band broadening is due to dispersion inside and outside the chromatographic column (undesirable band broadening) and to the polydispersity of the sample (desirable SEC selectivity). The various contributors to band broadening are discussed. Integrity plots are introduced as a tool to evaluate the performance of specific SEC columns at given experimental conditions. For narrow polymer standards on single SEC columns the observed peak width is dominated by the chromatographic dispersion. MALDI-ToF-MS is demonstrated as an alternative to determine the PDI of narrowly distributed samples. The plate heights encountered at very high reduced velocities are found to be lower than expected. This is advantageous for fast separations by SEC.     

Off-line size-exclusion chromatographic fractionation-matrix-assisted laser desorption ionization time-of-flight mass spectrometry for polymer characterization. Theoretical and experimental study

The parameters affecting the fractionation performance in size-exclusion chromatography (SEC) of broad polymer samples were investigated. Some equations were derived which enable the prediction of polydispersity (PD) in an SEC fraction. Good agreements were obtained between the calculated data and the experimental values. Based on these equations, SEC fractionation conditions were optimized. In the off-line SEC-matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS), two different modes can be employed, i.e., using MALDI-MS to provide an absolute calibration curve for SEC, or using SEC as a sample preparation step for MALDI-MS measurements. It was demonstrated that it is more reliable to use the latter combination, because most problems inherent in SEC can be circumvented. Some guidelines for the optimization of off-line SEC fractionation-MALDI-TOF-MS were given. It was found that under optimized conditions normally only a few SEC fractions are already sufficient to separate a highly polydisperse sample into portions of low PD that can accurately be measured by MALDI-TOF-MS.     

Polymer fractionation using chromatographic column packed with novel regenerated cellulose beads modified with silane

Novel microporous beads with the particle size of about 90 microm were prepared, for the first time, from cellulose and konjac glucomannan (RC/KGM3) in 1.5 M NaOH/0.65 M thiourea aqueous solution by emulsification method. The microporous beads were then modified with silane to avoid the adsorption of polymers containing hydroxyl groups, coded as RC/KGM3-Si. A preparative size-exclusion chromatographic (SEC) column (500 mm x 20 mm) was packed with RC/KGM3-Si, and its exclusion limit and fractionation range of the stationary phase were, respectively, weight-average molecular masses (Mw) of 4.8 x 10(5) g/mol and 5.3 x 10(3)-4.8 x 10(5) g/mol for polystyrene in tetrahydrofuran. The preparative SEC column was used to fractionate poly(epsilon-caprolactone) (PCL, Mw = 8.31 x 10(4) g/mol polydispersity index d= 1.55) in tetrahydrofuran and a polysaccharide PC3-2 (Mw = 1.21 x 10(5) g/mol, d= 1.70) in 0.05 M NaOH aqueous solution, respectively. The Mw values of the fractions determined by analytical SEC combined with laser light scattering were from 1.2 x 10(4) to 1.84 x 10(5) for PCL and from 8.5 x 10(4) to 2.13 x 10(5) for PC3-2, as well as d from 1.2 to 1.5. The results indicated that the preparative SEC has good fractionation efficiency in both organic solvent and alkaline aqueous solution for the various polymers.     

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.     

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.

Use of polymeric reversed-phase columns for the characterization of polypeptides extracted from human pancreata. I. Effect of the mobile phase

The high-performance liquid chromatographic (HPLC) behaviour of two different styrene-divinyl-benzene-based reversed-phase (RP) columns was evaluated using crude acetic acid extracts from normal and diabetic human pancreata as samples. Acetic acid gradients in water and acetonitrile gradients in triethylammonium phosphate (TEAP) and trifluoroacetic acid (TFA) were used as mobile phases, and comparisons were made with a silica-based C4 column. When two different polymeric RP columns were eluted with acetic acid gradients in water, surprisingly similar HPLC profiles of the pancreatic extracts were obtained. Elution of the polymer-based columns with acetonitrile gradients in TFA or TEAP resulted in changes in the polypeptide selectivity of these columns, in parallel with that of a silica-based C4 column eluted under similar conditions, indicating the general usability of polymeric columns for RP-HPLC of peptides and proteins. The pronounced difference in composition between normal and diabetic samples, which also was demonstrated after size-exclusion chromatography (SEC) on a silica-based and an agarose-based high-performance SEC column, was found to be related to the different ischaemia times for the two types of pancreata.     

Columns switch recycling size exclusion chromatography for high resolution protein separation

Columns switch recycling size exclusion chromatography (csrSEC) was proposed to achieve high resolution protein separation with good biocompatibility. Proteins were firstly separated by two serially coupled SEC columns, and fractions were in sequence switched back to the first column by two-position valves for further separation in terms of close-loop recycling until satisfactory resolution was achieved. Compared to SEC, the separation window was broadened by increasing column length via cycling without further increase on back pressure. Compared to recycling SEC (rSEC), the overtaking of later eluted components by early eluted ones after several cycles could be avoided for complex sample analysis, by parking fractions in the second SEC column before transferred in turn back to the first one for cycling ordinally. In our experiments, the baseline separation of five proteins with molecular weight ranging from 10 kDa to 80 kDa was achieved by csrSEC. Furthermore, a multidimensional csrSEC–RPLC platform was constructed, and peak capacity up to 3600 was obtained for protein separation. All these results demonstrated that csrSEC is a promising protein separation mode with good biocompatibility, broadened separation window and improved resolution.     

Computer simulation of particle separation based on non-equilibrium swelling

Steric/hyperlayer field-flow fractionation (FFF) is an established analytical technique for separating and characterizing particles in the 1-100 microns diameter range. The separation can be based on differences in size, density, shape and mechanical properties of the particles. In the course of an analysis of the water transporter system of Chinese hamster ovary (CHO) cells and one of their high permeability mutants, the first successful attempt was made to use the steric/hyperlayer FFF system for the purpose of separating particles based on a time-dependent property, namely, the differential swelling of the two cell types. The present study was undertaken to simulate numerically the separation and suggest selection of operating conditions to minimize repetitive experiments. The computer simulation was developed using Maple V, a symbolic computing environment. It is shown that the model is able to predict an optimal velocity of carrier buffer that maximizes resolution. Predicted velocity/resolution pairs are in good agreement with available experimental data. Empirical models for the lift forces encountered in such FFF experiments, and for the zone broadening observed in work with cell sized particles, form the basis for this model.     

聚合离子液体毛细管气相色谱固定相的性能评价

离子液体作为毛细管气相色谱固定相的选择性和热稳定性是近年人们关注的课题。本文合成了1-烯基-3-苄基咪唑-二(三氟甲基磺酰)亚胺盐(VBIm-NTf2)离子液体并经毛细管柱内聚合制得了聚合离子液体PVBIm-NTf2色谱柱。与VBIm-NTf2色谱柱相比,PVBIm-NTf2色谱柱具有良好的色谱选择性和热稳定性。PVBIm-NTf2色谱柱对Grob试剂、醇混合物、酯混合物和苯系物等都表现了很好的分离能力,并且色谱峰峰形窄且对称。该聚合色谱柱在250 ℃下老化6 h后仍具有良好的分离能力和选择性。本文还测定了PVBIm-NTf2的Abraham溶剂化参数,解析了该固定相与溶质间的相互作用,相关研究国内尚未见报道。研究表明,对常规离子液体进行柱内聚合是改善常规离子液体气相色谱固定相的热稳定性和选择性的有效途径。     

Preparative SEC column packed with microporous particles prepared from cellulose

New microporous particles with large pore size (mean pore diameter of 820 nm) are successfully prepared from a mixture of cellulose and konjac glucomannan (RC-KGM3) in 1.5 M NaOH-0.65 M thiourea aqueous solution by coagulating with 5 weight percentage (wt%) CaCl(2), and then 2 wt% HCl aqueous solution. A preparative size-exclusion chromatographic (SEC) column packed with the gel particles is used for the fractionation of a dextran in water. The exclusion limit and fractionation range of the stationary phase are molecular masses of 125 yen 104 g/mol and 5.6 yen 104 to 125 yen 104 g/mol, respectively. The dextran [dextran 50, weight-average molecular mass (M(w)) = 40.1 yen 104 g/mol, polydispersity index (d) = 3.5] is fractionated by the preparative SEC column to obtain six fractions, and four of them are refractionated twice by the same preparative SEC column. The refractionated samples F-3-3 and F-4-3 are characterized by analytical SEC combined with laser light scattering and light scattering to obtain M(w) of 91.8 and 61.9 yen 104 g/mol, as well as d of 1.3 and 1.4, respectively. The results indicate that the fractions having narrow molecular mass distribution are satisfactorily prepared with the SEC column. The described SEC column can be successfully used to fractionate polymers in aqueous solution.     

Improved performance of protein separation by continuous annular chromatography in the size-exclusion mode.

In size-exclusion chromatography (SEC), proteins and peptides are separated according to their molecular size in solution. SEC is especially useful as an effective fractionation step to separate a vast amount of impurities from the components of interest and/or as final step for the separation of purified proteins from their aggregates, in a so-called polishing step. However, the throughput in SEC is low compared to other chromatographic processes as good resolution can be achieved only with a limited feed volume (i.e., maximal approximately 5% of the column volume can be loaded). This limitation opposed widespread application of conventional SEC in industry despite its excellent separation potential. Therefore a continuous separation process (namely preparative continuous annular chromatography) was developed and compared to a conventional SEC system both using Superdex 200 prep grade as sorbent. An immunoglobulin G sample with a high content of aggregates was chosen as a model protein solution. The influence of the feed flow-rate, eluent flow-rate and rotation rate on the separation efficiency was investigated. The height equivalent to a theoretical plate was lower for preparative continuous annular chromatography which could be explained by reduced extra column band broadening. The packing quality was proved to be identical for both systems. The productivity of conventional batch SEC was lower compared to continuous SEC, consequently buffer consumption was higher in batch mode.     

Brush-type chiral stationary phase for enantioseparation of acidic compounds. Optimization of chiral capillary electrochromatographic parameters

The capillary electrochromatographic separations of three acidic enantiomers (carprofen, coumachlor and warfarin) were studied on a capillary column packed with 5 microm (3R,4S)-Whelk-O 1 chiral stationary phase. The influence of several experimental parameters (mobile phase pH, type of background electrolyte, acetonitrile ratio, temperature, applied voltage and ionic strength) on electroosmotic flow velocity, retention factor, selectivity factor, efficiency, resolution and effectiveness of chiral separation was evaluated. It was notable that the optimum resolution of the acidic enantiomers was achieved at pH 3.0 phosphate buffer, suggesting that capillary electrochromatography in the ion-suppressed mode can be applied for chiral separations of a range of acidic compounds.     

A sub-two minutes method for monoclonal antibody-aggregate quantification using parallel interlaced size exclusion high performance liquid chromatography

In process development and during commercial production of monoclonal antibodies (mAb) the monitoring of aggregate levels is obligatory. The standard assay for mAb aggregate quantification is based on size exclusion chromatography (SEC) performed on a HPLC system. Advantages hereof are high precision and simplicity, however, standard SEC methodology is very time consuming. With an average throughput of usually two samples per hour, it neither fits to high throughput process development (HTPD), nor is it applicable for purification process monitoring. We present a comparison of three different SEC columns for mAb-aggregate quantification addressing throughput, resolution, and reproducibility. A short column (150 mm) with sub-two micron particles was shown to generate high resolution (~1.5) and precision (coefficient of variation (cv)<1) with an assay time below 6 min. This column type was then used to combine interlaced sample injections with parallelization of two columns aiming for an absolute minimal assay time. By doing so, both lag times before and after the peaks of interest were successfully eliminated resulting in an assay time below 2 min. It was demonstrated that determined aggregate levels and precision of the throughput optimized SEC assay were equal to those of a single injection based assay. Hence, the presented methodology of parallel interlaced SEC (PI-SEC) represents a valuable tool addressing HTPD and process monitoring.     

Fast size-exclusion chromatography at high temperature

We report on the size-exclusion chromatography (SEC) operated at high column temperature to reduce the analysis time. The column temperature was raised beyond the normal boiling point of the eluent and a sufficient column backpressure was applied to prevent the mobile phase from boiling by inserting a narrow bore tubing between the separation column and the detector. The narrow bore tubing also functions to cool the effluent down to the room temperature before it reaches the detector. Therefore, normal SEC detectors can be used without any modification. It was confirmed that the SEC analysis time could be shortened significantly by the high-temperature operation without serious deterioration in the resolution.     

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).     

Dispersive mixing and intraparticle partitioning of protein in size-exclusion chromatographic refolding

Refolding enables bioprocesses predicated on proteins expressed as inclusion bodies in Escherichia coli. Optimization of size-exclusion chromatography (SEC) refolding is a significant challenge because a wide range of factors, including the choice of gel media, the column dimensions and configuration, affect the final yield in a protein-specific manner. In this study, we investigated these factors by relating them to dispersive mixing and partitioning of refolding molecules within the SEC pore structure. Lysozyme was refolded using SEC resins giving different column dispersion and chromatography resolution. Despite a low separation resolution, the desalting SEC resin Sephadex G-25 resulted in a refolding yield that was 12-30% higher than those obtained with Superdex 75 and Superdex 200. This finding supported the notion that SEC refolding was enhanced by dispersive mixing, which was increased by a wide particle size distribution of the Sephadex G-25 used. Column dispersion was further improved by strategically placing an inlet gap before the packed resin beds, leading to a 20% increase in refolding yield. Refolding yield in Superdex 75 was 20% higher than that in Superdex 200 under conditions giving similar dispersive mixing. This yield enhancement is expected to be protein-specific since Superdex 75 was chosen to specifically maximize partitioning of lysozyme molecules within the resin particles, reducing the likelihood of aggregation during refolding. The highest refolding yield (65%) was achieved using a Sephadex G-25 column with a 15 mm inlet gap, suggesting that desalting systems optimized for dispersive mixing might be an economical and generic alternative for preparative SEC protein refolding.     

High temperature thermal field-flow fractionation of polyethylene and polystyrene

In this paper the high-temperature thermal field flow fractionation method is exploited for the analysis of polyethylene (PE). The experimental apparatus set-up, obtained by simply modifying a commercial instrument, is presented. The numerical procedure for deriving retention calibration plot versus molecular weight is discussed with reference to the specific polymer-solvent pair, PE-o-dichlorobenzene (ODCB), here employed. Different methods for computing the physicochemical data set of the solvent, necessary for calibration, are compared. The selectivity of the checked PE-ODCB system proves comparable with respect to the values currently found in thermal field-flow fractionation (ThFFF) analysis. Differences are found between PE and polystyrene (PS) analysis in the same solvent. The conditions for high temperature ThFFF operation in PE analysis and their advantages are discussed with respect to the standard SEC technique for PE, PS, and PE-PS copolymer analysis. Molecular weight distributions obtained by ThFFF of two PE commercial samples agree with those obtained by SEC. © 1995 John Wiley & Sons, Inc.