Conventional Size Exclusion Chromatography Research Articles

A New Functional Role of the Fibrinogen RGD Motif as the Molecular Switch That Selectively Triggers Integrin αIIbβ3-dependent RhoA Activation during Cell Spreading

A number of RGD-type integrins rely on a synergistic site in addition to the canonical RGD site for ligand binding and signaling, although it is still unclear whether these two recognition sites function independently, synergistically, or competitively. Experimental evidence has suggested that fibrinogen binding to the RGD-type integrin alphaIIbbeta3 occurs exclusively through the synergistic gamma(400-411) sequence, thus questioning the functional role of the RGD recognition site. Here we have investigated the respective role of the fibrinogen gamma(400-411) sequence and the RGD motif in the molecular events leading to ligand-induced alphaIIbbeta3-dependent Chinese hamster ovary (CHO) cell or platelet spreading, by using intact fibrinogen and well characterized plasmin-generated fibrinogen fragments containing either the RGD motif (fragment C) or the gamma(400-411) sequence (fragment D), and CHO cells expressing resting wild type (alphaIIbbeta3wt), constitutively active (alphaIIbbeta3T562N), or non-functional (alphaIIbbeta3D119Y) receptors. Our data provide evidence that the gamma(400-411) site by itself is able to initiate alphaIIbbeta3 clustering and recruitment of intracellular proteins to early focal complexes, mediating cell attachment, FAK phosphorylation, and Rac1 activation, while the RGD motif subsequently acts as a molecular switch on the beta3 subunit to trigger cell spreading. More importantly, we show that the premier functional role of the RGD site is not to reinforce cell attachment but, rather, to imprint a conformational change on the beta3 subunit leading to maximal RhoA activation and actin cytoskeleton organization in CHO cells as well as in platelets. Finally, alphaIIbbeta3-dependent RhoA stimulation and cell spreading, but not cell attachment, are Src-dependent and phosphoinositide 3-kinase-independent and are inhibited by the Src antagonist PP2.

Characterization of alkyd resins by size exclusion chromatography coupled with a multi-angle light scattering photometer

An on-line multi-angle light scattering detector (MALS) coupled with size exclusion chromatography (SEC) has been used for the determination of molar mass and size distribution of alkyd resins. Molar mass distribution of alkyd resins is usually determined by SEC using calibration by polystyrene standards (PS). Since the molecules of alkyd resins are significantly more compact than those of linear polystyrene the molar mass determined by conventional SEC is incorrect. The correct molar mass can be obtained using an on-line MALS detector that eliminates the column calibration. In addition, it allows sample differentiation in respect to their degree of branching. It was found that highly branched alkyd molecules have abnormal elution behavior.

On the Estimation of Flow Properties of Polyolefins Based on Fast and Conventional Size Exclusion Chromatography

AbstractSummary: The present article highlights the possible perdiction of flow properties of polyolefins by fast and conventional size exclusion chromatography measurements. By using a rheological model based on double reptation the viscosity function under shear can be predicted from the molecular mass distribution. The predicted flow curves for polyolefins of narrow molecular mass distributions agree fairly well with the measured data.Comparison of predicted and measured complex viscosity functions of polyethylenes with narrow molecular mass distribution.magnified imageComparison of predicted and measured complex viscosity functions of polyethylenes with narrow molecular mass distribution.

Using light scattering to determine the stoichiometry of protein complexes.

The stoichiometry of a protein complex can be calculated from an accurate measurement of the complex's molecular weight. Multiangle laser light scattering in combination with size-exclusion chromatography and interferometric refractometry provides a powerful means for determining the molecular weights of proteins and protein complexes. In contrast to conventional size-exclusion chromatography and analytical centrifugation, measurements do not rely on the use of molecular weight standards and are not affected by the shape of the proteins. The technique is based on the direct relationship between the amount of light scattered by a protein in solution and the product of its concentration and molecular weight. A typical experimental configuration includes a size-exclusion column to fractionate the sample, a light-scattering detector to measure scattered light, and an interferometric refractometer to measure protein concentration. The determination of the molecular weight of an anthrax toxin complex will be examined to illustrate how multiangle laser light scattering can be used to determine the stoichiometry of protein complexes.

Metallo-celluloses, 1 Synthesis and solution properties of 4-ferrocenylbutylsubstituted cellulose derivatives

Abstract An efficient method is described for the simultaneous introduction of 4-ferrocenylbutyl and n-hexyl side groups into cellulose. For this purpose, cellulose triacetate is treated in dimethyl sulfoxide/NaOH solution with mixtures of 4-bromobutylferrocene and n-hexyl bromide. An almost quantitative conversion of the cellulose hydroxyl groups was reached when less than one ferrocene substituent was attached to the cellulose repeating units on average. At higher degrees of ferrocene substitution, some of the cellulose hydroxyl groups seem to remain unsubstituted. This conclusion has been drawn on the basis of size exclusion chromatography (SEC) with online light-scattering detector where coil contraction was observed with increasing ferrocene content in the cellulose sample. This finding was further supported by IR spectroscopy, viscometry, and conventional SEC investigations. We assume that steric hindrance of the bulky side groups is responsible for incomplete conversion.

Analysis of aggregates of human immunoglobulin G using size-exclusion chromatography, static and dynamic light scattering

Large aggregates ( M r: 10 6–10 7 g/mol) of human immunoglobulins are present in extremely small concentrations in IgG preparations (<0.1%). Traces of large protein aggregates cannot be determined by conventional size-exclusion chromatography (SEC) using UV detection due to limitations in sensitivity. The conventional analysis of IgG by SEC is limited to dimers and oligomers. Using light scattering it is possible to determine significant differences concerning the aggregate composition and the extent of protein aggregation in samples of different process steps. Two different pilot preparations were analyzed by SEC with UV and static light scattering detection and compared to dynamic light scattering in the batch mode. The change of large aggregates could be monitored and data were corroborated by dynamic light scattering.

Effect of UV irradiation on organic matter extracted from treated Ohio River water studied through the use of electrospray mass spectrometry.

Ohio River water was treated by settling, sand filtration, and granular activated carbon filtration. It was then irradiated by low-pressure (monochromatic) and medium-pressure (polychromatic) UV lamps to investigate the effects of UV irradiation on the extracted organic matter (EOM). When the EOM, collected by solid phase extraction cartridges, was analyzed by conventional UV spectroscopy and size exclusion chromatography (SEC), no significant changes in the EOM were revealed for various UV doses. Positive and negative electrospray ionization mass spectrometry (ESI-MS) of the EOM produced mass spectra that vary significantly with UV dose. The UV dosage conditions also appear to affect the reactivity of the EOM to subsequent chlorination. The magnitude of the spectral changes is generally greater for medium-pressure lamps than for low pressure and increases with UV exposure. Based on the observed MS peaks, the changes may be due to the presence of lignin, resulting perhaps from photooxidation and/or photo rearrangement of macromolecules in the sample. When chlorination is used for secondary disinfection, these results suggest that it may be important to consider the effects of UV irradiation on the organic matter in the water before applying UV disinfection technology to a particular source water.

Separation of mixed solutes using reciprocating size exclusion chromatography: Computer simulation based upon experimental parameters

Mixed solutes of different molecular size were separated by reciprocating size exclusion chromatography and the results were compared with conventional size exclusion chromatography with repeated feeds using computer simulation. Operational conditions, such as overlapping between elution curves and diffusivities of solutes, were varied in simulation. Simulation showed the separation efficiency was higher in RSEC than SEC with repeated feeds in the region of high diffusivity of either solutes at low degree of overlapping. Further study to improve RSEC by employing temperature swing is being made.

Multielement trace-element speciation in metal-biomolecules by chromatography coupled with ICP-MS.

The state-of-the-art of multi-element speciation in biological materials by hybrid techniques with inductively coupled plasma-mass spectrometric (ICP-MS) detection is critically reviewed with special attention to multi-metal speciation in metallothioneins (MT) and metallothionein-like proteins (MLP). The most common separation techniques used for the purpose include conventional size-exclusion chromatography (SEC) (with off-line detection) and anion-exchange (AE) or reversed-phase (RP) HPLC coupled on-line to ICP-MS. Advantages and limitations of the different commercial element-selective ICP-MS detectors, including the quadrupole (Q), double focusing (DF), and time-of-flight (TOF) mass analysers, are discussed. Procedures reported for speciation analysis, by use of hybrid techniques, for multi-metal speciation in mussel MLP and rabbit liver MT are illustrated with special emphasis on work performed in the authors' laboratory.

Determining the molar mass of a plasma substitute succinylated gelatin by size exclusion chromatography–multi-angle laser light scattering, sedimentation equilibrium and conventional size exclusion chromatography

The clinical effectiveness of succinylated gelatin as a plasma substitute depends strongly on its molar mass, determined conventionally by size exclusion chromatography (SEC). This study evaluates different SEC calibration standards in comparison with two independent “absolute” methods for determining the weight average molar mass ( M w) of a succinylated gelatin sample. SEC calibrated using succinylated gelatin fractions correlated well with size exclusion chromatography–multi-angle laser light scattering (SEC–MALLS) and sedimentation equilibrium whereas SEC calibrated with unmodified gelatin, sodium polystyrene sulfonates or pullulans overestimated M w by over 20%. Universal calibration was equivocal. The problems associated with the preparation of succinylated gelatin fractions suggest that an absolute method such as SEC–MALLS may be a more suitable choice for determining the M w in succinylated gelatins.

Chromatographic characterization of synthetic peptides: SPf66 malaria vaccine

The development and validation of a quantitative size-exclusion chromatography (SEC) method for SPf66 malaria vaccine was achieved. The results show the reliability of the analytical method for the intended use. SPf66 malaria vaccine characterization was performed using both relative techniques such as the conventional SEC and absolute techniques: mass spectrometry and multi-angle laser-light scattering detection. The relative and absolute molecular masses were in the 4600–18 000 Da range. The results clearly indicate the presence of the monomer and dimer species, whereas the third species could be the trimer or tetramer.

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.

Local polydispersity detection in size exclusion chromatography: Method assessment

AbstractLocal polydispersity is the term describing the variety of molecules present at the same retention volume in size exclusion chromatography (SEC) analysis. In the analysis of a linear homopolymer, local polydispersity is generally attributed to the effect of axial dispersion: it can cause molecular size variety (i.e., imperfect resolution) at each retention volume and thus local polydispersity in the molecular weight. In the analysis of polymer blends (copolymers and branched polymers), it is possible to have local polydispersity, even when the resolution is perfect, because molecules of different compositions (or degrees of branching) can have the same molecular size in solution. Conventional SEC interpretation assumes no local polydispersity if the axial dispersion effects are negligible. Three methods are currently available for detecting local polydispersity by using a combination of differential refractive index, light scattering, and viscometer detectors: the chromatogram comparison method, the conventional calibration curve comparison method, and the universal calibration comparison method. Here we experimentally assess these three methods using polymer blends and emphasize the chromatogram comparison method. All three are shown to be useful for assessing triple detector systems, but they are capable of detecting local polydispersity due to molecular heterogeneity only for very large differences in specific refractive index increments in the blend components. © 2001 John Wiley &amp; Sons, Inc. J Appl Polym Sci 81: 370–383, 2001

Characterization of novel liquid crystalline polymers of polyacetylenes having a comb-like structure via SEC/RI/RALLS/DV

Novel liquid crystalline polymers comprised of polyacetylenes having a comb-like structure were studied by size exclusion chromatography (SEC) with refractive index, right angle laser light scattering, and differential viscometry detectors (RI/RALLS/DV). This combination of SEC detectors is known as triple detector SEC, or simply SEC3. Based upon the data generated for these materials, the relationship between molecular size and the polymer structure was investigated, SEC universal calibration was shown to be valid, Flory's characteristic ratio (C∞) was estimated, and the effect of the size of the side group on chain flexibility was elucidated. These results demonstrate that the technique of SEC3 is a powerful mean for characterizing polymers that cannot be effectively characterized by conventional SEC, such as the liquid crystalline polymers of this study.

Size exclusion chromatography of step-growth polymers with cyclic species: theoretical model and data analysis methods

The measurement of the molecular weight averages and the molecular weight distribution (MWD) of many step-growth polymers is complicated due to the presence of cyclic oligomers formed during polymerization. If size exclusion chromatography (SEC) is used to determine the MWD, the cyclic oligomers are generally only partly resolved from the linear polymer, and hence distort the measured linear MWD. Further, the cyclic oligomers require a different calibration curve from the linear species and hence, in general, their molecular weights are not accurately measured. In order to clarify the effect of cyclic species on the measured MWD, a model of the SEC separation of step-growth polymers with cyclic species was developed. In this article, this model is described and used to illustrate aspects of the characterization of these polymers using both conventional SEC and multi-detector SEC. The results from the model are used to develop methods for estimating the MWD of the linear polymer and to determine the weight fraction of cyclic species. The results of the model are compared with experimental data for nylon 6, nylon 6,6 and poly(ethylene terephthalate).

Molecular characterization of α,β-poly(asparthylhydrazide) a new synthetic polymer for biomedical applications

α,β-Poly(asparthylhydrazide) (PAHy) is a new synthetic polymer that exhibits interesting properties and is a candidate for biomedical applications. In this article the characterization of PAHy polymer by multi-angle laser light scattering (MALS) and single-capillary viscometer (SCV) detectors on-line to a size exclusion chromatography (SEC) system is reported. The SEC–MALS–SCV system furnishes exhaustive and consistent characterization of the PAHy polymer. Further, it is possible to characterize the PAHy polymer through conventional SEC and universal calibration. The universal calibration method gives intrinsic viscosity and dispersity very close to those measured by the absolute detectors; instead the weight-average molar mass is approximately 8% lower. This finding means that the chromatographic separation of PAHy polymer is fundamentally based on its hydrodynamic volume. By the SEC–MALS–SCV system the constants of the Mark–Houwink–Sakurada equation were also estimated.

Differential size‐exclusion chromatography for the analysis of gelatin–polyelectrolyte complexes

Differential size-exclusion chromatography (SEC) is used to characterize complexes formed between gelatin and two synthetic polyelectrolytes, sodium poly(styrenesulfonate) and sodium poly(2-acrylamido-2-methylpropanesulfonate). The analysis is performed under aqueous, low-salt conditions where maximum complexation between gelatin and the polyelectrolytes occurs. The adsorption effects that are commonly encountered in conventional SEC for gelatin and other charged polymers chromatographed under these solution conditions are minimized, because the columns are constantly equilibrated with the analytes in the mobile phase. Analyte solutions of identical composition, but of higher or lower concentration than that contained in the mobile phase, are injected, resulting in positive or negative detector responses, respectively. This method can separate the complexes from individual components, and can be used to determine relative sizes and stoichiometries of the complexes as a function of both the input ratio of gelatin to polyelectrolyte and the molecular weight of the polyelectrolyte.

Molar Mass Distribution of a Polymer-Drug Conjugate Containing the Antitumor Drug Paclitaxel by Size Exclusion Chromatography and Universal Calibration

The development of a Size Exclusion Chromatography (SEC) method for the characterization of the polymer-drug conjugate PNU 166945 is presented. PNU 166945 is a conjugate between poly[N-(2-hydroxypropyl) methacrylamide)] and the antitumor drug paclitaxel. This study follows a previous paper on a similar polymer conjugate, FCE 28068, containing the same polymeric carrier and the antitumor drug doxorubicin. The aim was an accurate and reproducible, yet relatively simple and rapid method for the routine quality control of production batches. Thirteen PNU 166945 narrow fractions were separated, by semi-preparative SEC, and characterized by on-line SEC-Viscometry and off-line Light Scattering. The fractions allowed us to verify that PNU 166945 follows the universal calibration constructed with PEO/PEG narrow standards. So a conventional SEC method that utilizes just a refractive index detector and universal calibration with commercial narrow standards was found suitable. The study also reports data on accuracy and reproducibility of the SEC measurement.

Characterization of polydisperse synthetic polymers by size-exclusion chromatography/matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Synthetic polymers having a polydispersity greater than 1.1 cannot be reliably characterized by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry alone. This complication has been overcome by off-line coupling of size-exclusion chromatography (SEC) with MALDI. Data are presented for a wide variety of synthetic polymers having polydispersities from 1.7 up to 3.0. Polystyrene (15 kDa and 48 kDa), polybutylacrylate (62 kDa), polycarbonate (28 kDa), aromatic polyester resin (8 kDa) and a methyl methacrylate–methacrylic acid copolymer (34 kDa) were injected on a conventional SEC system with refractive index (RI) or UV absorbance detection and 40 fractions collected over the polymer distribution. Ten fractions were analyzed by a MALDI time-of-flight mass spectrometer operated in the continuous extraction linear mode. The m/z thus obtained were used as calibration points for the SEC-RI system and allowed accurate calculation of the molecular weight averages Mw, Mn, Mz and the polydispersity for all the polymers tested. The data for these entirely different polydisperse polymers were in good agreement with reference data from, for example, SEC with intrinsic viscosity detection. In addition, the choices of matrix and cation for individual types of polymers are commented on. The end groups of the polymers were determined from isotopically-resolved oligomer spectra recorded by MALDI time-of-flight in the delayed-extraction reflectron mode. End groups proposed are compared with reference data from, amongst others, NMR and MALDI Fourier transform ion cyclotron resonance-MS measurements. © 1997 John Wiley & Sons, Ltd.

High performance liquid chromatography characterization of macromolecules

AbstractA new method, temperature gradient interaction chromatography(TGIC) is employed for the characterization of macromolecules. Fine and reproducible control of interaction between polymer chains and the alkyl chain bonded silica packing material can be achieved by varying the temperature of the column. This method provides a far superior resolution to the conventional size exclusion chromatography. In addition, this method has a high sample loading capacity to be effective for preparative purpose. Furthermore, this method can be used to characterize binary polymer mixtures, where one component of a polymer mixture is separated by the size exclusion mechanism and the other is by the interaction mechanism simultaneously from single isocratic elution.