Aqueous Size Exclusion Chromatography Research Articles

Aqueous size exclusion chromatography applied to polymer analysis: experimental conditions and molecular weight calibration curves

Aqueous mode size exclusion chromatography (SEC) was employed for the analysis and construction of molecular weight (MW) calibration curves of three water-soluble polymers, namely, polyethylene glycol, polyethylene oxide, and polyacrylic acid sodium salt. Several calibration curves were obtained, varying chromatographic conditions such as columns arrangement, ionic strength, temperature and pH, in addition trends in polymeric chromatographic behavior were examined. The variation in SEC distribution coefficients at different temperatures was found to be below 10 %, indicating that the studied polymers follow an ideal SEC mechanism under the tested conditions. Thus, differences in chromatographic behavior were ascribed to changes in polymer configuration induced by media and/or temperature. These variations in morphology were consistent with the observed SEC behavior.Regarding MW calibration, polynomial regression models ranging from first to fifth order were applied, and the most adequate ones were selected based on their fit and prediction capabilities. Third order polynomials were the preferred models for polyethylene glycol and polyacrylic acid sodium salt, independently of chromatographic conditions. Meanwhile for polyethylene oxide, either third or fifth-order polynomial models were optimal depending on the chromatographic conditions. All the selected regression models presented coefficients of multiple determination (R2) above 0.990, while achieving relative errors of prediction (REP%) in MW ranging from 0.3 to 4 % for cross-validation.

Molecular size distribution in pentavalent (A, C, Y, W, X) meningococcal polysaccharide conjugate vaccine by HPSEC-UV-MALS-RI method- a conceivable stability indicating parameter

Molecular size distribution (MSD) of polysaccharides serves as a key parameter that directly correlates to the immunogenicity of vaccine. MSD at meningococcal polysaccharide (A, C, Y and W) or conjugate bulk level is well established under detailed pharmacopeial and WHO guidelines. We report here, a newly developed method for determination of molecular size distribution of pentavalent Meningococcal conjugate vaccine comprising of A, C, Y, W and X (MenFive). Although serogroup specific molecular size could not be estimated here; lot to lot consistency monitoring, molecular aggregates distribution in final lot, are key takeaways of this method. Determination of MSD in pentavalent fill finished product was quite challenging. Various columns/detectors combination, buffers, physico-chemical conditions (temperature, 2–8 °C, 25 °C, 40 °C and 60 °C; flow rate, 0.3 mL to 0.8 mL), liquid/lyophilized formulations, were explored. Polymer-based packed columns were explored for estimation for MSD by aqueous size exclusion chromatography, using combinations of- Shodex OHPAK SB 807 HQ, Shodex OHPAK SB 806 HQ, G6000 PWXL, coupled with guard Shodex OHPAK SB-G-6B. MenFive showed heterogenous distribution of molecules ranging from 200 to 19000 kDa, indicating its complex nature. However, 1000–8000 kDa was dominant range, comprising of ≥ 50 % distribution of molecules, in both liquid as well as lyophilized formulations, with average molecular weight around 6000–6500 kDa. The molar mass distribution after slicing would provide an insight to the conformation of molecules through its presentation as HMW, LMW, aggregates and subsequently, the presence of dominant population of molecules of a particular molecular weight and its total contribution in the sample.

Universal calibration for natural polysaccharides in aqueous Size-exclusion chromatography

This article shows the validity of the Universal calibration concept in aqueous Size-exclusion chromatography for arabinogalactan and carboxymethylchitosans is presented. The electrostatic effects of these polysaccharides during separation process were eliminated by utilizing of a water-salt eluent as a mobile phase, and the size-exclusion separation mechanism of this method was realized.

Tailor-Made Poly(vinylamine) via Purple LED-Activated RAFT Polymerization of N-vinylformamide.

Photo-iniferter reversible addition-fragmentation chain transfer (PI-RAFT) polymerization of N-vinylformamide (NVF) is demonstrated by using purple light. PNVFs with predetermined molar masses and narrow molar mass distributions are obtained. High RAFT chain-end fidelity is confirmed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS), and chain extension experiment. To demonstrate the potential of this approach, an original poly(N-vinylpyrrolidone)-b-poly(N-vinylformamide) (PVP-b-PNVF) diblock copolymer is synthesized and characterized by aqueous size-exclusion chromatography (SEC), asymmetric flow field-flow fractionation (A4F), and 1 H diffusion-ordered spectroscopy nuclear magnetic resonance (1 H DOSY NMR). Finally, selective hydrolysis of PNVF block to corresponding pH-responsive poly(N-vinylpyrrolidone)-b-poly(N-vinylformamide) (PVP-b-PVAm) is performed.

Development of aqueous size exclusion chromatography conditions to characterize polyzwitterion-block-N-isopropyl acrylamide copolymers.

Block copolymers that exhibit both an upper critical solution temperature and a lower critical solution temperature are difficult to characterize due to inherent solubility difference between the two blocks. For example, accurate determination of both the molar mass and molar mass distribution is challenging for polyzwitterion-block-N-isopropyl acrylamide (NIPAM) copolymers in aqueous solutions due to self-assembly. However, there are a few examples of using size exclusion chromatography (SEC) for characterization, in which hexafluoro isopropanol (HFIP) is used in all cases. Yet, researchers are hesitant to use this solvent due to how expensive and hazardous HFIP is. Therefore, alternatives to HFIP for SEC analysis would be desirable. Here, a systematic methodology featuring aqueous SEC is demonstrated using several solvent conditions to enable the elution of polyzwitterion-block-NIPAM copolymers on Agilent PolarGel† and Tosoh TSKgel† column sets. These SEC conditions include 0.2M KI in water on the PolarGel columns and 0.2M KI/ 30% DMF in water on the PolarGel and TSKgel columns. These aqueous systems can be utilized for the characterization of similar water-soluble block copolymers that are relevant for drug delivery and other biomedical applications.

Evaluation of particle and bed integrity of aqueous size-exclusion columns packed with sub-2 µm particles operated at high pressure

The performance of columns packed with 1.7 µm particles for aqueous size-exclusion chromatography was assessed at high-pressure conditions and linked to particle- and column-bed integrity. Decreasing the particle size from 3.5 µm to 1.7 µm increases the resolution due to the improved mass-transfer characteristics, allowing to significantly speed-up analysis without compromising the selectivity. A sub-minute separation of intact proteins was realized on a 4.6 mm i.d × 75 mm long column packed with 1.7 µm SEC particles applying a flow rate of 1.8 mL/min, corresponding to a column pressure of 530 bar. Ultra-high pressure operation (exceeding manufacturer's recommendations) resulted in peak deformation, a shift towards earlier retention times, and an alteration in selectivity. To gain insights in the mechanisms of column deterioration, short 30 mm long columns were operated at UHPLC conditions, maximizing the pressure drop over individual particles. This resulted in the presence of fractured particles situated at the column outlet, as verified by scanning electron micrographs. Mercury-intrusion porosimetry and argon-adsorption measurements did not reveal significant differences in intraparticle volume between particle batches sampled before and after pressure stress testing. As particles at the column outlet fracture (but not collapse) at high pressure operation, a void was formed at the column inlet. The degradation of the separation performance appeared to be the result of a decrease in interparticle pore volume.

Affinity profiling of monoclonal antibody and antibody-drug-conjugate preparations by coupled liquid chromatography-surface plasmon resonance biosensing

Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are highly potent biopharmaceuticals designed for targeted cancer therapies. mAbs and ADCs can undergo modifications during production and storage which may affect binding to target receptors, potentially altering drug efficacy. In this work, liquid chromatography was coupled online to surface plasmon resonance (LC-SPR) to allow label-free affinity evaluation of mAb and ADC sample constituents (size and charge variants), under near-native conditions. Trastuzumab and its ADC trastuzumab emtansine (T-DM1) were used as a test sample and were analyzed by aqueous size-exclusion chromatography (SEC)-SPR before and after exposure to aggregate-inducing conditions. SEC-SPR allowed separation of the formed aggregates and measurement of their affinity towards the ligand-binding domain of the human epidermal growth factor receptor 2 (HER2) receptor immobilized on the surface of the SPR sensor chip. The monomer and aggregates of the mAb and ADC were shown to have similar antigen affinity. Conjugation of drugs to trastuzumab appeared to accelerate the aggregate formation. In addition, cation-exchange chromatography (CEX) was coupled to SPR enabling monitoring the maximum ligand-analyte binding capacity (Rmax) of individual charge variants present in mAbs. Deamidated species and lysine variants in trastuzumab sample were separated but did not show different binding affinities to the immobilized HER2-binding domain. In order to allow protein variant assignment, parallel MS detection was added to the LC-SPR setup using a column effluent split. The feasibility of the LC-MS/SPR system was demonstrated by analysis of trastuzumab and T-DM1 providing information on antibody glycoforms and/or determination of the drug-to-antibody ratio (DAR), while simultaneously monitoring binding of eluting species to HER2.Graphical abstractᅟ

Separation and identification of polymeric dispersants in detergents by two-dimensional liquid chromatography

Polymeric dispersants are an important ingredient in many consumer products. Their separations and identifications in final product formulation can be very challenging due to the presence of multiple polymeric dispersants at different levels and the presence of other polymeric and small-molecule components. In this study, using nearly comprehensive two-dimensional liquid chromatography (2D-LC), various water-soluble polymer and co-polymer dispersants were separated with aqueous size exclusion chromatography (SEC) in the first dimension (1D) and gradient elution reversed-phase liquid chromatography (RPLC) in the second dimension (2D). Detection of the polymeric dispersants was accomplished by evaporative light scattering detector (ELSD). A large ID (8.0 mm) SEC column used in common one-dimensional SEC practices was directly adopted in the 2D setup for rapid method development. A close representation of fully comprehensive 2D separation was achieved even with 60% of 1D eluent diverted to waste, demonstrating the flexibility and versatility of having SEC in 1D for two dimensional separation of polymers. Important method parameters, such as 2D column dimensions and flow rate, gradient conditions, and buffer pH were studied. Practical aspects of routine industrial applications such as solvent consumption and analysis time were also considered. This method was exploited for quick identification of polymeric dispersants in commercial detergent samples. Nine detergent samples were screened and polymeric dispersants and additional polymer features were detected in the samples.

Evaluation of charged molecules content of plant polysaccharides by aqueous size exclusion chromatography

Aqueous size-exclusion chromatography (SEC) equipped with refractive index (RI) and multiangle laser light scattering (MALLS) detectors were applied to evaluation of charged molecules content in a series of polysaccharide derivatives with different average molar mass (MM) values. It was shown that a minor amount of residual charged groups in the polysaccharide chains caused a polyelectrolyte expansion effect and neutral fractions of birch xylans, larch arabinogalactans (AG) and galactoglucomannans (GGM) were separated from neutral molecules in various concentrations of the injected samples

Aqueous size-exclusion chromatography of polyelectrolytes on reversed-phase and hydrophilic interaction chromatography columns

The size-exclusion separation of a water-soluble polyelectrolyte polymer, sodium polystyrene sulfonate (NaPSS), was demonstrated on common reversed-phase (C18, C4, phenyl, and cyano) and hydrophilic interaction chromatography (HILIC) columns. The effect of common solvents – acetonitrile (ACN), tetrahydrofuran (THF), and methanol (MeOH), used as mobile phase modifiers – on the elution of NaPSS and the effect of column temperature (within a relatively narrow range corresponding to typical chromatographic conditions, i.e., 10 °C–60 °C) on the partition coefficient, KSEC, were also investigated. Non-size-exclusion chromatography (non-SEC) effects can be minimized by the addition of an electrolyte and an organic modifier to the mobile phase, and by increasing the column temperature (e.g., to 50 °C or 60 °C). Strong solvents such as THF and ACN are more successful in the reduction of such effects than is the weaker solvent MeOH. The best performance is seen on medium polarity and polar stationary phases, such as cyanopropyl- and diol-modified silica (HILIC), where the elution of the NaPSS polyelectrolyte is by a near-ideal SEC mechanism. Hydrophobic stationary phases, such as C18, C4, and phenyl, require a higher concentration of a strong solvent modifier (THF) in the mobile phase to reduce non-SEC interactions of the solute with the stationary phase.

Advances in native high-performance liquid chromatography and intact mass spectrometry for the characterization of biopharmaceutical products.

The characterization of biotherapeutics represents a major analytical challenge. This review discusses the current state-of-the-art in analytical technologies to profile biopharma products under native conditions, i.e., the protein three dimensional conformation is maintained during liquid chromatographic analysis. Native liquid-chromatographic modes that are discussed include aqueous size-exclusion chromatography, hydrophobic interaction chromatography, and ion-exchange chromatography. Infusion conditions and the possibilities and limitations to hyphenate native liquid chromatography to mass spectrometry are discussed. Furthermore, the applicability of native liquid-chromatography methods and intact mass spectrometry analysis for the characterization of monoclonal antibodies and antibody-drug conjugates is discussed.

Kinetic plots in aqueous size exclusion chromatography of monoclonal antibodies and virus particles

The growing importance of monoclonal antibodies and virus particles has led to a pressure for faster size exclusion chromatography. In recent years, numerous small particle columns for size exclusion chromatography of biologicals have been introduced. Small particles are a strategy to reduce analysis time. In the following study, opportunities of small particles in size exclusion chromatography of large biomolecules are investigated. Poppe plots reveal that the lower particle size limit depends on the size of the sample molecule. Hydrodynamic radii of monoclonal antibody monomer, aggregates and H1N1 as well as the diffusion coefficients were determined. Considering this sample compound dependency, kinetic plots referring to the resolution of a distinct compound pair instead of the plate number of a single analyte are more meaningful. Plate times were found to be equivalent with 4 and 2μm particles for a monoclonal antibody aggregate separation at resolutions smaller than 1.8. Quantification of a H1N1 in clarified cell culture can be accomplished with 17μm and 13μm particles at equal plate times at resolutions smaller than 2.5. Virus polydispersity is likely to be affected by run times of several hours at room temperature and shear forces resulting from particles smaller than 10μm. Comparatively high flow rates should be applied in size exclusion chromatography of the 100nm H1N1 virions.

Influence of Counterion on Thermal, Viscoelastic, and Ion Conductive Properties of Phosphonium Ionenes

SummaryAnion metathesis enabled a systematic study focused on the thermal, viscoelastic, and conductivity properties of a 4P,12 phosphonium ionenes with various counterions. Aqueous size exclusion chromatography confirmed the well‐defined synthesis of 4P,12‐Br from the step‐growth polymerization of 1,4‐bis(diphenylphosphino) butane and 1,12‐dibromododecane at a 1:1 stoichiometric ratio. Subsequent anion‐exchange employing a dialysis method exchanged the Br‐ counterion to trifluoromethanesulfonate (TfO‐), bis(trifluoromethane) sulfonimide (Tf2N‐), and tetrafluoroborate (BF4‐) counterion. 1H nuclear magnetic resonance spectroscopy of the 4P,12 ionenes showed a distinct upfield chemical shift for methylene protons adjacent to the phosphonium cation after anion‐exchange. Thermal characterization using thermogravimetric analysis and differential scanning calorimetry probed the thermal properties of the phosphonium ionenes. Counterion exchange to more bulky and delocalized anions led to improved thermal stabilities and lower glass transition temperatures. Rheological characterization facilitated the generation of time‐temperature superposition (TTSp) master curves and pseudo‐master curves for each 4P,12 ionene. TTSp revealed two distinct relaxation modes attributed to long‐range segmental motion and electrostatic interactions. Anion‐exchange resulted in a shift of these two modes of relaxation to higher shear rates. The calculated melt flow activation energy and thermal expansion coefficients were also observed to decrease and increase, respectively. Melt rheological characterization also probed the temperature dependence of the storage and loss moduli and suggested that the counterions have a plasticizing effect on the viscoelasticity of the 4P,12 ionene. Ionic conductivity increased with increasing size of the counterion (Br‐ < BF4‐< TfO‐< Tf2N‐) and demonstrated the viability of these novel materials as potential anion‐exchange ionomeric membranes.

Aqueous size-exclusion chromatographic method for the quantification of cyanobacterial native glycogen

Cyanobacterial glycogen has gained interest as a valuable biomass feedstock for biofuel production. However, an ideal method for native glycogen quantification has not been developed. Here, we have proposed a simple methodology that enables the quantitative determination of cyanobacterial glycogen concentration with high repeatability using aqueous size-exclusion chromatography with a differential refractive index detector (SEC/RID). Our SEC/RID system also allows size distributions for native glycogen based on hydrodynamic volumes (Vh), which is proportional to the product of the molecular mass (M) and intrinsic viscosity [η], obtained by universal calibration using linear homopolymers of known M with Mark-Houwink-Sakurada parameters. The universal calibration curve achieved a broad linear range (Vh parameter [η]M=2×10(2)-8×10(8)mLg(-1)) with a high correlation coefficient (R(2)=0.9942), because the developed system is equipped with an OHpak SB-806M HQ aqueous column containing four types of polyhydroxy methacrylate-based particles with different particle and pore sizes. Based on the SEC/RID system, response of molecular size distribution of glycogen in microalgae to the cultivation condition was first observed. Our established SEC/RID method has several advantages over conventional techniques, including the simultaneous quantitative and size distribution analyses of glycogen, and represents a potentially useful tool to elucidate the relationship between structural properties and the roles of glycogen in metabolism.

Linear-, Cyclic-, and Multiblock Amphiphilic Polyelectrolytes as Surfactants in Emulsion Polymerization: Role of Topological Structure

Combining atom transfer radical polymerization (ATRP) and “click” chemistry, a set of well-defined amphiphilic block copolymers poly(n-butyl acrylate)-b-poly(acrylic acid) (PnBA20-PAA85) with a similar chemical component, but different topological structures, i.e., linear-, cyclic-, and multiblock structures, were successfully prepared, characterized (size exclusion chromatography (SEC), FT-IR and 1H NMR), and used as surfactants in emulsion polymerization. Our further transmission electron microscopy (TEM) and laser light scattering (LLS) characterization of the resultant latex particles demonstrates all the surfactants with different topologies can effectively stabilize the latex particles but no significant difference among the solids contents was observed. Moreover, we have, for the first time, experimentally established the quantitative relation between the final number of latex particles (Np) and the concentration of polymeric surfactant with different topologies (C), i.e., Np = kCα, and the order of our measured exponent α is as follows: αmulti(1.10) > αlinear(0.81) ≥ αcyclic(0.73), indicating cyclic surfactant molecules behave more like small-molecule surfactants attributed to its strongest unimers extraction and diffusion ability; in contrast, multiblock surfactant molecules can act as seeds to directly nucleate to create latex particles. In addition, Np,multi > Np,linear ≥ Np,cyclic at higher concentration, and Np,linear > Np,cyclic ≥ Np,multi at lower concentration was observed. Similar results (αmulti(1.02) > αlinear(0.65) ≥ αcyclic(0.58)) were also observed when polystyrene-b-poly(acrylic acid) (PS9–PAA60) copolymers were used as surfactants. Further aqueous SEC characterization shows the broad size distribution of our micellar solution has no effect on obtaining narrowly distributed latex particles. Finally, interfacial tension measurement of the micellar solution indicates, compared to multiblock structure, the rate of adsorption at a hydrophobic interface is much faster for linear and cyclic-block structures, agreeing with our observed order of exponent α.

Phosphonium ionenes from well-defined step-growth polymerization: thermal and melt rheological properties

Step-growth polymerization of ditertiary phosphines with dibromoalkanes enabled the synthesis of novel phosphonium ionenes. In situ FTIR spectroscopy monitored the increase in absorbance as a function of time at 1116 cm−1, which corresponded to the polymeric P+–Ph stretch. Aqueous size-exclusion chromatography (SEC) provided absolute molecular weights and confirmed expected molecular weight growth for difunctional, step-growth polymerization. Phosphonium ionenes exhibited improved thermal and base stability compared to ammonium ionenes, which was attributed to the propensity of the ammonium cation towards Hofmann elimination. Melt rheology examined phosphonium ionene viscous flow and the influence of charge density on melt viscosity as a function of shear rate and temperature. Time–temperature superposition (TTS) resulted in both master curves and pseudomaster curves depending on phosphonium ionene composition. Two primary relaxations occurred: (1) onset of long-range segmental motion at Tg, and (2) relaxation attributed to electrostatic interactions. Higher charge densities shifted these two relaxations to longer time scales and increased flow activation energies. Phosphonium ionenes also readily bound pDNA effectively (± ratios of 1), and base stability suggested applications in energy generation.

Detailed characterization of hyaluronan using aqueous size exclusion chromatography with triple detection and multiangle light scattering detection

Hyaluronan (HA) has attracted great interest and attention from ophthalmic surgical and eye care companies owing to its unique properties. A more complete understanding of HA biopolymers has, therefore, become increasingly critical as thorough characterization of raw materials helps promote product quality and process control. Often, such detailed information requires the use of a combination of analytical techniques. In this study, we compared size exclusion chromatography (SEC) with online multiangle light scattering (SEC-MALS) and SEC with triple detection (SEC-TD) experiments for HA analysis. Three lots of commercially available eye drop grade HA were characterized by SEC-MALS and SEC-TD. The absolute molecular weight averages, molecular weight distribution, radius of gyration, and solution conformation of the three HA lots were determined and compared by the two techniques. In addition, the intrinsic viscosity and intrinsic viscosity distribution were measured by SEC-TD.

Synthesis of imidazolium ABA triblock copolymers for electromechanical transducers

Nitroxide-mediated polymerization enabled the synthesis of cationic, imidazolium-containing triblock copolymers as a membrane for an electromechanical transducer. Nitroxide-mediated polymerization afforded poly(styrene-b-[1-ethyl-3-(4-vinylbenzyl)imidazolium bis(trifluoromethane sulfonyl)imide)]-b-styrene) in a controlled fashion as confirmed using aqueous size exclusion chromatography and 1H NMR spectroscopy. Dynamic mechanical analysis revealed a modulus of approximately 100 MPa for the triblock copolymer at 23 °C, which was suitable for fabrication of an electromechanical actuator. Evaluation of electromechanical actuators revealed device curvatures over twice the curvatures for Nafion® in both the presence and absence of a conductive network composite. Addition of the ionic liquid (IL) 1-ethyl-3-methylimidazolium trifluoromethane sulfonate selectively reduced the glass transition temperature (Tg) of the central block and increased overall ionic conductivity. Normalizing temperature with the central block Tg caused the ionic conductivity for the IL-incorporated polymers to collapse onto a single curve, which was an order of magnitude higher than the block copolymer in the absence of added IL.

A comprehensive study on the size exclusion chromatography of kappa‐carrageenan for the identification of after‐peaks

AbstractAqueous size exclusion chromatography (SEC) of polysaccharides in general and carrageenans in particular is complicated by a number of factors. The chromatograms of carrageenans which are sulfated anionic natural polymers contain a number of after‐peaks depending on the occlusion, adsorption, or association of various ionic species either naturally present or evolved during their processing. A systematic SEC analysis of after‐peaks appearing in the chromatograms was made to identify the species responsible for their formation. The five after‐peaks constantly appearing in the aqueous (0.1M NaNO3) SEC of kappa‐carrageenan are attributed to sulfate, chloride, and nitrate anions whereas the first three and the fourth are due to divalent cations, mostly, and the fifth appears to result from the unknown impurities. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013

Controlled Radical Polymerization of 4-Vinylimidazole

We report the unprecedented controlled radical polymerization of 4-vinylimidazole (4VIM). Reversible addition–fragmentation chain transfer (RAFT) of 4VIM in glacial acetic acid afforded homopolymers with well-defined molecular weights and narrow polydispersity indices (PDIs). The polymerizations in acetic acid mediated with 4-cyano-4-(ethylsulfanylthiocarbonylsulfanyl)pentanoic acid (CEP) displayed linear pseudo-first-order kinetics and linear molecular weight growth with monomer conversion. Systematic variation of numerous polymerization parameters included solvent composition, initiator concentration, monomer concentration, and the calculated degree of polymerization. Aqueous size exclusion chromatography (SEC) confirmed linear molecular weight growth to number-average molecular weights (Mn) of 65 000 g/mol with low PDIs (<1.20). Subsequent monomer addition confirmed the presence of the trithiocarbonate functionality at the chain ends, which provided further evidence of controlled polymerization conditi...