Aqueous SEC Research Articles

Thiol–ene addition enables tailored synthesis of poly(2‐oxazoline)‐graft‐poly(vinyl pyrrolidone) copolymers for binder jetting 3D printing

AbstractThe continued interest in graft copolymer architectures arises from their unique solution properties and potential for a myriad of applications ranging from drug delivery to adhesives. Poly(vinyl pyrrolidone) (PVP) represents a popular amorphous, water‐soluble polymer used as a polymeric binder in binder jetting additive manufacturing, as fillers in cosmetic products, and for subcutaneous drug delivery systems. This report describes the synthesis of poly(2‐oxazoline) and PVP graft copolymers using a ‘grafting to’ methodology with an efficient thiol–ene ‘click’ reaction. Copolymerization of 2‐methyl‐2‐oxazoline and 2‐(3‐butenyl)‐2‐oxazoline introduced pendent vinyl grafting sites with a predictable absolute number‐average molecular weight. In parallel, reversible addition‐fragmentation chain‐transfer polymerization and subsequent aminolysis yielded well‐defined, oligomeric, thiol‐terminated PVP. Thiol–ene click chemistry enabled the formation of poly(2‐oxazoline)‐graft‐poly(vinyl pyrrolidone) (PMeOx‐g‐PVP) copolymers with varying mole percent grafting sites and PVP graft length. 1H NMR spectroscopy, aqueous SEC with multiangle light scattering (SEC‐MALS), and bromine titrations confirmed chemical structure, and DSC with TGA elucidated thermal transitions. Aqueous SEC‐MALS and 1H NMR spectroscopy also determined absolute number‐ and weight‐average molecular weights and average grafting levels, which revealed optimal reaction conditions. Zero‐shear viscosities of 5 and 10 wt% solutions in deionized water for each graft copolymer compared to their linear analogs demonstrated a significant (ca 31%) decrease in viscosity at the same number‐average molecular weight. This decrease in solution viscosity suggested PMeOx‐g‐PVP copolymers as exceptional alternatives to linear analogs for aqueous‐based, binder jetting additive manufacturing.

Complementary light scattering and synchrotron small-angle X-ray scattering studies of the micelle-to-unimer transition of polysulfobetaines.

AB and ABA di- and triblock copolymers where A is the hydrophilic poly(oligoethylene glycol methacrylate) (POEGMA) block and B is a thermo-responsive sulfobetaine block [2-(methacryloyloxy) ethyl] dimethyl-(3-sulfopropyl) ammonium hydroxide (PDMAPS) were synthesised by aqueous RAFT polymerisation with narrow dispersity (ĐM ≤ 1.22), as judged by aqueous SEC analysis. The di- and triblock copolymers self-assembled in salt-free water to form micelles with a PDMAPS core and the self-assembly of these polymers was explored by SLS and TEM analysis. The micelles were shown, by DLS analysis, to undergo a micelle-to-unimer transition at a critical temperature, which was dependent upon the length of the POEGMA block. Increasing the length of the third, POEGMA, block decreased the temperature at which the micelle-to-unimer transition occurred as a result of the increased hydrophilicity of the polymer. The dissociation of the micelles was further studied by SLS and synchrotron SAXS. SAXS analysis revealed that the micelle dissociation began at temperatures below that indicated by DLS analysis and that both micelles and unimers coexist. This highlights the importance of using multiple complementary techniques in the analysis of self-assembled structures. In addition the micelle-to-unimer morphology transition was employed to encapsulate and release a hydrophobic dye, Nile Red, as shown by fluorescence spectroscopy.

Aqueous sec analysis of cationic polymers as gene carriers

Cationic polymers have been receiving much attention as non-viral gene vectors. The aqueous mobile phase was optimized in combination with Shodex OHpak SB columns for size-exclusion chromatography (SEC) analysis of disulfide-containing poly(ethylene imine) (PEI) derivatives used in gene delivery. Addition of acetonitrile in mobile phase was shown to be able to suppress the hydrophobic interactions between polymer analytes and the stationary phase. The absolute molecular weights and distributions of the cationic polymers were determined directly from online SEC-MALS (multi-angle light scattering)/RI (refractive index detector). The results demonstrate that a good SEC separation of disulfide-containing PEI derivatives used in gene delivery with little band broadening was achieved.

Characterization of hydroxypropylmethylcellulose (HPMC) using comprehensive two-dimensional liquid chromatography

Various hydroxyl-propylmethylcellulose (HPMC) polymers were characterized according to size and compositional distributions (percentage of methoxyl and hydroxyl-propoxyl substitution) by means of comprehensive two-dimensional liquid chromatography (LC × LC) using reversed-phase (RP) liquid chromatography in the first dimension and aqueous size-exclusion chromatography (aq-SEC) in the second dimension. RP separation was carried out in gradient-elution mode applying 0.05% TFA in water and 1-propanol, while 0.05% TFA in water was used as mobile phase in aqueous SEC. A two-position ten-port switching valve equipped with two storage loops was used to realize LC × LC. Detection of HPMC was accomplished by charged-aerosol detection (CAD). Data processing to visualize chromatograms was carried out using Matlab software. The significant influence of the LC × LC temperature on (the retention of) HPMC was studied using a column oven which allowed accurate temperature control. Due to the phenomenon of thermal gelation, which is a result of methyl and hydroxypropyl substitution of anhydroglucose units from the cellulose backbone, we were able to obtain additional, specific information on compositional characteristics of various HPMC samples. As the retention behaviour of gelated and non-gelated polymer proved to be different, the fraction of the polymer that is gelated in the chromatographic column could be monitored at different temperatures. Moreover, the temperature at which half of the polymer is gelated could be correlated with the cloud-point temperature. As a result, differences in inherent cloud points of modified cellulose can be used as a further distinguishing property in “temperature-responsive” LC × LC.

SEC Assay for Polyvinylsulfonic Impurities in 2-(N-Morpholino)ethanesulfonic Acid Using a Charged Aerosol Detector

A quantitative method for measuring polyvinylsulfonic acid (PVS) concentrations in 2-(N-morpholino)ethanesulfonic acid (MES) is described here. Utilizing a TSK-GEL G2500PWxl SEC column, PVS elutes in the void volume as a sharp peak, yielding group separation between PVS and MES. 20 mM formic acid is used as the mobile phase, as its volatility is compatible with a charged aerosol detector. The low pH mobile phase also suppresses residual negative charges on the stationary phase, reducing the ion exclusion effect commonly observed in aqueous SEC. This method was qualified to measure PVS concentration in MES per International Conference on Harmonization guidelines, using multiple MES lots and other Good’s buffers such as N-(2-acetamido)-2-aminoethanesulfonic acid and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

Characterization of galactomannans isolated from legume endosperms of Caesalpinioideae and Faboideae subfamilies by multidetection aqueous SEC

Galactomannans isolated from legume seed endosperms, including those of commercial interest, have been characterized by multidetection aqueous SEC. Galactomannans derived from seeds of the Faboideae subfamily had substantially higher M w than those from Caesalpinioideae seeds ( M w,Fab = 2.4–3.1 × 10 6 g/mol, M w,Caes. = 0.86–2.1 × 10 6 g/mol) and within the latter botanical subfamily, an apparent correlation between M w and the degree of galactose substitution DG was found. The molar mass distributions were unimodal and differed primarily by a scale factor, with distributional widths narrower than a true Flory ‘most-probable distribution’; good fits to Schulz–Zimm model were obtained. Across subfamilies no differences were found in the exponents of [ η]– M and R v– M relationships (0.61 ± 0.02, 0.54 ± 0.01, respectively), the Flory chain stiffness ratio ( C ∞ = 20 ± 1 (BSF analysis)), or the persistence length ( L p = 5.5 ± 0.2 nm) obtained from SEC fraction data. However, it was found that prefactors in the [ η]– M and R v– M relationships as well as the unperturbed parameter K Θ decrease in proportion to DG and therefore chain density. Generalized relationships incorporating galactose-dependent prefactors were therefore developed to model SEC fraction data of native galactomannans ([ η] GM = (1800 ± 200) × M o −1.61 × M 0.61±0.02, R v,GM = 0.63 ± 0.05 × M o −0.54 × M 0.54±0.01) as well as lower- M fractions obtained by ultrasonication ([ η] GM = (730 ± 100) × M o −1.71 × M w 0.71±0.02, R v,GM = 0.49 ± 0.05 × M o −0.57 × M w 0.57±0.01, M ≈ 1 × 10 5-native). As a consequence of this dependence and the observed patterns in molar mass variation, [ η] varies within a narrow range for galactomannans as a whole despite substantial M w differences.

Hyphenation of aqueous liquid chromatography to pyrolysis-gas chromatography and mass spectrometry for the comprehensive characterization of water-soluble polymers

A recently developed hyphenated system for “organic” size-exclusion chromatography–pyrolysis-gas chromatography–mass spectrometry (SEC–Py-GC–MS) is adapted to allow the use of aqueous LC eluents as applied in the characterization of water-soluble polymers. The system uses syringe-based transfer of multiple LC-fractions to the GC instrument with solvent elimination and subsequent pyrolysis in a programmed temperature vaporization injector. The problems of the large-volume injections of aqueous, salt containing eluents into the Py-GC–MS are solved by using a ‘sintered-bed liner’ for elimination of the water at a high temperature, a volatile salt and the installation of a back-flush option. After optimization, the system was applied for the determination of the combined molecular weight–chemical composition of a polyethylene glycol–polypropylene glycol block copolymer. This analysis was done with the system in the aqueous SEC–Py-GC–MS mode. Also demonstrated is the automated at-line characterization of a random polystyrene–polymethylmethacrylate copolymer, now with the system in the gradient reversed-phase LC–Py-GC–MS mode. The methods proposed in the present work are very useful for the detailed characterization of water-soluble copolymers.

New Chromatographic and Hyphenated Techniques for Hydrophilic Copolymers

AbstractMost synthetic polymers are distributed in more than one parameter of molecular heterogeneity. For hydrophobic copolymers there are different chromatographic techniques available to analyse these distributions. As a result of the increasing interest in hydrophilic polymers and copolymers new chromatographic techniques are developed for the characterization of these polymers as well. However, very frequently these polymers contain highly polar or charged functional groups making them soluble only in aqueous mobile phases. There are several problems related to the use of aqueous mobile phases in polymer chromatography. Even the SEC analysis of such copolymers is not straightforward. As for HPLC in aqueous mobile phases, there are only a few applications in the literature so far. In addition to the fact that only a very limited number of stationary phases is available for aqueous HPLC of polymers, the interactions of polyelectrolytes in such chromatographic systems are not well understood.The present paper addresses the problems related to the application of SEC and HPLC in aqueous mobile phases. For graft copolymers with a polyethylene oxide backbone, e.g. PEG‐g‐polymethacrylic acid and PEG‐g‐polyvinyl alcohol, it will be shown that methods can be developed that give accurate molar mass and chemical composition information. Two‐dimensional chromatography where aqueous HPLC and SEC are coupled on‐line will be shown to be the most powerful analysis tool for the analysis of such copolymers. The hyphenation of the chromatographic separation techniques with spectroscopic detection techniques provides further insight into the molecular complexity of these copolymers.

Development of direct size-exclusion chromatography separation for the determination of molar mass of native procyanidins in the phenolate form

A size-exclusion chromatographic method using an aqueous solvent as eluent was developed for the determination of the molecular mass of procyanidins has been developed. Size-exclusion chromatography is a chromatographic technique used to determine molecular weight distribution of polymers. During the development work, PL™ aquagel-OH columns and several different eluent compositions were investigated. The best separation of procyanidins was obtained with an eluent consisting of water–methanol (50:50, v/v), NaNO 3: 0.18 M, pH < p K a + 2. The results from this aqueous SEC method were compared with results obtained using SEC with organic solvents.

HEMA/MMA/EDMA packing material evaluation for size exclusion chromatography (SEC)

HEMA/MMA/EDMA (2-hydroxyethyl methacrylate/methyl methacrylate/ethyleneglycol methacrylate) copolymer beads have been synthesized for use in aqueous SEC. This porous chromatographic support consisted of a large average particle size (250 µm) and contained macropores with a median pore size of approximately 880 A. The material was packed in glass and stainless columns to assess their chromatographic performance. The packed columns were calibrated using narrow molecular weight (MW) distribution standards (dextran) and exhibited a range of molecular weight separation between 40,000 and 2,000,000 daltons. The packing material showed the ability to separate large molecules through the size exclusion mechanism.

Affinity Chromatography as a Tool to Analyze Polyanion–Polycation Complexes: the Case of Poly(Llysine Citramide)–Poly(L-lysine) Systems

Artificial polymeric carriers for bioactive compounds, such as drugs and genes, are increasingly being reported in the literature. Most are polyelectrolytes aimed at being injected into body fluids that are composed of charged macromolecules (proteins, glycoproteins, poly(glycosamino glycane)s, polynucleotides, etc.). Many interactions can occur and lead to dramatic phenomena, such as polyelectrolyte complexation or substitution, cell aggregation and hemolysis. For the purpose of modeling and better understanding these interactions and their effects, a library of polyanions of the poly(L-lysine citramide)-type was interacted with the polycation poly(L-lysine) in aqueous media. Different fractions of complexes were generated by successive additions of aliquots of the polycation solution to the polyanion library solution by a titrating process. The two components, the polycation and the complex population of polyanions, were separated from the collected complex fractions by affinity chromatography and then analyzed by aqueous SEC. Molecular weight selectivity was shown, the high molecular weight polyanion macromolecules precipitated first, while complexes involving smaller molecules were found to remain in solution. The data show the potential of affinity chromatography for studying interpolyelectrolyte complexes and emphasizes the critical role that polydispersity can play when therapeutic charged macromolecules are injected into blood.

Characterization of METHOCEL cellulose ethers by aqueous SEC with multiple detectors

Initial evaluation of a multiple-detector, aqueous SEC technique has shown:•that it is capable of high precision and accuracy;•applicability to the analyses of METHOCEL cellulose ethers;•ability to generate consistent values of RgW, IVW, M̄W,M̄N and polydispersity;•ability to generate dependable Mark–Houwink–Sakurada parameters.Cellulose ethers in this study were commercial products, used ‘as received’, solutions were not dialyzed, corrections were not made for salt content or moisture content and batches were not specially selected. The robustness, convenience and reproducibility of the technique are impressive.

Molecular Characterization of Corn Starch Using an Aqueous HPSEC‐MALLS‐RI System Under Various Dissolution and Analytical Conditions

ABSTRACTMolecular characteristics based on absolute weight‐average molecular weight (Mw) and z‐average radius of gyration (Rg) of normal corn starch were analyzed by high‐performance size‐exclusion chromatography (HPSEC) attached to multiangle laser‐light scattering (MALLS) and refractive index (RI) detectors under different starch dissolution and analytical conditions. Autoclaving (121°C, 20 min) or microwave heating (35 sec) provided better HPSEC recovery and higher Mw for starch molecules than simple dissolution in hot water. The Mw for the autoclaved corn amylopectin and amylose fractions separated with a TSK G5,000 column at 60°C were 201 × 106 and 3.3 × 106, respectively. The specific volume for gyration (SVg) calculated from Mw and Rg could be used for the comparison of molecular compactness which was inversely related to the degree of branching. The SVg values of amylopectin and amylose fractions in the chromatogram (TSK G5,000, autoclaved for 20 min) were 0.092 and 0.529, respectively. But a portion (20–30%) of large amylopectin molecules did not pass the injection membrane filter (3.0 μm) and the SEC column, resulting in incomplete recovery. The unfiltered portion varied according to the dissolution treatment. Homogenization (7,000 rpm, 5 or 10 min) of the starch solution improved the recovery of the amylopectin fraction, but significantly increased the Mw of the amylose fraction (17 × 106). Sonication for 5 min degraded starch molecules. For accurate analysis of a native starch using an aqueous SEC, the starch should be fully dissolved with proper treatment such as autoclaving or microwaving, and the column should be improved for full recovery of large amylopectin molecules.

Degradation of poly(?-malic acid)-monitoring of oligomers formation by aqueous SEC and HPCE

Poly (β-malic acid) is a functional aliphatic polyester which can serve as parent compound to make systems aimed at temporary therapeutic applications. Degradation of the polyester backbone of poly(β-malic acid) leads to the formation of oligomers which can be hardly separated by conventional Size Exclusion Chromatography. In order to monitor the formation of oligomeric degradation by-products and to quantify their relative amount, the potential of two separation techniques has been investigated, namely Aqueous Size Exclusion Chromatography using a Bio-Gel P2 gel column and High Performance Capillary Electrophoresis. Aqueous SEC allowed separation of oligomers smaller than hexamer whereas HPCE successfully separated oligomers smaller than pentadecamers. These techniques were used to control the efficiency of oligomer fractionation by the dissolution/precipitation method.applied to the sodium salt of poly(β-malic acid).

In vivo Fate of Repeat-Unit-Radiolabelled Poly(β-malic acid), a Potential Drug Carrier

In order to compare the fates of end-chain-radiolabelled and repeat-unit-radiolabelled poly(β-malic acid)s after intravenous injection in mice, repeat-unit 14C-radiolabelling of this biodegradable water-soluble poly carboxylic acid polymer was achieved starting from 14C-aspartic acid. The ac tivity of the resulting radioactive poly(β-malic acid) (sodium salt) ( Mw ∼ 20,000 as determined by aqueous SEC) was 4.5 μCi·g -1. Aliquots of a neutral 0.3 monoM (4.14% w/v) solution of poly(β-malic acid) (sodium salt) were given intravenously to mice through a lateral tail vein. Intravenous and intra peritoneal toxicity was checked in order to show whether injection of this polymer can affect significantly the normal behavior of experimental animals. Radioactivity was counted in liver, kidney, intestine, lung, brain, spleen, heart, muscle, urine and blood for various post-injection times up to 24 h. Data con firmed the occurrence of predominant and fast urinary excretion (70% after 1 h) already observed with the end-chain-radiolabelled homologue. Although the polymer is basically metabolizable, the fast elimination of radioactivity via urinary tract is most likely to be due to the excretion of polymeric molecules and not of metabolic by-products because end-chain- and repeat-unit-labellings led to similar excretion patterns.

Limitation and evaluation of the two‐broad standard (TBS) method of calibration of aqueous SEC

AbstractFew years ago, the two broad standard method of molecular weight (MW) calibration was proposed1 and the presence of negative σ2 (peak dispersion coefficient) was found to be somewhat disturbing. The limitation of the method was not specified. Since large negative values of σ2 cannot be tolerated, herein is reported another type of instrumental spreading function for which the method may seem to apply, and an evaluation of this method. In its evaluation, plots of loge (intercept of a linear molecular weight calibration curve), that is, loge(D1) vs. the corresponding slope of the molecular weight calibration curve, D2, which were found to be linear, were used. The systems employed were Dextran/Corning controlled porous glass (CPG‐10) packing in well‐chosen mobile phase.

Universal Calibration in Aqueous Size Exclusion Chromatography with On-Line Differential Viscometry Using Commercial TSK-PW Columns

Abstract Poly(ethylene oxide), poly(ethylene glycol), and poly(saccharide) exhibit universal calibration under aqueous SEC conditions with commercial TSK-PW columns using on-line, differential viscometry. The product of log(intrinsic viscosity molecular weight) versus retention volume converges to a single curve for these polymers. Mobile-phase compositions of 0.1 N sodium nitrate and 20% aqueous acetonitrile at 35° C are used to examine this behavior. Mark-Houwink constants for these polymers are determined directly from on-line specific viscosity measurements.

Polysaccharide Characterization by Aqueous Size Exclusion Chromatography and Low-Angle Laser Light Scattering

Abstract Aqueous size exclusion chromatography coupled with on-line low-angle laser light scattering (SEC/LALLS) is a valuable analytical tool for characterization of polysaccharides and other important biopolymers. This work reviews the fundamental size separation mechanism of polymers chromatographed via SEC, the development of SEC/LALLS methods for characterization of eluted polymers, and applications of this technique to determine polysaccharide physical and chemical properties. Important nonsize exclusion effects encountered in aqueous SEC of polysaccharides are discussed and attributed to intramolecular and polymer-support interactions, as well as flow-related anomalies. The necessity of absolute molecular weight detection as a direct means of calibration is presented. Low-angle laser light scattering coupled to SEC provides a simple method of direct calibration and allows determination of polymer molecular weight and molecular weight distribution. Recent applications of SEC/LALLS to determine polysaccharide branching characteristics are detailed. The combined knowledge of molecular weight distributions and branching distributions provides insight into the molecular kinetic events of polysaccharide processing operations.

Characterization of Dextrans by Size Exclusion Chromatography Using DRI/LALLSP Detector System

Abstract Herein is reported an experimental investigation of the molecular weight characterization of dextrans by aqueous SEC with a DRI/LALLSP detector system. Methodology for the determination of the molecular weight calibration curve and peak broadening parameter [sgrave]2 (variance of a Gaussian instrumental spreading function) across the chromatogram have been developed. A blend of dextran standards permits, with one injection, measurement of the molecular weight calibration curve over a wide range of retention volumes. Measurements with salt-free water as mobile phase have confirmed that some dextran chains may have a negative charge.

Characterization of Nonionic Polyacrylamides by Aqueous Size Exclusion Chromatography Using a DRI/LALLSP Detector System

Abstract Herein is reported an experimental investigation of the molecular weight characterization of nonionic polyacrylamides by aqueous SEC with a DRI/LALLSP detector system. Methodology for the use of the DRI/LALLSP detector responses to determine the molecular weight calibration curve and the peak broadening parameter, [sgrave]2 (variance of a Gaussian instrumental spreading function) over a wide molecular weight range has been developed. The method is based on the use of a broad MWD standard made by blending Polysciences broad MWD standards and a generalized analytical solution of Tung's integral equation for the detector response corrected for peak broadening. Molecular weight averages measured by SEC/DRI/LALLSP are in excellent agreement with those measured offline by LALLSP.