Viscometric Detection Research Articles

Probing macromolecular architectures of nanosized cyclic structures of (1→3)-β- d-glucans by AFM and SEC-MALLS

Comb-like branched (1→3)-β- d-glucans dissolve in water as stiff triple-helical structures. Dissociation followed by re-association leads to the formation of a blend of various macromolecular topologies, where the cyclic species make up a significant fraction. In this study, the molecular properties of these nanosized cyclic structures of (1→3)-β- d-glucans were probed using a combination of AFM and SEC-MALLS. The cyclic structures were obtained by subjecting linear triple-helical molecules of (1→3)-β- d-glucans to a denaturation–renaturation cycle, and the fraction of cyclic structures in the renatured sample was determined by AFM. Samples containing different known fractions of linear and circular molecules were studied by SEC with online multi-angle laser-light scattering and viscometric detectors. The molecular weight and the radius of gyration of the molecules eluting from the SEC column, as well as the concentration and the intrinsic viscosity, were determined simultaneously. By extrapolating the results to a situation of only circular species, the results allowed to determine the linear mass per unit length ( M L) of not only the linear but also the circular morphologies of the (1→3)-β- d-glucans. The values obtained were M L = 2140 ± 180 g mol −1 nm −1 for the circular species and 2045 ± 80 g mol −1 nm −1 for the linear species. This is the first direct determination of the M L parameter of the circular topology, and the results indicate that the reassociation of the individual chains yield a triplex structure also for the circular morphology, similar to the initial triple helix.

Round Robin Test on the Molecular Characterization of Epoxy Resins by Liquid Chromatography

ABSTRACT This paper reports results acquired during the IUPAC Project No. 1999-021-1-400 Round Robin Test on the Molecular Characterization of Epoxy Resins by Liquid Chromatography. The results obtained by size exclusion chromatography (SEC) with column calibration are compared with those determined by SEC combined with a light scattering or a viscometric detector and absolute methods of molar mass determination. The influence of SEC experimental conditions on the obtained results is studied.

High resolution high-performance liquid chromatography separation of polyglutamic acids

Poly-L-glutamic acids can exist in random coil, ibility because of its poor pH stability and organic helix, or sheet depending on the environmental conditions. Therefore, they have been used as molecular models for a better understanding of macromolecular stereochemistry [1]. Polyglutamylation is also the major posttranslational modiWcation of neuronal tubulin [2,3], and foliates are polyglutamylated in most organisms, with pentaglutamates being the predominant forms of foliate polyglutamylation in a number of species [4]. Poly-L-glutamic acids were also found to be related to a variety of biological functions such as the enhancement of the transgene expression for plasmids [5] and the preferential delivering of paclitaxel [6,7], a widely used anticancer agent, to tumor sites, thereby improving its eVectiveness and reducing its toxicity. Polyglutamic acids are highly charged in physiological pH solution. Many of the properties of polyglutamic acids in solution vary considerably with molecular weight. The accurate determination of molecular weights and the molecular weight distribution of polyglutamic acids used in chemical and biochemical experiments are essential. The methods for the estimation of the range of the molecular weight and the molecular weight distribution of polyglutamic acids were mainly based on viscometry and light-scattering determination. An improvement in accuracy was achieved with the introduction of size exclusion chromatography combined with online diVerential viscometer, diVerential refractometer, and light-scattering detectors [8]. The separation of oligomers of glutamic acid was also achieved by high-performance liquid chromatography (HPLC) using RPC-5 chromatography [9]. However, RPC-5 chromatography suVered from poor reproduc-

Linear or branched structure? Probing molecular architectures of fullerene–styrene copolymers by size exclusion chromatographs with online right-angle laser-light scattering and differential viscometric detectors

The poly(C 60- co-styrene)s prepared by radical copolymerizations of C 60 and styrene in bulk were characterized by multiple-detector size-exclusion chromatographs consisting of a refractometer, a differential viscometer, and/or a right-angle laser-light scattering photometer. The multidetector systems enabled the determinations of reliable, absolute molecular weights of the copolymers. The plots of intrinsic viscosity vs. molecular weight and radius of gyration vs. molecular weight offered valuable information about the molecular architectures of the copolymers. The slopes of the plots reveal that the structure of the copolymer changes with its C 60 content: the copolymer with a low C 60 content of 0.58 wt% possesses a linear structure, whereas its conger with a high C 60 content of 1.14 wt% possesses a branched structure.

Linear or branched structure? Probing molecular architectures of fullerene?styrene copolymers by size exclusion chromatographs with online right-angle laser-light scattering and differential viscometric detectors

Linear or branched structure? Probing molecular architectures of fullerene?styrene copolymers by size exclusion chromatographs with online right-angle laser-light scattering and differential viscometric detectors

Evaluation of radius of gyration and intrinsic viscosity molar mass dependence and stiffness of hyaluronan.

Nine hyaluronan (HA) samples were fractionated by size-exclusion chromatography, and molar mass (M), radius of gyration (Rg), and intrinsic viscosity ([eta]) were measured in 0.15 M NaCl at 37 degrees C by on-line multiangle light scattering and viscometer detectors. Using such method, we investigated the Rg and [eta] molar mass dependence for HA over a very wide range of molar masses: M ranging from 4 x 10(4) to 5.5 x 10(6) g/mol. The Rg and the [eta] molar mass dependence found for HA showed a meaningful difference. The Rg = f(M) power law was substantially linear in the whole range of molar masses explored with a constant slope of 0.6. In contrast, the [eta] = f(M) power law (Mark-Houwink-Sakurada plot) showed a marked curve shape, and a linear regression over the whole range of molar masses does not make sense. Also the persistence length (stiffness) for HA was estimated. The persistence length derived by using both the Odijk's model (7.5 nm from Rg vs M data) and the Bohdanecky's plot (6.8 nm from [eta] vs M data) were quite similar. These persistence length values are congruent with a semistiff conformation of HA macromolecules.

Electrostatically Enhanced Second and Third Virial Coefficients, Viscosity, and Interparticle Correlations for Linear Polyelectrolytes

The use of an automatic, continuous mixing (ACM) technique, coupled with multiangle static light scattering, refractometric, and viscometric detectors allowed detailed measurements of electrostatic effects in polyelectrolyte solutions to be made while varying ionic strength [I], and polyelectrolyte concentration. A linear polyelectrolyte, sodium hyaluronate (HA) was used. The behavior of second and third virial coefficients, A2 and A3, the angular scattering envelope, and the reduced viscosity were determined simultaneously. The crossover regime from where interparticle effects dominate the scattering to where single particle form factors dominate is clearly seen and is rationalized by a simple pair correlation function. Good agreement is found for polymer dimensions and A2 using a combination of electrostatic persistence length and electrostatic excluded volume theories, with no adjustable parameters. The contribution to [I] from the HA counterions is observed via the electroviscous effect, a reduced viscosity crossover phenomenon, and an unusual scattering minimum vs polyelectolyte concentration at low [I].

Enzymatic modification of pectin to increase its calcium sensitivity while preserving its molecular weight.

A commercial high-methoxy citrus pectin was treated with a purified salt-independent pectin methylesterase (PME) isozyme isolated from Valencia orange peel to prepare a series of deesterified pectins. A series of alkali-deesterified pectins was also prepared at pH 10 under conditions permitting beta-elimination. Analysis of these pectins using high-performance size exclusion chromatography (HPSEC) with on-line multiangle laser light-scattering, differential viscometer, and refractive index (RI) detectors revealed no reduction in weight-average molecular weight (M(w); 150000) in the PME-treated pectin series, whereas a 16% reduction in intrinsic viscosity (IV) occurred below a degree of esterification (DE) of 47%. In contrast, alkali deesterification rapidly reduced both M(w) and IV to less than half of that observed for untreated pectin. PME treatment of a non-calcium-sensitive citrus pectin introduced calcium sensitivity with only a 6% reduction in the DE. Triad blocks of unesterified galacturonic acid were observed in (1)H nuclear magnetic resonance spectra of this calcium-sensitive pectin (CSP). These results demonstrate that the orange salt-independent PME isozyme utilizes a blockwise mode of action. This is the first report of the preparation of a CSP by PME treatment without significant loss of the pectin's M(w) due to depolymerization.

Star Polymer Synthesis from ε-Caprolactone Utilizing Polyol/Protonic Acid Initiator

Three- and four-armed star-shaped poly(ε-caprolactone)s were synthesized from trimethylolpropane and pentaerythritol as initiators using fumaric acid as an activator of the monomer, respectively. Star-shaped poly(ε-caprolactone)s could be satisfactorily obtained, which was confirmed by size exclusion chromatography equipped with low-angle laser light scattering and viscometer detectors. Comparing the melting points among the polymers with similar molecular weights, the order was as follows: linear poly(ε-caprolactone) > three-armed poly(ε-caprolactone) > four-armed poly(ε-caprolactone). On the other hand, poly(ε-caprolactone)s with similar arm length showed similar melting points irrespective of the arm numbers. Thermal decomposition temperature of poly(ε-caprolactone)s increased with the arm number.

CHARACTERIZATION OF POLYTHIIRANES USING MULTIDETECTION SIZE EXCLUSION CHROMATOGRAPHY

Series of polythiiranes with pendant Oxyethylene groups havebeen characterised. Size Exclusion Chromatography (S.E.C.) analysis of polymerswith such polar groups needs some precautions. Accurate measurement of dn/dcis used in order to check the amount of recovery of the injected samples.Polymers with short side chains are normally eluted, but an irreversible adsorptionoccurs for long side chains. There appears to be an intermediate situationfor which adsorption is reversible and produces an important dependence ofelution volume on injected concentration. Using a combined Light Scattering, a Viscometric detector, and additionaloff-line Dynamic Light Scattering (D.L.S.) measurements, it was possible tostudy, more completely, the relationship between size and molecular weightfor such polymers. When some of these polymers do not exhibit any adsorptionphenomenon, universal calibration is observed and a constant correction factoris available, allowing the conversion of relative molecular weights into absolutemolecular weights.

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 & Sons, Inc. J Appl Polym Sci 81: 370–383, 2001

Characterization of Hemicellulose B from Corn Fiber by High-Performance Size Exclusion Chromatography with On-Line Molar Mass and Viscometric Detection

Hemicellulose B (arabinoxylan, corn fiber gum, CFG) was prepared from corn fiber (CF) by alkaline extraction with 2 meq/g of CF using either NaOH, Ca(OH)2 or an equimolar mixture of the two. Prior to CFG extraction, oil was removed from CF by extraction with hexane and starch was removed from CF by treatment with α-amylase. The solutions of alkali-extracted CFG were then bleached with alkaline H2O2. The effect of extractant composition on the weight-average molar mass M w, weight-average intrinsic viscosity [η]w, and z-average root-mean-square radius of gyration R gz on CFG was determined. Depending on extractant composition and method of calculation, M w ranged from 2.7–6.96 × 105, [η]w from 1.87 to 2.00 dL/g and R gz from about 31 to 50 nm. Substituting Ca(OH)2 for NaOH reduced M w and R gz but had little or no effect on [η]w. Chromatographic analysis in conjunction with molar mass detection revealed that molar mass distributions were bimodal. M w for the high-molar-mass component ranged from about 8.4–16.1 × 105, whereas M w ranged from about 1.1–2.1 × 105 for the low-molar-mass component. The high-molar-mass component comprised about 15–43% by weight of CFG and was much more compact in shape than the low-molar-mass component.

Flow Irregularities due to Polymer Elution in Size Exclusion Chromatography and its Effect on Viscometric Detection

A new experimental setup for detecting flow-rate fluctuations caused by the passage of polymer through a separation system, including a differential refractometer and a singlecapillary on-line viscometric detector in size exclusion chromatography (SEC) is demonstrated. Numerical methods for correction of elution volume and the baseline of the viscometric output, including the “Lesec effect”, are presented on SEC data of (2-hydroxy-ethyl) cellulose in 50% methanol/50% water 10 mM NaCl (mobile phase). The effect of flow-rate fluctuations on experimental molecular weight averages is discussed.

Uniform Regular and Irregular Three-Arm Star Poly(methyl methacrylate)s

Isotactic (it-) and syndiotactic (st-) uniform poly(methyl methacrylate)s (PMMAs) with hydroxyl group at the chain end (PMMA-OH's) were prepared by fractionating the corresponding non-uniform PMMAs by means of supercritical fluid chromatography (SFC). The uniform PMMA-OH's with degrees of polymerization (DPs) of 16, 26 and 33 were each allowed to react with a trifunctional coupling agent, 1,3,5-benzenetricarbonyl trichloride, to obtain three-arm star polymers with uniform PMMA arms. Intrinsic viscosities of a series of the star PMMAs were measured in tetrahydrofuran at 40°C by using a differential viscometric detector connected with a size exclusion chromatograph. Tacticity dependence was observed not only in the viscosity but also in shrinking factor g η . A three-arm star PMMA with one arm with DP=16 and two arms with DP=31 (irregular star PMMA) was also prepared in a similar manner with the aid of SFC fractionation. The viscosity of the irregular star PMMA was found larger than that of the regular star PMMA with the same total number of monomer units.

Assessment of errors in the determination of Mark-Houwink-Sakurada and Stockmayer-Fixman constants using size-exclusion chromatography with on-line viscometric detection: Analyses of poly(p-substituted styrenes) in tetrahydrofuran

In this work, we present values for the Mark-Houwink-Sakurada (MHS) and Stockmayer-Fixman (SF) constants for a series of homopolymers of para-substituted styrenes (4-X-styrene; X = OCH3, OCH2CH3, CH3, F, Cl, and Br) in THF at room temperature. The respective values of K (in 10(-5) dL/g) and alpha were: 0.685 and 13.2; 0.662 and 14.1; 0.740 and 8.41; 0.781 and 5.24; 0.726 and 8.95; 0.700 and 7.79. The respective values for K-theta (in 10(-4) dL/g) and K' (in 10(-7) dL/g) were: 6.01 and 16.1; 6.22 and 9.07; 7.64 and 17.4; 5.59 and 23.7; 6.29 and 17.3; 4.44 and 10.3. These constants were measured using size-exclusion chromatography with on-line viscometry. As part of this work, we investigate the applicability of common model fitting procedures to this method of measuring MHS/SF constants and the effect of uncertainties in their estimated values on the accuracy of molecular weight analysis. (C) 1999 John Wiley & Sons, Inc.

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.

Viscometric detection of polymer inclusion complexes

The reduced viscosity of poly(ethylene glycol) (PEG) is increased in solutions of (2-hydroxypropyl)-α-cyclodextrin (HPACD) significantly more than in the solution of (2-hydroxypropyl)-β-cyclodextrin (HPBCD) in agreement with the fact that a crystalline complex of PEG is formed in solutions of natural α-cyclodextrin but not in solutions of natural β-cyclodextrin. In analogy with that, the viscometry indicates formation of a complex between HPBCD with poly(vinyl alcohol) because the reduced viscosity is markedly increased in solutions of HPBCD but only slightly in solutions of HPACD. On the other hand, the increase in the viscosity of poly(N-vinylpyrrolidone) (PVP) was identical in HPACD and HPBCD solutions; therefore, the viscometry did not provide support for the suggested side-chain complexation between PVP and cyclodextrins.

Data Reduction in Multidetector Size Exclusion Chromatography

A comprehensive analysis of multidetector size exclusion chromatography (SEC) is presented. The system examined consists of differential refractometer, capillary viscometer, and light-scattering detector combination. New approaches for interpreting multidetector data have been developed. The first one consists in eliminating the concentration chromatogram from the data reduction process. The traces of two molecular-weight-sensitive detectors alone allow calculating molecular weight and intrinsic viscosity distributions of an unknown polymer, assuming the validity of the universal calibration concept for this polymer. This approach can dramatically improve the characterization of polymers with long high-molecular-weight tails, when small amounts of high-molecular-weight fractions are detected by the light-scattering and viscometer detectors, but the concentration is too low to be registered by the refractometer. Another opportunity discovered is the calculation of the hydrodynamic volume of each fraction of a chromatographed polymer across its distribution, using measured signals from three on-line detectors. This calculation can be performed for any complex polymers, e.g., copolymers or oligomers, even in situations when refractive index increments of molecules depend on retention volume, or columns do not provide full recovery of injected polymer mass. The verification of the universal calibration hypothesis for any unknown polymer is accomplished now by the routine single chromatographic run. The method developed helps to analyze different reasons responsible for the deviation from universal calibration, such as polymer-polymer interactions in solution, band-broadening phenomena, non-steric interactions with a stationary phase or the structural or chemical heterogeneity of a solute. Using triple detection with multiangle light-scattering detector allows, also, to calculate the Flory's viscosity factor across the distribution for polymers with different configurational and conformational structure. The experimental data supporting the theoretical predictions are presented.

Evaluation of a single‐capillary viscometer detector on line to a SEC system used with a new pulse‐free pump

A single-capillary viscometer (SCV) used as an on-line viscosity-sensitive detector for size exclusion chromatography (SEC) presents considerable interest. Unfortunately, the commercially available version, which uses a traditional HPLC pump and a pulse dampener, presents serious problems and can give poor performance. Flow rate fluctuations are the main problems in the use of the on-line SCV detector. The pulse dampener resolves the flow fluctuations problems that are intrinsic to a reciprocating pump. Unfortunately, the pulse dampener introduces secondary, yet variable, flow fluctuations that make the SCV unreliable. In the past, several solutions to the problem have been described. Our proposal is to use the SCV on-line detector with a new commercial pulse-free pump without a pulse dampener. A detailed evaluation of this new SEC–SCV system has been performed using various polymers. Molar mass distribution, intrinsic viscosity distribution, and constants of the Mark–Houwink–Sakurada relationship have been determined. The used polymers were soluble both in organic solvent (PS, PMMA, PVAc, PVC, polyalkylthiofene) and in aqueous solvent (PEO, PEG, pullulan, and hyaluronan). The results obtained are encouraging. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 68: 1651–1659, 1998

Evaluation of a single-capillary viscometer detector on line to a SEC system used with a new pulse-free pump

Evaluation of a single-capillary viscometer detector on line to a SEC system used with a new pulse-free pump