Molar mass distribution by size exclusion chromatography: Comparison of multi-angle light scattering and universal calibration

Series of polymers of various molar mass, chemical composition, and molecular architecture was analyzed by size exclusion chromatography (SEC) coupled with a multi-angle light scattering (MALS) photometer and an online viscometer. The molar mass averages were determined from the signal of MALS or calculated from the intrinsic viscosity and universal calibration. The comparison of the obtained results showed significant differences between the two methods. The MALS detection was shown to be more accurate for the determination of the weight-average molar mass and less vulnerable to the spreading of polymer peak by band broadening. The universal calibration can yield more accurate estimation of the number-average molar mass of branched polymers. It is also significantly more accurate for the characterization of fluorescent polymers than MALS with a regular laser of 660 nm. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47561.     

Improved methods for evaluating the molar mass distributions of cellulose in kraft pulp

Multi‐angle laser light scattering (MALLS) was used to characterize birch kraft pulps with respect to their absolute molecular mass distributions (MMDs). The pulps were dissolved in lithium chloride/N,N‐dimethylacetamide and separated by size exclusion chromatography (SEC). The weight‐average and number‐average molecular masses of the cellulose fractions of the pulps obtained from the absolute MALLS measurements were compared with the molar masses obtained by direct‐standard‐calibration relative pullulan standards. Discrepancies between the two detection methods were found, and two ways of correlating the relative pullulan molar masses to the absolute molar masses were examined. In the first method, the correlation was made over a large range of molecular masses. The second method correlated the molecular masses of the standards to the molecular masses of samples by the calculation of fictitious, cellulose‐equivalent molar masses of the standards. With the preferred second method, a more correct MMD of kraft pulp samples could, therefore, be obtained from an SEC system calibrated with narrow standards. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 88: 1170–1179, 2003     

Characterization of bisphenol A–based epoxy resins by HPLC,GPC, GPC–MALLS,VPO, viscometry,and end group analysis: On the identification of 2,3‐dihydroxypropyl‐containing compounds,determination of molar mass distribution,and branching

Reversed‐phase high performance liquid chromatography (HPLC), conventional gel permeation chromatography (GPC), and gel permeation chromatography coupled with a multiangle laser light scattering photometer (GPC–MALLS) were used for the analysis of epoxy resins based on bisphenol A. Compounds containing 2,3‐dihydroxypropyl group were identified in HPLC chromatograms by means of the derivatization of sample by acetone. The presence of branched molecules was proved by GPC–MALLS using a molar mass versus root mean square (RMS) radius plot or molar mass versus elution volume plot. The molar mass distribution determined by HPLC was compared with that obtained by GPC–MALLS. Molar mass averages measured by means of GPC, GPC–MALLS, vapor pressure osmometry (VPO), and end group analysis (EG) were compared and the differences of results obtained by particular methods were discussed. An appropriate GPC calibration was found on the basis of literature data and the comparison of molar mass averages measured by GPC, VPO, and GPC–MALLS. The refractive index increment of epoxy resins was determined. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 74: 2432–2438, 1999     

Molar mass distributions of polymers from size exclusion chromatography and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry: Methods for comparison

Equations are presented for calculating molar mass averages and molar mass distributions from matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) data and from size exclusion chromatography (SEC) data. The utility of polydispersity is examined as an indicator of the expectation of MALDI‐TOF MS mass discrimination effects. Cumulative distributions are found to be rich in information for comparing the two techniques and are easily obtained from both SEC and MALDI‐TOF MS data. Analyses of a series of narrow molar mass distribution poly(methyl methacrylate) (PMMA) standards and one polydisperse sample have been performed with both methods. MALDI‐TOF MS did not detect dimer and trimer in the PMMA samples, and it often indicated lower amounts of high‐molar‐mass polymers than did SEC. The results showed that the distribution breadth, as evidenced by the standard deviation of the distribution (calculated from the polydispersity and number‐average molar mass), correlated well with the molar mass range observed in the MALDI‐TOF MS spectra, whereas the polydispersity alone did not. Ratioing the extremes in the molar mass concentrations measured with the SEC differential refractometer, which were necessary to adequately define molar mass distributions, showed that detector dynamic range values as high as approximately 370,000 were required for the polydisperse samples. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 97: 627–639, 2005     

Characterization of poly[di(carboxylatophenoxy)-phosphazene] by an aqueous gel permeation chromatography

A water-soluble phosphazene polyelectrolyte, poly[di(carboxylatophenoxy)phosphazene] (PCPP), was characterized using aqueous gel-permeation chromatography (GPC) with concentration (UV and RI) and molecular weight sensitive (multiangle laser light-scattering) detectors. Agreement was observed between the weight-average molecular weights determined by GPC with a light-scattering detector, conventional GPC using fractionated narrow PCPP standards, and also by static light-scattering measurements. The effect of chromatography conditions, such as ionic strength of the mobile phase, column resolution, and injection volume was investigated. Mark–Houwink constants of PCPP in aqueous solution (phosphate buffer, pH 7.4, 0.42MNaCl) were determined. The validity of the universal calibration curve and the occurrence of a secondary nonexclusion mechanism of separation in aqueous GPC of phosphazene polyelectrolytes are discussed. © 1996 John Wiley & Sons, Inc.     

Analysis of the accuracy of determination of molar mass distribution by the GPC with the on-line light-scattering detector

The effect of errors of the concentration (c-) and the light-scattering molar mass (M-) detector in gel permeation chromatography (GPC) on the accuracy of the determination of the molar mass distribution (MMD) was analyzed. Model calculations showed that the reliability of MMD obtained from c-detector data and from a carefully performed calibration dependence is higher than that obtained by combining data of the M- and c-detectors. A criterion of the uncertainty of the MMD determination by the GPC method with the M-detector was suggested, and its dependence on MMD parameters of the analyzed polymer and on the magnitude of injection was investigated.     

Compositional heterogeneity in partially hydrolysed poly(vinyl alcohol) by reversed phase liquid chromatography

Reversed phase high performance liquid chromatography (HPLC) was employed to elucidate the composition distribution of partially hydrolysed samples of poly(vinyl alcohol) (PVOH), demonstrating that elution across a chromatogram proceeded from higher to lower degree of hydrolysis (DoH). Fractions isolated by preparative HPLC fractionation and characterised by ¹H nuclear magnetic resonance spectroscopy (NMR) were used as HPLC standards to construct a calibration curve of retention time versus DoH, allowing for DoH determination of any PVOH sample once its chromatogram was available. Plots of cumulative and differential distributions as a function of DoH were determined, allowing for comparisons of samples having average DoH in the range 70–90 mol%. A second set of fractions originating from a parent polymer having different molar mass was also isolated to confirm that calibration was not influenced by molar mass or size exclusion effects.     

Determination of molecular weight for poly(vinyl butyral) using size exclusion chromatography/low-angle laser light scattering (SEC/LALLS) in hexafluoroisopropanol

Size exclusion chromatography/low-angle laser light-scattering (SEC/LALLS) determinations of molecular weight for poly(vinyl butyral) (PVB) using hexafluoroisopropanol (HFIP) as the polymer solvent and chromatographic mobile phase are described. The use of HFIP provided aggregate-free SEC/LALLS chromatograms from which molecular weight distribution averages were calculated. Comparison of SEC/LALLS molecular weights determined in HFIP with similar data obtained using tetrahydrofuran as the polymer solvent and chromatographic mobile phase indicated that the former is more generally applicable for PVB of different vinyl alcohol content.     

Laser light-scattering study of polyacrylamides with and without hydrolyzation in 0.35M KH2PO4 aqueous solution

Both the hydrolyzed and unhydrolyzed polyacrylamides with different molar masses were characterized in 0.35M KH₂PO₄ aqueous solution at 25°C by laser light scattering (LLS). The Laplace inversion of precisely measured intensity-intensity time correlation function leads to an estimate of the characteristic line-width distribution G(Γ) which can be further reduced to a translational diffusion coefficient distribution G(D). A combination of the measured weight-average molar mass M_w and G(D) enables us to establish a calibration of D (cm²/s) = (4.46 ± 0.02) × 10⁻⁵M^{−0.50±0.003}. Using this calibration, we convert each G(D) into a corresponding molar mass distribution. The calculated M_w from such a molar mass distribution is reasonably close to the measured M_w from static LLS. Most important is that when 0.35M KH₂PO₄ aqueous solution is used as solvent, both the hydrolysed and unhydrolysed polyacrylamides can be represented by an identical calibration; namely, in 0.35M KH₂PO₄ aqueous solution, the polyelectrolytes effect in the hydrolysed polyacrylamides has been suppressed. Therefore, the hydrolysed polyacrylamides can be characterized as a normal neutral polymer in 0.35M KH₂PO₄ aqueous solution, which makes a routine characterization of the hydrolysed polyacrylamides much easier. © 1997 John Wiley & Sons, Inc. J Appl Polym Sci 63: 1755–1760, 1997     

IR spectroscopy as a quantitative and predictive analysis method of phenol–formaldehyde resol resins

A set of resin samples was characterized by IR and ¹³C-NMR spectroscopy. The suitability of IR spectroscopy for the quantitative analysis of resins was evaluated by statistical methods using the NMR reference data as calibration. The values of interesting properties, for example, the amount of free phenol and the formaldehyde-to-phenol (F/P) molar ratio, of the resins being similar to the calibration resins were predicted from the IR spectra. Also, the predicted results were compared with the ones observed by ¹³C-NMR spectroscopy. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 69: 2175–2185, 1998     

Gelpermeationschromatographie und Kalibrierung an resolischen Phenol-Formaldehyd-Kondensaten

Several resols with a molar ratio of formaldehyde to phenol of 2.5 and 1.7, respectively, and a mass content of sodium hydroxide of 8 or 4 wt.-% were synthesized and were analysed by size exclusion chromatography. Hereby, tetrahydrofuran with 0.4 wt.-% trichloroacetic acid was used as solvent and eluent, which is capable of dissolving even highly alkaline resols. In the lower molar mass range, these resins were separated in up to eight peaks; comparison of the chromatograms of pure substances, which are expected to be found during resol synthesis, allowed detailed interpretation. In the range of higher molar mass, calibration was attempted by polystyrene and poly(ethylene oxide) standards; for the fractions with the highest molar mass, a molar mass of 150 kg mol^-1 was estimated. Comparison of the signals from a refractive index (RI) detector and a u.v. detector showed that the u.v. detector is less disturbed by traces of low molar mass impurities.     

Evaluation of light-scattering detectors for size exclusion chromatography. I. Instrument precision and accuracy

A systematic evaluation of two types of light-scattering detectors for size exclusion chromatography (SEC) was completed. The two detectors were the low-angle laser light scattering photometer (LALLS) and the multiangle laser light-scattering photometer (MALLS). Instrument evaluations were performed at both room (30–40°C) and high (135–145°C) temperatures using the polystyrene standard, NBS 706, at room temperature and the polyethylene standard, SRM 1476, at high temperature. Results of the evaluation showed that when experimental uncertainties were taken into account LALLS and MALLS demonstrated equivalent precision and accuracy for molecular weight determination. The main source of inaccuracy found (particularly for SRM 1476) was the sensitivity difference between the light-scattering and the concentration (DRI) detectors; i.e., the DRI detector was unable to measure very low concentrations of very high molecular weight material present in SRM 1476, whereas the light-scattering detectors respond strongly. It was shown that for LALLS the overall weight-average molecular weight (M?_w) for the whole polymer calculated using an equation that did not require the DRI detector output circumvented this sensitivity problem while assuming that the low angle used was sufficiently close to zero. Use of this equation for MALLS is possible by extrapolating data from all angles used to obtain a light-scattering chromatogram at zero angle. However, this possibility was not examined here. A particular advantage of MALLS over LALLS is that MALLS can provide the z-average root mean square radius (commonly referred to here and in other light-scattering literature as the “radius of gyration”) values from the same data as those used to obtain molecular weight values. Although the radius of gyration values at each retention volume were not as precise as the corresponding weight-average molecular weights, at room temperature, precision was better than 2% for a significant portion of the chromatogram. © 1993 John Wiley & Sons, Inc.     

Molar mass analysis of polyamides-11 and -12 by size exclusion chromatography in HFiP

The SEC analysis of polyamide-11 and polyamide-12 can be conducted free of association and aggregation phenomena when hexafluoroisopropanol + 0.05 mol/L potassium trifluoroacetate are used as the mobile phase. The calibration of the SEC system can be conducted in different ways. As stationary phases non-polar polystyrene and polar perfluoro silicagel were tested. The investigations showed that the polystyrene gel exhibits hydrophobic interactions with the polyamides while with the silicagel selective interactions were not found. Investigating different options for SEC calibration it was found that conventional PMMA calibration does not yield correct results. The universal calibration approach based on PMMA calibration did not work either. Correct molar masses were obtained when the PMMA calibration curve was corrected with data from polyamide blends using a simplex algorithm. Alternatively, calibration can be conducted with broadly distributed polyamides that were first fully characterized by SEC-MALLS. The resulting molar mass distributions for different sets of polyamides were compared with molar masses that were determined directly by SEC-MALLS and excellent correlation was obtained.     

Correction method for a concentration effect in the calculation of molecular weight averages from GPC chromatograms

In the calculation of molecular weight averages by GPC, the traditional method uses the calibration curve obtained at the same concentration as the samples, which results in a large degree of disagreement between molecular weight averages at several concentrations. Because of the concentration dependence of peak elution position in gel permeation chromatography of polymers, correct molecular weight averages cannot be obtained if calibration concentrations are the same as sample concentrations. A computation approach which uses calibration curves at finite and zero concentrations and can correct concentration effects is shown. The concentration used varied from 0.1% up to 0.4%. The elution chromatogram was divided into several parts, and concentration of species at each elution point was obtained from a concentration–peak height calibration curve. Molecular weight at the point was obtained from a molecular weight–elution volume calibration curve corresponding to a concentration of species at the point, and molecular weight averages were calculated by using the usual method. Nearly identical values for molecular weight averages could be obtained at different concentrations, and additional support for this approach is that these values for molecular weight averages were in fair agreement with NBS data.     

Molar mass determination of poly(octadecene-alt-maleic anhydride) copolymers by size exclusion chromatography and dilute solution viscometry

The number average molar mass M_n of poly(octadecene-alt-maleic anhydride) (PODMA) copolymers calculated from data obtained by size exclusion chromatography (SEC) using a polystyrene (PS) calibration was found to be inaccurate. The use of SEC combined with dilute solution viscometry enabled a method to be developed using an iterative approach, which does not require knowledge of the Mark-Houwink constants for PODMA samples. A new calibration curve was constructed as a plot of molar mass M_u for PODMA. True number-average molar masses M_n (true) calculated using the new calibration are approximately twice the apparent molar mass M_n (app) based on a PS calibration for higher molar mass samples (>10?000 g mol⁻¹).     

On-Line Coupling of Thermal Field-Flow Fractionation with Low-Angle Laser Light Scattering

Abstract

The analysis of a polymethyl methacrylate sample dissolved in dimethylformamide is performed by using a low-angle laser light scattering photometer attached to a thermal field-flow fractionation channel and a differential refractometer. Relevant theoretical light scattering equations for flow-through operation are outlined. It is shown that the calibration curve of the separation system can be constructed in situ during the course of separation, without using any calibration standard. The average molecular weights as well as the molecular weight distribution curves of the polymer have been determined. The sensitivity of the light-scattering photometer has been measured, and it is compared to that of the differential refractometer in terms of signal-to-noise ratios. The various sources of errors in the molecular weight determination are discussed, and the potential of the coupling for physicochemical studies on the thermal diffusion of polymers is indicated. In spite of some inherent problems, this coupling is expected to have a very bright future if reliable low-angle light-scattering instruments can be made available at moderate prices.     

Truths and myths about the determination of molar mass distribution of synthetic and natural polymers by size exclusion chromatography

This article discusses various aspects of the determination of molar mass distribution by means of size exclusion chromatography (SEC) in various application modes. The effects of erroneous specific refractive index increment (dn/dc), branching, column performance, and enthalpic interactions on the results obtained by different SEC techniques are discussed. Combination of SEC and a light scattering detector represents the most direct way to the molar mass distribution of all natural and synthetic polymers as it completely eliminates the need for column calibration and to a certain extent eliminates the dependence of the obtained results on some operational variables such as flow rate, temperature, or injected mass. A multiangle light scattering (MALS) photometer has become the most frequently used light scattering detector capable of determination of molecular size as another important polymer characteristic. This article contrasts SEC‐MALS method with other application modes of SEC from the viewpoint of some frequent confusions and misunderstandings. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40111.     

Determination of molecular weight averages and molecular weight distribution by g.p.c. of N-trifluoroacetylated polyamides

To determine their molecular weight averages and molecular weight distribution, N-trifluoroacetylated (TFA) polyamides are studied using gel permeation chromatography in tetrahydrofuran at 25°C. Several calibration methods (calibration with polydisperse standards, universal calibration) are tested; among these, direct calibration of M_w of polydisperse samples vs. peak maximum of the corresponding g.p.c. chromatogram leads to the best results. Calculated molecular weight averages of nylon 6-TFA and Trogamid^R-TFA are compared with data from light scattering, viscometry and osmometry.     

Molecular mass calibration in size-exclusion chromatography of coal derivatives

A number of procedures have been investigated for the calibration of a size-exclusion chromatography (s.e.c.) column in the determination of molecular mass (MM) distributions of coal derivatives. The behaviour of narrow fractions of coal extracts in the MM range 200–3000 was compared with a variety of the more generally available calibration standards. Calibration with preparative s.e.c. subfractions of materials similar to those under study has been recommended. Polystyrene standards are satisfactory, however, for MM < 1000, but above this range other polymer standards should be sought. Universal and molar volume calibration do not apply to coal-derived materials.     

Optimization of pullulan production from synthetic medium by Aureobasidium pullulans in a stirred tank reactor by response surface methodology

The production of pullulan from synthetic medium by Aureobasidium pullulans P56 in a stirred tank fermenter was investigated. The kinetics of polysaccharide, pullulan and biomass production was determined. Response surface methodology was used to investigate the effects of three factors (initial sugar concentration, aeration rate and agitation speed) on the concentration of pullulan in batch cultures of A pullulans. In the experiments, the range of values used for the three variables described were; 30–70 g dm^?3 initial sugar concentration, 200–600 rpm agitation speed and 1.0–3.0 vvm aeration rate. No previous work has used statistical analysis in determining the interactions among these variables in pullulan production. Results of the statistical analysis showed that the fit of the model was good in all cases. Aeration rate, agitation speed and sugar concentration had a strong linear effect on pullulan concentration. Moreover, pullulan concentration was significantly influenced by the negative quadratic effects of the given variables and by their positive or negative interactions with the exception that the interaction between agitation speed and aeration rate was insignificant (P > 0.05). Maximum pullulan concentration of 17.2 g dm^?3 was obtained at the optimum levels of process variables (initial sugar concentration 51.4 g dm^?3, aeration rate 2.36 vvm, agitation speed 345.3 rpm). These values were obtained by fitting of the experimental data to the model equation. Scanning electron microscope (SEM) photographs of polysaccharide particles containing different concentrations of pullulan were also taken to observe the morphological differences of the samples. Copyright © 2005 Society of Chemical Industry