Specific Refractive Index Increment Research Articles

Characterizing Styrene Monomer and Oligomers by SEC/MALS/VISC/DRI

Worldwide polystyrene (PS) production in 2020 was approximately 27 million metric tons, distributed among many nations, making it one of the most heavily imported and exported chemicals. Commercially produced PS usually possesses a broad molar mass distribution, often with a substantial oligomeric component. The latter can significantly affect processing and end-use, in addition to having potentially hazardous health effects and to impacting the polymer’s export classification by regulatory agencies. Quantitation of the oligomeric region of polymers by size-exclusion chromatography with concentration-sensitive and/or static light scattering detection is complicated by the non-constancy of the specific refractive index increment (∂n/∂c) in this region, which affects the calculated amount (mass fraction) of oligomer in a polymer, molar mass averages, and related conclusions regarding macromolecular properties. Here, a multi-detector SEC approach including differential refractometry, multi-angle static light scattering, and differential viscometry has been applied to determining the ∂n/∂c of n-butyl terminated styrene oligomers at each degree of polymerization from monomer to hexamer, and also of a hexadecamer. Large changes in this parameter from one degree of polymerization to the next are observed, including but not restricted to the fact that the ∂n/∂c of the monomer is less than half that of PS polymer at identical experimental conditions. As part of this study, the individual effects of injection volume, flow rate, and temperature on chromatographic resolution were examined. Incorporation of the on-line viscometer allowed for accurate determination of the intrinsic viscosity and viscometric radius of the monomer and oligomers.

Polymer Characterization by Size-Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS): A Tutorial Review.

This tutorial review presents the theory and application of SEC-MALS with minimal equations and a focus on synthetic polymer characterization, serving as an entry point for polymer scientists who want to learn more about SEC-MALS. We discuss the principles of static light scattering, outline its capability to generate absolute weight-average molar mass values, and extend its application to SEC-MALS. Practical elements are emphasized, enabling researchers to appreciate how values for , and are determined in an SEC-MALS experiment and how experimental conditions and input values, such as the specific refractive index increment ( ), influence the results. Several illustrative SEC-MALS experiments demonstrate the impact of separation quality on (as opposed to ), the appearance of contaminants in SEC chromatograms from sample preparation, the influence of concentration on data quality, and how polymer topology affects molecular weight characterization in SEC. Finally, we address practical considerations, common issues, and persistent misconceptions.

Characterization and Calibration of Cold-Alcohol Plasma Fractionation Intermediates by Kjeldahl Digestion and Digital Refractometry

Background Almost 80 years after implementation of the industrial ethanol fractionation process, based on the pioneering work of Edwin J. Cohn's research group, it has not lost its importance in providing life-saving biotherapies to patients. The focus has shifted from albumin, which was first used to treat intensive care patients, to immunoglobulin G preparations. Nowadays the latter enables effective, life-long treatment of patients suffering from immunodeficiencies. Ethanol concentration, pH, temperature, protein, and salt concentration are diligently varied during the Cohn fractionation process to finally yield fractions in which the main plasma proteins are enriched at adequate purity. Total protein concentration of the intermediates, probably not as important as ethanol levels and pH, has nevertheless an indisputable influence on the process’ performance Objectives Total protein measurement is therefore a valuable in process-control for versatile process performance monitoring. For this purpose, we investigated the application of analytical digital refractometry. Design For our pilot study, we used six consecutive batches of nine relevant intermediates covering Takeda's whole industrial-scale Cohn Fractionation process. Methods Total protein concentrations of the intermediates were measured with the method of Kjehldal to obtain reference data and digital analytical refractometry, using 10-kDa ultrafiltration to obtain a protein free permeate serving as a sample-specific blank. Results and Conclusions Specific refractive index increment dn/dc values, allowing the calculation of the total protein concentrations by refractive index measurement, were obtained for the Cohn Fractionation intermediates investigated. Thus, analytical digital refractometry, a simple, fast, and robust technique, was shown to be fit for this purpose.

Molecular characterization of a galactomannan extracted from Tara (Caesalpinia spinosa) seeds

Tara gum (TG) is a polysaccharide extracted from the seeds of a South American tree called Tara (Caesalpinia spinosa). TG is a galactomannan with many applications in the food industry, mainly as an emulsifier and stabilizer agent. In addition, it is also used in the paper and cosmetic industries. In the present study, we performed a molecular characterization based on chemical composition and physicochemical properties to understand the properties behind TG applications. TG was extracted and purified from Tara seeds distributed in different ecoregions of Bolivia. The monosaccharide composition analysis was determined by high-performance anion-exchange chromatography/pulsed amperometric detection (HPAEC-PAD). At the same time, their molecular characteristics, such as molar mass, root-mean-square radius, hydrodynamic radius, conformation, and densities, were studied by asymmetrical flow field-flow fractionation coupled to multi-angle light scattering refractive index (AF4-MALS-dRI), also the specific refractive index increment (dn/dc) was determined for the first time using AF4 for TG. The results revealed that the gum samples are galactomannans composed of mannose (Man) and galactose (Gal) in a ratio of 3.37 (Man/Gal), with an average molar mass range from 2.460 × 107 to 3.699 × 107 Da, distributed in a single population. The root-mean-square radius range from 260.4 to 281.6 nm, and dn/dc is 0.1454. The Kratky plots based on 14 scattering angles indicated that the conformation of all samples corresponds to random coil monodisperse, while their gyration radius/hydrodynamic radius ratio (ρ) is high. All these results suggest that the chains have a low branched density, consistent with the Gal/Man composition. To the best of our knowledge, we report for the first time an integrated physicochemical study of TG relevant to developing emulsifier and stabilizer formulations.

What is the diameter of a fibrin fiber?

BackgroundAltered fibrin fiber structure is linked to pathologic states, including coronary heart disease, ischemic stroke, and atherosclerosis. However, several different techniques are commonly utilized for studying fibrin structures, and comparison of results obtained using different techniques can be challenging due to lack of standardization. ObjectivesThis study provides a path toward standardization by comparing fibrin fiber diameters for a range of physiologic fibrinogen and thrombin concentrations using multiple different complementary experimental methods. MethodsWe determined fiber diameter using scanning electron microscopy (SEM), superresolution (stochastic optical reconstruction microscopy) fluorescence microscopy, and 4 commonly utilized turbidimetric approaches to determine the congruence between the results and the conditions under which each should be used. ResultsWe found that diameter values obtained using SEM and superresolution imaging agree within 10% for nearly all conditions tested. We also found that when a wavelength range of 500 to 800 nm was used for measurements and accounting for the wavelength dependence of the refractive index and specific refractive index increment, diameters obtained using the corrected Yeromonahos turbidimetric approach agree with SEM within 20% for most conditions. ConclusionWe performed a systematic, multitechnique survey assessing fibrin fiber diameters under a range of biochemical conditions. The similarity in the diameter values obtained using SEM and superresolution imaging suggests that drying and fixation during SEM sample preparation do not dramatically alter fiber cross-sections. Congruence, under certain conditions, between diameter values obtained using SEM, superresolution fluorescence imaging, and turbidimetry demonstrates the feasibility of a fibrin diameter standardization project.

Measuring dn/dc for Polysaccharide Microgels of Varying Crosslinking Density

Static light scattering (SLS) is a powerful, noninvasive experimental method that yields the molecular weight (average molecular weight, Mw), the size (radius of gyration, Rg), and the interactions between the scatters (the second virial coefficient, A2). However, proper SLS measurements require determination of the specific refractive index increment (dn/dc) for the samples studied. While tables of dn/dc values are available for various substances, they are not generally available for microgel particles, which in our case are crosslinked chains of an amphiphilic polymer. This paper is focused on measuring dn/dc for microgel samples of varying crosslinking concentrations. Microgel dn/dc values were found to be different from the value of the parent polymer given in literature and found to have a temperature dependence as well as a crosslinker concentration dependence at higher crosslinker concentrations. Using the measured values of microgel dn/dc instead of tabulated parent polymer values on estimates of microgel Mw, Rg, and A2 highlights the importance of direct dn/dc measurements for samples studied by SLS.

Fractionation and Absolute Molecular Weight Determination of Organosolv Lignin and Its Fractions: Analysis by a Novel Acetone-Based SEC─MALS Method

Accurate and reliable structural characterization of technical lignins is still challenging and inhibits industrial utilization as only poor understanding of structure–property relationship is available. Especially, molar mass analysis of technical lignins is of paramount interest; however, the usage of conventional size exclusion chromatography (SEC) and synthetic polymer standards as a consequence of inaccessible lignin standards is predominantly found in academia. This leads to a huge discrepancy in molecular weight analyses of lignins between different laboratories and consequently to fragile comparability. In this study, organosolv (OS) lignin from beech wood was exemplarily used to develop and evaluate a new acetone–water-based SEC method with the ability to be coupled to an electrospray ionization-mass spectrometer (ESI-MS) or to a multi-angle light scattering detector (MALLS or short MALS). The eluent system used shows very good solubility for the lignins investigated and hence lowers the probability for molecular aggregation of lignin molecules in solution. Solvent fractionation in acetone–water mixtures was conducted to acquire molecular weight classes. The starting lignin (parent lignin) was initially dissolved in 80 wt % acetone–water. Various fractions of the parent lignin were produced by a stepwise reduction of the acetone content. Molecular weights based on narrow polyethylene glycol (PEG)/polyethylene oxide (PEO) standards and absolute molar masses by coupling SEC with MALS were obtained, and the drawback of using polymer standards is discussed in detail. At a lower acetone content, low-molecular-weight fractions were found. Additionally, the specific refractive index increments (dn/dc) were determined for the parent lignin and its fractions. The impact of dn/dc on the final molecular weight (MW) was evaluated considering the chemical composition obtained by nuclear magnetic resonance spectroscopy (NMR) analysis. Furthermore, light scattering revealed that the absorption behavior for this OS lignin is low and neglectable. This article proposes a new acetone-based analytical method for direct determination of absolute molar masses of OS lignin molecules.

Detection of the Butyl Acrylate–Vinyl Acetate (BA–VA) Copolymer in Soil Binders Using Gel Permeation Chromatography (GPC) and Nuclear Magnetic Resonance (NMR)

Dust palliatives are commonly used when airborne dust is problematic. Dry soils are treated with a mixture of polymers, such as butyl acrylate vinyl acetate (BA–VA), emulsified in water. Eventual spread through surface runoff or leaching may transport palliative away from the application area. This paper reports a novel method developed for the determination of BA–VA using gel permeation chromatography (GPC) and nuclear magnetic resonance spectroscopy (1H NMR) to provide qualitative and quantitative measurements allowing further investigation of polymer-based soil palliatives. The results showed that the average specific refractive index increments (dn/dc) for BA–VA copolymer in two dust palliatives were 0.055 and 0.038 mL/g. The masses of extracted BA–VA were 1400 and 2900 mg from these products. The corresponding limits of quantification (LOQ) for the extracted BA–VA polymer using a refractive index (RI) detector were 11.63 and 17.04 µg, respectively.

"Goldberg Validations": Refractometric Protein Concentration Calibration of Pooled Source Plasma and Validation of Clinical Serum Protein Refractometers

Conflicting historical data for the specific refractive index increment dn/dc of human plasma/serum protein have warranted an experimental re-calibration for citrated source plasma for fractionation. Measurements by <em>Dumas</em> combustion and digital refractrometry applied the pharmacopeial protein-nitrogen ratio of 6.25. The non-protein matrix was separated by centrifugal ultrafiltration. Refractometric data agreed excellently with <em>Dumas</em>-based protein concentrations.

A practical method for estimating specific refractive index increments for flexible non-electrolyte polymers and copolymers in pure and mixed solvents using the Gladstone-Dale and Lorentz-Lorenz equations in conjunction with molar refraction structural constants, and solvent physical property databases

Equations for calculating the specific refractive index increment, ∂n/∂c, for polymer/solvent systems have been developed starting with either the Gladstone-Dale equation or the Lorentz-Lorenz equation for the refractive index of a dielectric medium. The resulting equations can be used to estimate ∂n/∂c as a function of polymer concentration, temperature, solvent composition, and, for copolymers, monomer composition. Simplifying assumptions have been made to generalize these equations and make them more usable: 1) molar refractions are treated as constants, 2) the density of the polymer solution, in the limit of low polymer concentration, can be approximated via the additivity of volumes approach; the densities of polymer-free solvents and solvent mixtures are not necessarily subject to this approximation, 3) the mass density of solvents and solvent mixtures can be estimated by any one of several models developed specifically for this purpose; we will consider the Rackett equation, the COSTALD equation, and the standard assumption of mass and volume additivity, and 4) the mass density of a copolymer can be obtained from a weight fraction average of the mass densities of the homopolymers from which it is comprised. Comparisons to experimental data show that (∂n/∂c)GD and (∂n/∂c)LL both yield average absolute deviations of approximately 0.009 ml/gm.

Rapid quantification and characterization of the pyrolytic lignin fraction of bio-oils by size exclusion chromatography coupled with multi-angle laser light scattering detector (SEC-MALS)

A rapid method was developed to characterize the Pyrolytic Lignin (PL) fraction of flash pyrolysis bio-oils, based on size exclusion chromatography (SEC) coupled to differential refractive index (DRI) and multi-angle laser light scattering (MALS) detectors. Two beech wood bio-oils with different PL content were used in the study. The first was produced with a single-stage condensation system (BO), while the second was an organic fraction collected in the first stage of a two-stage condensation system (F1). PL was isolated from both the BO and F1 bio-oils by the water precipitation method. Our results suggested that quantification of the pyrolytic lignin fraction of bio-oils can be performed by the SEC-MALS-DRI method provided that the specific refractive index increment (dn/dc) is known, and the integration interval is carefully chosen. Average molar mass (Mn) values for the BO and F1 samples were 580 ± 50 Da and 890 ± 50 Da, respectively. Our results indicated that the condensation system and the water precipitation method affect the average molar mass of isolated PL oligomers.

Determining the Chemical-Heterogeneity-Corrected Molar Mass Averages and Distribution of Poly(styrene-co-t-butyl methacrylate) Using SEC/MALS/UV/DRI.

Chemical heterogeneity, defined as the change (or lack thereof) across the molar mass distribution (MMD) in the monomeric ratio of a copolymer, can influence processing and end-use properties such as solubility, gas permeation, conductivity, and the energy of interfacial fracture. Given that each parent homopolymer of the copolymer monomeric components has a different specific refractive index increment (∂n/∂c) from the other component, chemical heterogeneity translates into ∂n/∂c heterogeneity. The latter, in turn, affects the accuracy of the molar mass (M) averages and distributions of the copolymers in question. Here, employing size-exclusion chromatography coupled on-line to multi-angle static light scattering, ultraviolet absorption spectroscopy, and differential refractometry detection, the chemical heterogeneity (given as mass percent styrene) was determined for a poly(styrene-co-t-butyl methacrylate) copolymer. Also determined were the chemical-heterogeneity-corrected M averages and MMD of the copolymer. In the present case, the error in molar mass incurred by ignoring the effects of chemical heterogeneity in the M calculations is seen to reach as high as 53,000 g mol-1 at the high end of the MMD. This error could be much higher, however, in copolymers with higher M or with larger difference among component ∂n/∂c values, as compared to the current analyte.

Theoretical and experimental study of mirrorless fiber optics refractometer based on quasi-Gaussian approach

A mirrorless refractometer was studied and analyzed using the quasi-Gaussian beam approach. The Fresnel equation for reflectivity at the interface between two mediums with different refractive indices was used to calculate the directional reflectivity, R. Various liquid samples from 1.3325 to 1.4657 refractive indices units were used. Experimentally, a fiber bundle probe with a concentric configuration of 16 receiving fibers and a single transmitting fiber was employed to verify the developed models. The sensor performance in term of sensitivity, linear range, and resolution, were analyzed and calculated. It has been shown that the developed theoretical models are capable of providing quantitative guidance of the output of the sensor with high accuracy. The highest resolution of the sensor was 4.39 × 10−3 refractive indices units, obtained by correlating the peak voltage along the refractive index. The resolution is sufficient for determining the specific refractive index increment of most polymer solutions, certain proteins, and also in monitoring bacterial growth. The accuracy, simplicity, and effectiveness of the proposed sensor over a long period of time while having non-contact measurements reflect a good potential for commercialization.

Determination of length distribution of TEMPO-oxidized cellulose nanofibrils by field-flow fractionation/multi-angle laser-light scattering analysis

Aqueous nanocellulose dispersions were prepared from wood cellulose by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation. The obtained TEMPO-oxidized cellulose was converted into TEMPO-oxidized cellulose nanofibrils (TOCNs) of different lengths by controlling the nanofibrillation conditions in water or using dilute acid hydrolysis. The average lengths and length distributions of TOCNs have been measured from transmission electron microscopy (TEM) and atomic force microscopy (AFM) images. However, because the number of nanocelluloses observable in TEM and AFM images is limited, a more reliable method is needed to obtain the lengths/length distributions of TOCNs. In this study, the aqueous TOCN dispersions were subjected to a combination of field-flow fractionation (FFF) and multi-angle laser-light scattering (MALLS). The optimum FFF operation conditions for the acid-hydrolyzed TOCN were first established to obtain reasonable data. For TOCNs with average lengths > 400 nm, suitable separation could not be achieved using the FFF/MALLS system. In contrast, the TOCNs with average lengths of 170 and 270 nm were adequately separated according to their sizes by the FFF system. The TOCN length distribution patterns corresponded well to those obtained from TEM images. However, the amounts of TOCNs with lengths > 250 nm were underestimated compared with those determined from TEM images. For TOCNs with average lengths of 170 and 270 nm, the molar mass at each TOCN length was determined using the FFF/MALLS system combined with a refractive index detector, where a specific refractive index increment of 0.165 mL/g was used for TOCN.

Determination of molar masses of macromolecules by size exclusion chromatography-light scattering not requiring knowledge of refractive index increments

A new approach for the calibration of SEC-light scattering (SEC-LS) setups is proposed, which requires solely the molar mass of a reference polymer. Neither the specific refractive index increment of the calibrant nor of the analyte is required. Comparison of the molar masses derived in different solvents for a large number of chemically different polymers shows that the new approach yields the same molar masses as if molar masses were derived using dn/dc to calibrate the light scattering setup. The approach therefore allows easier determination of molar masses by SEC-LS.

Specific refractive index increment (∂n/∂c) of polymers at 660 nm and 690 nm.

The specific refractive index increment (∂n/∂c) is an essential datum for the accurate quantitation of molar mass averages and distributions (inter alia) of macromolecules when refractometry, static light scattering, and/or viscometry detection are coupled on-line to size-based separation techniques. The latter include methods such as size-exclusion and hydrodynamic chromatography, and asymmetric and hollow-fiber flow field-flow fractionation. The ∂n/∂c is also needed for accurate determination of the weight-average molar mass of polymers by off-line, batch-mode multi-angle static light scattering. However, not only does ∂n/∂c differ among chemical species, it also depends on experimental conditions such as solvent, temperature, and wavelength. For the last seventeen years, the author's laboratories have measured the ∂n/∂c of a variety of natural and synthetic polymers, at both 690 nm and, more recently, 660 nm, under a variety of solvent and temperature conditions. In all cases, this has been done by off-line, batch-mode differential refractometry, not by assuming 100% analyte column recovery and 100% accurate peak integration. Results of these determinations are presented here, along with the relevant experimental data.

Branching Analysis of Multivalent Conjugates Using Size Exclusion Chromatography-Multiangle Light Scattering.

Multivalent conjugates (MVCs) (conjugation of multiple proteins to a linear polymer chain) are powerful for improving the bioactivity and pharmacokinetics of a bioactive molecule. Since this effect is highly dependent upon the valency of the conjugated proteins, it is imperative to have a technique for analysis of the conjugation ratio. Studies of MVCs have used size exclusion chromatography-multiangle light scattering (SEC-MALS), which allows for the separate and individual analysis of the protein and biopolymer components based on their specific refractive index increment and UV extinction coefficient constants to determine the number of proteins bound per biopolymer molecule. In this work, we have applied traditional branching analysis to the SEC-MALS data, with the primary assumption that the polymer backbone can be used as the linear counterpart. We demonstrated good agreement between the branching values and the valency determined by traditional analysis, demonstrating that branching analysis can be used as an alternative technique to approximate the valency of MVCs. The branching analysis method also provides a more complete picture of the distribution of the measured values, provides important branching information about the molecules, and lowers the cost and complexity of the characterization. However, since MVC molecules are both conjugate molecules and branched molecules, the most powerful approach to their characterization would be to use both traditional multivalent conjugate analysis and branching analysis in conjunction.

Simultaneous measurement of the molecular weight distribution and 5‐ethylidene‐2‐norbornene content across the molecular weight distribution of ethylene–propylene–diene terpolymer via a new size exclusion chromatography–ultraviolet–refractive index method

ABSTRACTA size exclusion chromatography (SEC)–UV–refractive index (RI) method was developed to measure the 5‐ethylidene‐2‐norbornene (ENB) content across the molecular weight distribution (MWD) in ethylene–propylene–diene terpolymer (EPDM) at room temperature. The ratio of the UV and RI signals at the same effective elution volume was converted to ENB content. The feasibility of using this method to measure the ENB content across the MWD in EPDM at high temperature was also demonstrated. Prior understanding was that ENB had insufficient UV absorbance relative to high‐temperature SEC solvents to allow for useful measurements. We demonstrated this by using high‐boiling‐point solvents, such as decalin, with a low UV absorbance in the UV wavelength range of interest for ENB. These solvents also gave rise to a high enough specific RI increment (dn/dc) for EPDM that a suitable RI detector response was obtained. Additionally, this methodology could be readily applied to other polymers soluble at high temperature as long as the polymers contained a UV chromophore. These include polymers containing vinyl, conjugated vinyl, aromatic ring, carbonyl, or halocarbon groups. This UV‐absorption‐based detection concept might also be extended to high‐temperature thermal‐gradient interactive chromatography‐UV, high‐temperature solvent‐gradient interactive chromatography‐UV (high‐temperature liquid chromatography‐UV), temperature‐rising elution fractionation‐UV, crystallization analysis fractionation‐UV, and crystallization elution fractionation‐UV. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 43911.

Extinction coefficients and refractive index increments of complex salts between cetylpyridinium cations and poly(styrenesulfonate) anion

Abstract Polyelectrolyte–surfactant complexes, PSCs, are widely used and studied. In this data article, we report an easy preparation procedure of the complex salt formed between cetylpyridinium cations and poly(styrenesulfonate) anion complemented by values of its molar extinction coefficient and specific refractive index increment in aqueous solutions at various charge ratios between the surfactant (S) and the polyion (P) charges (S/P). These complexes have superior solubility in water. The molar extinction coefficient increases linearly with S/P, whereas the specific refractive index increment does not depend on S/P. These data are useful for the spectrophotometric determination of concentration and for molar mass determination by light scattering, respectively. They were already used in measurements of several thermodynamic and transport properties (Kogej, 2010) and of molar mass values of the soluble PSCs (Prelesnik et al., 2011) where the knowledge of precise concentration is crucial.

Dendritic‐Linear Hybrid Multiarm Star Polymers: A Straightforward Synthesis of Polymer as Molecular Nanoparticles

Synthesis of multiarm star polymers via “core first” method but possibly through two different chain growth fashions has been described. Two distinct styrenyl‐TEMPO‐based G3 and G4 dendritic unimolecular initiators have been used for this method. Polymerization of styrene using these core‐cum‐initiators at 125 °C varying the monomer to initiator ratio and polymerization time yields multiarm star polymers with molecular weight (Mw) in the range of 1.71 × 105 to 4.75 × 105 g mol−1 and polydispersity in the range of 1.34–1.46. Hydrolysis leading to degradation of inner polyurethane core yields highly narrow dispersed—PDI 1.00 to 1.04—polystyrene chains, and the SEC‐MALLS data of these chains confirm the accurate control on predetermination in the number of arms up to 48 of star polymers. The specific refractive index increment (dn/dc) of the polymers is found to be decreased with decreasing weight fraction of core. The transmission electron microscope (TEM) and atomic force microscopy (AFM) images and hydrodynamic radius of chosen polymers confirm the two different chain growth fashions leading to the formation of star polymers as discrete nanoparticle in the case of initiator in which TEMPO is anchored and formation of peripherally low entangled short chains in the case of initiators in which TEMPO is end‐capped. image