Molar Mass Determination Research Articles

Potential of Benchtop NMR for the Determination of Polymer Molar Masses, Molar Mass Distributions, and Chemical Composition Profiles by Means of Diffusion-Ordered Spectroscopy, DOSY.

The determination of molar masses and their distributions is crucial in polymer synthesis and design. This work presents the current performance and limitations of diffusion-ordered spectroscopy (DOSY) on a low-field (benchtop) NMR spectrometer (at 90MHz) as an alternative to size exclusion chromatography (SEC) for determining diffusion coefficient distributions (DCDs) and molar mass distributions (MMDs). After optimization for narrowly distributed homopolymers, MMDs obtained with inverse Laplace transformation (ILT) and log-normal distribution are compared with average molar masses obtained with mono- and bi-exponential fits, as well as MMDs obtained from SEC. This approach enables ILT to determine DCDs and MMDs even for bimodal homopolymers with fully spectrally overlapping signals and block copolymers with various chemical compositions, for which chemical composition profiles are determined. The feasibility of low-field diffusion NMR with samples dissolved in non-deuterated solvents is further demonstrated and methods for solvent suppression are discussed.

Experimental teaching reform of polymer molar mass determination using gel permeation chromatography coupled with light scattering

Gel permeation chromatography coupled with light scattering (GPC-LS) is among the most common methods for determining the molar masses of polymers. GPC-LS is widely used in polymer science research and has been adopted for many industrial applications owing to its high sensitivity, accuracy, and precision. The determination of polymer molar masses using GPC-LS is an important experimental component of the "Polymer Physics Experiments" course. However, the present GPC-LS experimental teaching content tends to be overly simplistic and lacking in depth. Herein, the original experimental content is expanded and multiple sets of experiments are redesigned: (1) Using commercial polystyrene as an experimental sample, the molar mass, molar mass distribution, radius of gyration, and other molecular structure parameters are determined using GPC-LS; (2) Using two polyacrylonitriles with similar molecular structure parameters, subtle differences in the molar mass distributions of the samples are explored using differential mass distribution curves; (3) By comparing the chromatograms of a series of polyethylene glycols with different molar masses, the effect of molar mass on chromatographic peaks is investigated; and (4) For three different polymers (polyacrylonitrile, poly(methyl methacrylate), and poly(β-cyclodextrin)), the polymer chain conformations are analyzed using conformation plots (i.e., radius of gyration vs. molar mass). In addition, the experimental teaching method is modified to convert passive learning into active learning, thereby improving the students' self-directed learning ability. This experimental teaching reform will help students obtain a more comprehensive understanding of GPC-LS principles and applications, stimulate their enthusiasm for learning, and improve the teaching quality of the experimental course.

Characterization of biotinylated human ACE2 and SARS-CoV-2 Omicron BA.4/5 spike protein reference materials

Accurate diagnostic and serology assays are required for the continued management of the COVID-19 pandemic yet spike protein mutations and intellectual property concerns with antigens and antibodies used in various test kits render comparability assessments difficult. As the use of common, well-characterized reagents can help address this lack of standardization, the National Research Council Canada has produced two protein reference materials (RMs) for use in SARS-CoV-2 serology assays: biotinylated human angiotensin-converting enzyme 2 RM, ACE2-1, and SARS-CoV-2 Omicron BA.4/5 spike protein RM, OMIC-1. Reference values were assigned through a combination of amino acid analysis via isotope dilution liquid chromatography tandem mass spectrometry following acid hydrolysis, and ultraviolet–visible (UV–Vis) spectrophotometry at 280 nm. Vial-to-vial homogeneity was established using UV–Vis measurements, and protein oligomeric status, monitored by size exclusion liquid chromatography (LC-SEC), was used to evaluate transportation, storage, and freeze–thaw stabilities. The molar protein concentration in ACE2-1 was 25.3 ± 1.7 µmol L−1 (k = 2, 95% CI) and consisted almost exclusively (98%) of monomeric ACE2, while OMIC-1 contained 5.4 ± 0.5 µmol L−1 (k = 2) spike protein in a mostly (82%) trimeric form. Glycoprotein molar mass determination by LC-SEC with multi-angle light scattering detection facilitated calculation of corresponding mass concentrations. To confirm protein functionality, the binding of OMIC-1 to immobilized ACE2-1 was investigated with surface plasmon resonance and the resulting dissociation constant, KD ~ 4.4 nM, was consistent with literature values.Graphical

On-line coupling of hollow-fiber flow field-flow fractionation and depolarized multi-angle static light scattering (HF5/D-MALS). Proof of principle

Introduced here is the on-line coupling of hollow-fiber flow field-flow fractionation (HF5) to depolarized multi-angle static light scattering (D-MALS). HF5 is a size-based separation alternative to size-exclusion and hydrodynamic chromatography and asymmetric flow field-flow fractionation. HF5 can separate larger sizes than its chromatographic counterparts and provides several advantages over its fractionation counterpart, including reduced sample consumption and greater ease of operation. D-MALS is a variant of MALS in which the depolarized scattering from the analyte solution is measured at a variety of angles simultaneously. Measurements of depolarized scattering have previously been employed in studying the optical properties of solutions or suspensions, to determine the length of rod-like analytes, and to gain increased accuracy in the determination of analyte molar mass. The coupling HF5/D-MALS allows for the depolarization ratio of a solution or suspension to be measured continuously across the fractogram. This is demonstrated here for a Teflon latex the size range of which extends beyond that accessible to commercial size-exclusion columns. The results presented provide the first reported on-line HF5/D-MALS coupling, showing the feasibility of the technique as well as its realized potential for providing continuous depolarization measurements, inter alia.

Online coupling of liquid chromatography and two-dimensional diffusion ordered spectroscopy for the analysis of oligostyrenes

The aim of this study was to introduce a powerful coupling of Liquid Adsorption Chromatography (LAC) and Diffusion-Ordered Spectroscopy (DOSY) for comprehensive structure analysis. This new hyphenation approach facilitated the simultaneous separation of a polymer mixture and the determination of molar masses within a single 3D experiment. The online coupling of High-Performance Liquid Chromatography (HPLC) and two-dimensional DOSY-NMR will be called 3D-LAC-NMR-DOSY experiment. Our methodology enabled the chromatographic separation of analytes based on their chemical heterogeneity, and provided accurate molar masses of the analytes through 2D-DOSY. This new method was demonstrated on a polystyrene oligomer mixture. In this case, the oligostyrenes could be separated with LAC according to their tacticity and chain length in protonated acetonitrile as eluent and DOSY measurements provided the molar masses of each oligomer. In order to show the power of the 3D-LAC-NMR-DOSY method, the comparison to 2D-DOSY, 3D-DOSY and LAC-NMR was separately evaluated. Furthermore, the recently published solvent-independent molar mass calibration of diffusion coefficients was also successfully applied in our LAC-DOSY studies for molar mass predictions of the oligomers in acetonitrile. The predicted molar masses were in good agreement with the LAC-DOSY measurements and were verified by calibrations of diffusion coefficients and mass spectrometry. Finally, this pioneering 3D technique offers a powerful new tool for advancing structure analysis and enhancing our understanding of complex systems such as oligostyrenes.

Molar mass determination via linear regression from SEC measurements: The case of polylactide, polycaprolactone and poly(2,2‐dimethylthrimethylene carbonate)

AbstractMolar mass correction coefficients (cc) were determined for calculating the absolute molar masses of polylactide (PLA), polycaprolactone (PCL), and poly(2,2‐dimethyltrimethylene carbonate) (PDTC) in the range of 2,5–190 kg/mol, using calibration based on polystyrene standards (PS). Linear PLAs, PCLs, and PDTCs with a wide range of molar masses were synthesized using tin (II) octoate and diol as a catalyst/initiator system. Size Exclusion Chromatography (SEC) coupled with a Refractive Index detector (SEC‐RI) or a Multi‐Angle Laser Light Scattering detector (SEC‐MALLS) was utilized to determine the relative molar mass or absolute molar mass of the samples under investigation. Additionally, for comparison, the absolute molar mass of low molar mass PLA, PCL, or PDTC was calculated based on end‐group analysis using the 1H NMR method. Correction coefficients were subsequently calculated by comparing the molar mass obtained from SEC‐MALLS and SEC‐RI. The calculated correction coefficients ranged from 0.40 to 0.87 and increased with the rising molar mass of the polymers. The derived equation could be used to evaluate the absolute molar masses from PS standards over a broad range for the analyzed polymers.

Towards the universal use of DOSY as a molar mass characterization tool: temperature dependence investigations and a software tool to process diffusion coefficients

The temperature dependence of DOSY molar mass determination is discussed.

The Universal Calibration for Structure- and Solvent-Independent Molar Mass Determinations of Polymers Using Diffusion-Ordered Spectroscopy.

It will be shown how diffusion-ordered spectroscopy (DOSY) can produce a universal calibration of molar mass dependences of polymers compared to size exclusion chromatography (SEC) or recently published DOSY methods. Whereas SEC can deliver only structure-independent universal calibrations for a particular solvent, DOSY was used for creating solvent-independent calibrations for a certain polymer. Now, we can demonstrate a universal calibration method that generates both a structure- and solvent-independent molar mass calibration. Only one mathematical function describes the structure- and solvent-independent calibrations for DOSY by implementing the Mark-Houwink approach. The derived equation is tested on polystyrene (PS), poly(ethylene oxide), and poly(methyl methacrylate) of different molar masses and in different solvents. Altogether, 94 diffusion coefficients representing 16 molar mass calibrations of the diffusion coefficients in 10 different solvents could be perfectly matched to one universal calibration function with an average deviation of just 2.5%. It was also found that the Mark-Houwink parameters calculated by DOSY are very close to the SEC data. In any case, this new approach is a very useful tool for the determination of molar masses and new Mark-Houwink parameters via DOSY.

New Quaternary Ammonium Derivatives Based on Citrus Pectin.

New citrus pectin derivatives carrying pendant N,N-dimethyl-N-alkyl-N-(2-hydroxy propyl) ammonium chloride groups were achieved via polysaccharide derivatization with a mixture of N,N-dimethyl-N-alkyl amine (alkyl = ethyl, butyl, benzyl, octyl, dodecyl) and epichlorohydrin in aqueous solution. The structural characteristics of the polymers were examined via elemental analysis, conductometric titration, Fourier Transform Infrared spectroscopy (FTIR) and 1D (1H and 13C) nuclear magnetic resonance (NMR). Capillary viscosity measurements allowed for the study of viscometric behavior as well as the determination of viscosity-average molar mass for pristine polysaccharide and intrinsic viscosity ([η]) values for pectin and its derivatives. Dynamic light scattering measurements (DLS) showed that pectin-based polymers formed aggregates in aqueous solution with a unimodal distribution. Critical aggregation concentration (cac) for the hydrophobic pectin derivatives were determined using fluorescence spectroscopy. Atom force microscopy (AFM) images allowed for the investigation of the morphology of polymeric populations obtained in aqueous solution, consisting of flocs and aggregates for crude pectin and its hydrophilic derivatives and well-organized aggregates for lipophilic pectin derivatives. Antimicrobial activity, examined using the disc diffusion method, proved that all polymers were active against Staphylococcus aureus bacterium and Candida albicans yeast.

The significance of sample preparation of historical and contemporary poly(vinyl chloride) objects to investigate the distribution and changes of molar mass by SEC-MALS-dRI

The distribution of molar mass of historical and contemporary poly(vinyl chloride) objects was determined using size exclusion chromatography with multi-angle light scattering detection. The weight average molar mass was determined for a collection of 57 samples, ranging from 75 to 186 kg/mol with a median of 102 kg/mol. Rigid PVC objects were found to have a significantly lower weight average molar mass (Mw = 81 kg/mol) than plasticized ones (Mw = 102 kg/mol). Thin objects also exhibited significantly lower Mw than bulky objects. A substantial presence of aggregates was noted and characterized as an ‘aggregate ratio’ for the entire collection. Heating a solution of poly(vinyl chloride) in THF at 55 °C for 5 h proved successful in the dissociation of the aggregates and allowed for an accurate determination of molar mass. The uncertainty of the Mw determination was statistically evaluated and used to study the effects of accelerated degradation on Mw. No statistically significant changes in Mw were observed in samples artificially aged at 50 °C to 80 °C for up to 15 weeks, indicating that Mw does not decrease during degradation of a PVC heritage object and that accelerated degradation does not lead to polymer crosslinking.

Degradation of PET – Quantitative estimation of changes in molar mass using mechanical and thermal characterization methods

Polyethylene terephthalate (PET) films are used when mechanical strength, thermal and chemical stability, and barrier properties to atmospheric gases are required in combination with good processability. Hydrolysis leading to embrittlement of the material is a major concern as PET is used in a variety of applications with expected lifetimes of up to decades (e.g., for use in buildings, textiles, or photovoltaic backsheets). Therefore, a comprehensive understanding of the degradation processes and the effects on the molecular mass distribution is of great importance.Usually, the direct determination of molar mass and molar mass distribution involves high effort and sophisticated equipment. Therefore, the main objective of this work is to quantify molar mass changes due to accelerated aging using thermal and mechanical methods. Two stabilized PET films were subjected to seven different accelerated aging conditions (heat; combined heat-humidity). The samples were then characterized by size exclusion chromatography (SEC), tensile tests and differential scanning calorimetry (DSC).A linear correlation was found between crystallization temperature and average molar mass. The values of fracture stress from tensile tests indicate a ductile-brittle transition at a molar mass of 15 000 g mol−1. The study concludes that the crystallization temperature obtained from DSC measurements can be used to estimate changes in the average molar mass of PET after hydrolysis. Crystallization temperatures between 208 °C and 211 °C correspond to a critical reduction in molar mass and severe embrittlement.

On the Precise Determination of Molar Mass and Dispersity in Controlled Chain-Growth Polymerization: A Distribution Function-Based Strategy

Accurate information for the molar mass of polymers is a critical item in polymerization kinetic investigation and precision polymer synthesis. Size exclusion chromatography (SEC) is an irreplaceable technique to determine molar mass averages based on conventional calibration, which may nonetheless lead to inaccurate results due to the dissimilarity between the sample and the standard polymer. Herein, a facile distribution function-based strategy was proposed for the precise determination of molar mass average properties, aiming at rectifying the deviation of the standard-equivalent results from the true values. The number-average molar mass (Mn) can be recalculated involving the contribution of each molar mass component beyond the fundamental Mark–Houwink–Sakurada relation. In addition, the as-developed strategy is capable of converting the dispersity from the apparent to the true value, thereby re-estimating the uniformity of the rectified molar mass distribution. The strategy was successfully applied to the linear polymers with medium and low dispersities obtained by two well-controlled chain-growth polymerizations: reversible addition-fragmentation chain transfer polymerization and ring-opening metathesis polymerization, respectively. The rectified Mn closely match the benchmarks (i.e., absolute/theoretical Mn), showing a much higher accuracy than those conventionally calibrated SEC results. Attributing to the consideration of dispersity, the errors of the rectified results can be as low as zero. This work reduces the risk of experimental bias caused by conventionally calibrated SEC analysis to acquire precise mechanism insight and realistic polymerization process details.

Special Aspects of Nitrocellulose Molar Mass Determination by Dynamic Light Scattering.

The dynamic light scattering method was successfully applied to determine the molar mass of nitrocellulose. The methodology of nitrocellulose fractionation in acetonic solutions is described in detail; six polymer fractions with monomodal distribution were obtained. It was shown that the unfractionated colloxylin with polymodal molar mass distribution had mass average molecular mass values of 87.3 ± 14.1, 28.3 ± 7.3, and 0.54 ± 0.17 kDa when investigated by the dynamic light scattering method. The viscometric method only provided integral viscosity average molar mass equal to 56.7 ± 5.8 kDa.

Improvement and Application of the Device for Determination of Molar Mass by Freezing Point Depression

Improvement and Application of the Device for Determination of Molar Mass by Freezing Point Depression

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.

Absolute molar mass determination in mixed solvents. 2. SEC/MALS/DRI in a mix of two “nearly-isovirial” solvents

The accurate determination of polymer molar mass (M) averages and distributions via size-based separation methods employing mixed solvents remains one of the great macromolecular characterization challenges. This is due to the possibility for preferential solvation, whereby the region in the immediate vicinity of the polymer in solution becomes enriched in one of the solvents as compared to this solvent's fractional amount in the mix. In such cases, the chromatographic baselines of differential detectors such as light scattering photometers and differential refractometers no longer provide a quantitative reflection of the solvents' contribution to the heights of individual peak slices, thereby introducing error into molar mass calculations. The problem is addressed here through the use of a “nearly-isovirial” solvent pair. The second virial coefficient (A2) of both solvents being nearly equal for the polymers analyzed means that preferential solvation is obviated. The accuracy of this approach is shown via analysis by size-exclusion chromatography with on-line multi-angle static light scattering and differential refractometry detection (SEC/MALS/DRI), for a trio of narrow-dispersity polystyrene (PS) standards covering a nearly 40-fold range in M. In a mix of two nearly isovirial solvents, namely tetrahydrofuran (THF) and toluene, calculated molar mass averages and distributions are shown to be essentially identical across the range of solvent ratios. Polymer size is likewise shown to be constant with solvent ratio in this scenario. This is contrasted with the same polymers dissolved in a mix of the non-isovirial, non-isorefractive solvents THF and dimethylformamide (DMF). Results from this latter set of experiments show the large error in calculated M that results from preferential solvation, which can be as high as ≈ 1 × 105 g mol−1 for an 8 × 105 g mol−1 narrow-dispersity polystyrene. It is determined that, for a 25:75 THF:DMF mix, the solvent ratio in the immediate vicinity of the polymers examined “flips” to ≈75:25 THF:DMF due to preferential solvation.

Method for Improved Fluorescence Corrections for Molar Mass Characterization by Multiangle Light Scattering.

Multiangle light scattering (MALS) was used to determine the absolute molar mass of fluorescent macromolecules. It is standard protocol to install bandwidth filters before MALS detectors to suppress detection of fluorescent emissions. Fluorescence can introduce tremendous error in light scattering measurements and is a formidable challenge in accurately characterizing fluorescent macromolecules and particles. However, we show that for some systems, bandwidth filters alone are insufficient for blocking fluorescence in molar mass determinations. For these systems, we have devised a correction procedure to calculate the amount of fluorescence interference in the filtered signal. By determining the intensity of fluorescent emission not blocked by the bandwidth filters, we can correct the filtered signal accordingly and accurately determine the true molar mass. The transmission rates are calculated before MALS experimentation using emission data from standard fluorimetry techniques, allowing for the characterization of unknown samples. To validate the correction procedure, we synthesized fluorescent dye-conjugated proteins using an IR800CW (LI-COR) fluorophore and Bovine Serum Albumin protein. We successfully eliminated fluorescence interference in MALS measurements using this approach. This correction procedure has potential application toward more accurate molar mass characterizations of macromolecules with intrinsic fluorescence, such as lignins, fluorescent proteins, fluorescence-tagged proteins, and optically active nanoparticles.

An improved, less erroneous protocol for the classical \u201ccuen\u201d, \u201ccuoxam\u201d or \u201ccadoxen\u201d viscosity measurements of pulps

Correctness and reliability of molar mass data by viscometry in organometallic solvents (cuen, cuoxam, cadoxen) are compromised by the alkalinity of these solvents which causes immediate depolymerization especially in the case of pulps with higher carbonyl content (oxidative damage). The viscosity values thus correspond to the molar mass after the beta-elimination reactions that underly these degradative processes, which is sometimes significantly smaller than the molar mass determined by gel permeation chromatography (GPC) in the non-degrading solvent system DMAc/LiCl. Despite this well-known drawback, viscosity measurements have become a standard approach for molar mass measurements due to their ease and fastness, especially in the pulp and paper industries. A potential way to reduce the inherent error of these molar mass determinations via viscosity measurements is a reductive treatment prior to dissolution of the pulp in the organometallic solvents, which converts the labile, alkali-sensitive carbonyl structures back to the respective alcohols. Using sodium borohydride (NaBH4) on different types of cellulosic pulps, we demonstrate the beneficial effects of such a reduction step on the determined degree of polymerization (DP) for all three common solvents: cuen, cuoxam and cadoxen. Molar mass distributions and profiles of carbonyl groups were determined by GPC and by carbonyl selective fluorescence labeling (“CCOA method”). Such a reductive treatment was especially valuable for hemicellulose-containing pulps. While the decreased measurement error according to the new protocol is beyond doubt, an immediate acceptance in the pulp and paper industries is at least questionable, because the new, more correct data would not agree with the old – wrong, but consistent – numbers accumulated over years and decades. In the long run, however, the new, improved protocol will prevail here as well due to its lower error rate.

Determination of ultrahigh molar mass of polyelectrolytes by Taylor dispersion analysis

Taylor dispersion analysis (TDA) was successfully applied to obtain broadly distributed, ultrahigh molar masses of industrial anionic polyacrylamides (IPAMs) up to 25×106g/mol, far beyond the limits of Size Exclusion Chromatography (SEC) (about 7.3×106g/mol for anionic polyacrylamides standards (APAM)). Two protocols of TDA differing in capillary surface and rinsing procedure were employed: (i) bare fused silica capillaries under intensive between-run rinsing with 1M NaOH, and (ii) fused silica capillaries coated with polyelectrolyte multilayers composed of polydiallyldimethylammonium chloride polycation and sodium polystyrenesulfonate polyanion under simple rinsing with background electrolyte. Both cases led to similar results and in agreement with those obtained by static light scattering, the rinsing capillary step being much shorter in the second case (8min instead of 30min). The data processing of the obtained taylorgrams was realized using multiple-Gaussian fitting of the overall taylorgrams, by separating the contribution of low molar mass impurities from the polymeric profiles, and by determining the mean hydrodynamic radii and diffusion coefficients of the polymers. The molar masses of ultra-high molar mass industrial anionic polyacrylamides (IPAM) were derived from the hydrodynamic radii according to logRh versus logMw linear correlation established with APAM standards. Compared to capillary gel electrophoresis for which the size separation was only feasible up to Mw ∼ 10×106g/mol due to field induced polymer aggregation, TDA largely extended the range of accessible molar mass with easy-to-run and time saving assays.

Characterization of a SARS-CoV-2 spike protein reference material

Development of diagnostic testing capability has advanced with unprecedented pace in response to the COVID-19 pandemic. An undesirable effect of such speed is a lack of standardization, often leading to unreliable test results. To assist the research community surmount this challenge, the National Research Council Canada has prepared a SARS-CoV-2 spike protein reference material, SMT1-1, as a buffered solution. Value assignment was achieved by amino acid analysis (AAA) by double isotope dilution liquid chromatography–tandem mass spectrometry (LC-ID-MS/MS) following acid hydrolysis of the protein, in combination with ultraviolet–visible spectrophotometry (UV–Vis) based on tryptophan and tyrosine absorbance at 280 nm. Homogeneity of the material was established through spectrophotometric absorbance readings at 280 nm. Transportation and long-term storage stabilities were assessed by monitoring relative changes in oligomeric state by size-exclusion liquid chromatography (LC-SEC) with UV detection. The molar concentration of the spike protein in SMT1-1 was 5.68 ± 0.22 µmol L−1 (k = 2, 95% CI), with the native trimeric form accounting for ~ 94% of the relative abundance. Reference mass concentration and mass fraction values were calculated using the protein molecular weight and density of the SMT1-1 solution. The spike protein is highly glycosylated which leads to analyte ambiguity when reporting the more commonly used mass concentration. After glycoprotein molar mass determination by LC-SEC with multi-angle light scattering detection, we thus reported mass concentration values for both the protein-only portion and intact glycoprotein as 0.813 ± 0.030 and 1.050 ± 0.068 mg mL−1 (k = 2), respectively.