Preparative Size Exclusion Chromatography Research Articles

Comprehensive chromatographic profiling and structural analysis of key anticoagulant components in enoxaparin

Heparin is the most widely used anticoagulant in clinical practice, with enoxaparin being one of the most important low molecular weight heparins (LMWHs). In this study, an antithrombin III (ATIII) affinity column was used. Enoxaparin and its oligosaccharides of varying sizes, prepared using preparative size exclusion chromatography (SEC), were fractionated through the ATIII affinity column. The different affinity fractions from each oligosaccharide size were profiled using strong anion exchange (SAX) chromatography. Each peak was automatically transferred to an SEC column for desalting prior to mass spectrometry (MS) analysis, which enabled structural identification using a multiple heart-cut (MHC) 2D LC-MS system (SAX-SEC-MS). The high-affinity fraction from enoxaparin was further analyzed using the MHC 2D LC system (SEC-SAX). SAX profiles of the high-affinity oligosaccharides, prepared by both size and affinity fractionation, were consistent with those obtained by direct SEC-SAX analysis. The possible sequences of several high-affinity hexasaccharides and the domain compositions of high-affinity octa- and decasaccharides in enoxaparin were further elucidated by disaccharide analysis after manual collection of the oligosaccharides. This work advances the understanding of enoxaparin's structural features and offers a potential approach to improve the quality of enoxaparin, as well as to identify key structural motifs in heparin/LMWHs that contribute to protein binding.

Electron microscopy of stable electrophoretic fractions of natural humic acids – the key to the understanding of their structural organization

Transmission electron microscopy (TEM) with contrast staining by uranyl acetate solution was used to study morphological differences between soil humic acids (HAs) and their A, B and C + D fractions obtained using coupling preparative low-pressure size exclusion chromatography and analytical polyacrylamide gel electrophoresis. The electrophoretic mobility of fractions varied in order C + D B A. The distribution of various morphological elements between fractions showed that large structures such as vesicle-like formations 70–150 nm long and 30–80 nm wide with clear edges were found exclusively in fraction A and occupied ~55% of the TEM image area. On the other hand, long fibrils with a length of 60–100 nm, a width of 4–6 nm and a thickness of 2–3 nm, as well as their bundles with a length of 150 nm and a diameter of 30–70 nm were identified only in the C + D fraction and occupied ~59 % area of TEM images. Smaller morphological elements such as point particles with a diameter of 2–3 nm, ring particles with a diameter of 4–6 nm, worm-shaped short particles with a length of 20–30 nm, and spheroids with a diameter of 10– 30 nm were observed in all samples, but in varying quantities. Significant morphological differences between the fractions can be explained by their composition, previously established by using a few physico-chemical methods. The ratio Car(165–108 ppm)/Calk(108–0 ppm), or aromaticity index, calculated from 13C-NMR, could be one of the indicators of the various morphological structures formation. The obtained TEM results clearly confirm the supramolecular organization of soil HAs.

4,9- and 4,10-Substituted pyrenes: synthesis, successful isolation, and optoelectronic properties.

We report herein a way to prepare and purify optoelectronic functional 4,9- and 4,10-substituted pyrene isomers. By tuning the size of substituents, the designed 4,9- and 4,10-isomers can be successfully isolated by recycling preparative size-exclusion chromatography (SEC) and/or repeated recrystallization. The structure and purity of the isolated compounds 1-5 have been confirmed by 1H NMR, 13C NMR, and HRMS. The photophysical and electrochemical properties of compounds 1-5 have been studied in detail both experimentally and theoretically. The lowest transitions of these pyrenes, 1-5, are allowed, with moderate to high fluorescence quantum yields and radiative decay rates around 108 s-1. The differences between the electrochemical and photophysical properties of 4,9-, 4,10-, 1,6-, and 2,7-substituted isomers are compared and concluded.

Difficult-to-express antigen generation through a co-expression and disassociation methodology.

Extracellular domain (ECD) antigens are crucial components for antibody discovery, in vitro assays, and epitope mapping during therapeutical antibody development. Oftentimes, those antigens are difficult to produce while retaining the biologic function/activity upon extracellular secretion in commonly used expression systems. We have developed an effective method to cope with the challenge of generating quality antigen ECDs. In this method, a monoclonal antibody (Mab) or antibody fragment antigen-binding (Fab) region acts as a "chaperone" to stabilize the antigen ECD through forming an antibody:antigen complex. This methodology includes transient co-expression of the complex in Chinese hamster ovary cells and then dissociation of the purified complex into individual components by low pH treatment in the presence of arginine. The antigen is then separated from the chaperone on a preparative size exclusion chromatography (pSEC) followed by an optional affinity chromatography process to remove residual Mab or Fab. We demonstrate this co-expression/disassociation methodology on two difficult-to-express antigen ECDs from cluster-of-differentiation/cytokine family and were successful in producing stable, biologically active antigens when the common methods using Histidine-tagged and/or Fc-fused protein failed. This can be applied as a general approach for antigen production if a Mab or binding partner is available.

Unfolding of Lignin Structure Using Size-Exclusion Fractionation.

The heterogeneous and recalcitrant structure of lignin hinders its practical application. Here, we describe how new approaches to lignin characterization can reveal structural details that could ultimately lead to its more efficient utilization. A suite of methods, which enabled mass balance closure, the evaluation of structural features, and an accurate molecular weight (MW) determination, were employed and revealed unexpected structural features of the five alkali lignin fractions obtained with preparative size-exclusion chromatography (SEC). A thermal carbon analysis (TCA) provided quantitative temperature profiles based on sequential carbon evolution, including the final oxidation of char. The TCA results, supported with thermal desorption/pyrolysis gas chromatography-mass spectrometry (TD-Py-GC-MS) and 31P NMR spectroscopy, revealed the unfolding of the lignin structure as a result of the SEC fractionation, due to the disruption of the interactions between the high- and low-MW components. The "unraveled" lignin revealed poorly accessible hydroxyl groups and showed an altered thermal behavior. The fractionated lignin produced significantly less char upon pyrolysis, 2 vs. 47%. It also featured a higher occurrence of low-MW thermal evolution products, particularly guaiacol carbonyls, and more than double the number of OH groups accessible for phosphitylation. These observations indicate pronounced alterations in the lignin intermolecular association following size-exclusion fractionation, which may be used for more efficient lignin processing in biorefineries.

Microwave-Assisted Synthesis of Poly[bis(4-phenyl)(2,4,6-trimethylphenyl)amine] (PTAA) with Well-Defined End Groups and Narrow Dispersity

A series of PTAA (poly[bis(4-phenyl)(2,4,6-trimethylphenyl)amine]) polymers were synthesized using a Ni(0)-mediated Yamamoto coupling. Polymers with different molecular weights and only one series of well-defined end groups were obtained with various amounts of a tailormade endcapper. This level of structural uniformity has not yet been described for PTAA. By using microwave heating, it was possible to shorten the reaction time from at least 1 day to 30 min. The synthetic strategy allows the separation of single PTAA oligomers with up to 6 repeating units and polymer fractions with low dispersities from 1.06 to 1.17 by using preparative size exclusion chromatography. The carrier mobilities of the PTAA oligomers and polymers were derived from organic field-effect transistors. Mobilities increase with increasing molecular weight of the PTAAs and are higher compared to commercially available PTAA samples.

Transmission electron microscopy of electrophoretic humic acids fractions obtained by coupling size exclusion chromatography-polyacrylamide gel electrophoresis: The next step to understanding structural organization of soil humic matter

The transmission electron microscopy (TEM) with contrast staining by uranyl acetate solution was used to study morphological differences between soil humic acids (HAs) and their A, B and C + D fractions obtained using coupling preparative low-pressure size exclusion chromatography and analytical polyacrylamide gel electrophoresis. The electrophoretic mobility of fractions varied in order C + D > B > A. The distribution of various morphological elements between the bulk HAs and their fractions clearly showed that larger structures, such as smeared drops (70–150 nm long), were formed exclusively in fraction A, whereas elongated fibers (60–100 nm long) and their bundles (>150 nm long) were found only in fraction C + D. Smaller morphological elements such as point particles (2–3 nm in diameter), ring particles (4–6 nm in diameter) with hole in the center, worm-like short particles (20–30 nm long) and spheroids (10–30 nm in diameter) were observed in all samples, but in varying quantities. Significant morphological differences between the samples can be explained by their contrasting properties obtained earlier by different physical–chemical methods. The ratio Car(165–108 ppm)/Calk (108–0 ppm) or aromaticity derived from 13C NMR data appears to be one of the indicators for the formation of different morphological structures. This study provides the new information about morphological structures of soil organic matter that are consistent with the formation of different supramolecular aggregates, probably controlled by the association of smaller ring particles. The data obtained can clarify the role of humic acids in the environment and their involvement in physical–chemical processes in soils.

Co-aggregation between whey proteins and carotenoids from yellow mombin (Spondias mombin): Impact of carotenoids’ self-aggregation

The interaction between whey proteins and carotenoid is reported to improve carotenoid solubility and stability, however, the strong trend of carotenoids to aggregate when in polar systems is often neglected in papers addressing their molecular interaction. Therefore, this study focused on characterizing the aggregative behavior of the carotenoids from yellow mombin (Spondias mombin) and to understand how these carotenoids behave when added to aqueous dispersions of whey proteins. Carotenoids-rich extract, containing mainly β-cryptoxanthin and lutein, was obtained from freeze-dried yellow mombin pulp and its aggregative behavior in ethanol/water medium was studied. By increasing the medium polarity, carotenoids trend to form J-aggregation, causing a drop in the color intensity of the solution. When added to whey protein aqueous dispersions, rather than a protein-carotenoid bimolecular interaction, the formation of co-aggregates between carotenoids and whey proteins was evidenced by preparative size exclusion chromatography. These results may contribute to the developing functional food products.

Net anionic poly(β-amino ester)s: synthesis, pH-dependent behavior, and complexation with cationic cargo.

As hydrolytically-labile, traditionally-cationic polymers, poly(β-amino ester)s (PBAEs) adeptly complex anionic compounds such as nucleic acids, and release their cargo as the polymer degrades. To engineer fully-degradable polyelectrolyte complexes and delivery vehicles for cationic therapeutics, we sought to invert PBAE net charge to generate net anionic PBAEs. Since PBAEs can carry up to a net charge of +1 per tertiary amine, we synthesized a series of alkyne-functionalized PBAEs that allowed installation of 2 anionic thiol-containing molecules per tertiary amine via a radical thiol-yne reaction. Finding dialysis in aqueous solution to lead to PBAE degradation, we developed a preparative size exclusion chromatography method to remove unreacted thiol from the net anionic PBAEs without triggering hydrolysis. The net anionic PBAEs display non-monotonic solution behavior as a function of pH, being more soluble at pH 4 and 10 than in intermediate pH ranges. Like cationic PBAEs, these net anionic PBAEs degrade in aqueous environments with hydrophobic content-dependent hydrolysis, as determined by 1H NMR spectroscopy. Further, these net anionic PBAEs form complexes with the cationic peptide (GR)10, which disintegrate over time as the polymer hydrolyzes. Together, these studies outline a synthesis and purification route to make previously inaccessible net anionic PBAEs with tunable solution and degradation behavior, allowing for user-determined complexation and release rates and providing opportunities for degradable polyelectrolyte complexes and cationic therapeutic delivery.

Increase in cysteine-mediated multimerization under attractive protein–protein interactions

The CASPON enzyme became an interesting enzyme for fusion protein processing because it generates an authentic N-terminus. However, the high cysteine content of the CASPON enzyme may induce aggregation via disulfide-bond formation, which can reduce enzymatic activity and be considered a critical quality attribute. Different multimerization states of the CASPON enzyme were isolated by preparative size exclusion chromatography and analyzed with respect to multimerization propensity and enzymatic activity. The impact of co-solutes on multimerization was studied in solution and in adsorbed state. Furthermore, protein–protein interactions in the presence of different co-solutes were measured by self-interaction chromatography and were then correlated to the multimerization propensity. The dimer was the most stable and active species with 50% higher enzymatic activity than the tetramer. Multimerization was mainly governed by a cysteine-mediated pathway, as indicated by DTT-induced reduction of most caspase multimers. In the presence of ammonium sulfate, attractive protein–protein interactions were consistent with those observed for higher multimerization when the cysteine-mediated pathway was followed. Multimerization was also observed under attractive conditions on a chromatographic stationary phase. These findings corroborate common rules to perform protein purification with low residence time to avoid disulfide bond formation and conformational change of the protein upon adsorption.

A New Approach for Increasing Speed, Loading Capacity, Resolution, and Scalability of Preparative Size-Exclusion Chromatography of Proteins

Low speed, low capacity, and poor scalability make size-exclusion chromatography (SEC) unattractive for use in the preparative separation of proteins. We discuss a novel z2 cuboid SEC device that addresses these challenges. A z2 cuboid SEC device (~24 mL volume) was systematically compared with a conventional SEC column having the same volume and packed with the same resin. The primary objective of this study was to use the same volume of SEC medium in a much more efficient way by using the novel device. At any given flow rate, the pressure drop across the z2 cuboid SEC device was lower by a factor of 6 to 8 due to its shorter bed height and greater cross-sectional area. Under overloaded conditions, the peaks obtained during protein separation with the conventional column were poorly resolved and showed significant fronting, while those obtained with the z2 cuboid SEC device were much better resolved and showed no fronting. At any given flow rate, better resolution was obtained with the z2 cuboid SEC device, while for obtaining a comparable resolution, the flow rate that could be used with the z2 cuboid SEC device was higher by a factor of 2 to 3. Hence, productivity in SEC could easily be increased by 200 to 300% using the z2 cuboid SEC device. The scalability of the z2 cuboid SEC device was also demonstrated based on a device with a 200 mL bed volume.

Application of Quantum Cascade Laser-Infrared Spectroscopy and Chemometrics for In-Line Discrimination of Coeluting Proteins from Preparative Size Exclusion Chromatography.

An external-cavity quantum cascade laser (EC-QCL)-based flow-through mid-infrared (IR) spectrometer was placed in line with a preparative size exclusion chromatography system to demonstrate real-time analysis of protein elutions with strongly overlapping chromatographic peaks. Two different case studies involving three and four model proteins were performed under typical lab-scale purification conditions. The large optical path length (25 μm), high signal-to-noise ratios, and wide spectral coverage (1350 to 1750 cm–1) of the QCL-IR spectrometer allow for robust spectra acquisition across both the amide I and II bands. Chemometric analysis by self-modeling mixture analysis and multivariate curve resolution enabled accurate quantitation and structural fingerprinting across the protein elution transient. The acquired concentration profiles were found to be in excellent agreement with the off-line high-performance liquid chromatography reference analytics performed on the collected effluent fractions. These results demonstrate that QCL-IR detectors can be used effectively for in-line, real-time analysis of protein elutions, providing critical quality attribute data that are typically only accessible through time-consuming and resource-intensive off-line methods.

Hybrid Arborescent Polypeptide-Based Unimolecular Micelles: Synthesis, Characterization, and Drug Encapsulation.

This paper reports novel hybrid arborescent polypeptides based on poly(γ-benzyl l-glutamate)-co-poly(γ-tert-butyl l-glutamate)-g-polysarcosine [P(BG-co-Glu(OtBu))-g-PSar]. The synthesis is launched by ring-opening polymerization (ROP) of N-carboxyanhydride of γ-benzyl l-glutamate (BG-NCA) and γ-tert-butyl l-glutamate (Glu(OtBu)-NCA) to synthesize a random copolymer P(BG-co-Glu(OtBu)) serving as a precursor for the arborescent system, followed by deprotection of the tert-butyl (tBu) groups to afford free COOH moieties serving as coupling sites. Two copolymerization reactions were carried out to afford the side chains. One type of side chain was a random copolymer P(BG-co-Glu(OtBu)), while the other type was a triblock copolymer PGlu(OtBu)-b-PBG-b-PGlu(OtBu). The peptide coupling reactions were conducted between the COOH moieties on the precursor and the terminus amine on the chain end of the P(BG-co-Glu(OtBu)) random copolymer or the PGlu(OtBu)-b-PBG-b-PGlu(OtBu) triblock copolymer to obtain G0 polymers. Afterward, hydrolyzing the tBu moieties of the G0 substrates yielded randomly functionalized G0 and end-functionalized G0. Randomly functionalized G0 was used as a substrate for the next generation G1 (randomly functionalized and end-functionalized G1 after deprotection) or coated with polysarcosine (PSar) to gain G0-g-PSar. The G0 substrate prepared with the triblock copolymer PGlu(OtBu)-b-PBG-b-PGlu(OtBu) was only grafted with PSar after deprotection, resulting in G0-eg-PSar. Depending on the functionality mode of the G1 substrate, the PSar coating yielded two different graft polymers, G1-g-PSar and G1-eg-PSar, for randomly functionalized and end-functionalized G1, respectively. The PSar hydrophilic shell was decorated with the sequence of (arginine, glycine, and aspartic acid) tripeptides (RGD) as a targeting ligand to improve the potentiality of the arborescent unimolecular micelles as drug carriers. Preparative size exclusion chromatography (SEC) was used to fractionate these complex macromolecular architectures. Nuclear magnetic resonance (NMR), Fourier-transform infrared (FTIR), Raman spectroscopy, and SEC were used for molecular characterization of all intermediate and final products and dynamic light scattering (DLS), transmission electron microscopy (TEM), and atomic force microscopy (AFM) for micellar characterization. A comparison between randomly grafted (g) and end-grafted (eg) unimolecular micelles demonstrates that the former has an undefined core-shell structure, unlike its end-grafted analog. In addition, this study has proved that decoration of the shell with RGD contributed to avoiding micelle aggregation but limited chemotherapy agent encapsulation. However, more than their naked analog, the sustained release was noticeable in decorated micelles. Doxorubicin was utilized as a chemotherapy model, and loading was achieved successfully by physical entrapment.

Multi angle light scattering as a process analytical technology tool for real-time monitoring of molar mass of protein-polysaccharide conjugate fractions

Current polysaccharide conjugate vaccine manufacturing process involves conjugation of polysaccharide with protein followed by separation/purification of the conjugate from reaction components and free polysaccharide/protein, by preparative Size-Exclusion Chromatography (SEC). Each SEC fraction is analyzed and pooled the conjugate based on its molar mass and absence of free polysaccharide. In the present work, we have evaluated in-line Multi Angle Light Scattering (MALS) as a Process Analytical Technology (PAT) tool to eliminate the need for offline analysis of individual fractions after purification. The preparatory SEC unit is coupled to a MALS detector, i.e. preparatory SEC-MALS. In SEC, the molecules are separated based on size, where polysaccharide-protein conjugate elutes first followed by free polysaccharide and then free protein. The MALS detector monitors in-line weight-average molar mass (Mw) during SEC-fractionation. Except the first fraction, the molar mass distribution data from in-line MALS enables decision on preparative SEC fractions to be pooled. We found that offline analysis of preparative fractions using analytical SEC-MALS was in good agreement with the >2000 kDa cutoff criterion. Thus in-line process analysis is effective, time-saving, and simple to implement in manufacturing processes of conjugate vaccines.

High-Resolution Profiling of the Functional Heterogeneity of Technical Lignins.

In technical lignins, functionality is strongly related to molar mass. Hence, any technical lignin exhibits concurrent functionality-type distribution (FTD) along its molar mass distribution (MMD). This study combined preparative size-exclusion chromatography with offline characterizations to acquire highly resolved profiles of the functional heterogeneity of technical lignins, which represent crucial information for their material use. The shape of these profiles showed considerable dissimilarity between different technical lignins and followed sigmoid trends. Determining the dispersity in functionality (ĐF) of lignins via their FTD revealed a rather homogeneous distribution of their functionalities (ĐF of 1.00–1.21). The high resolution of the acquired profiles of functional heterogeneity facilitated the development of a robust calculation method for the estimation of functional group contents of lignin fractions based simply on their MMD, an invaluable tool to simulate the effects of intended purification processes. Moreover, a more thorough evaluation of separations based on functionality becomes accessible.

Increase in Speed, Loading Capacity, and Resolution of Preparative Size Exclusion Chromatography Using a Z2 Cuboid Packed-Bed Device

Increase in Speed, Loading Capacity, and Resolution of Preparative Size Exclusion Chromatography Using a Z2 Cuboid Packed-Bed Device

Glasses‐shaped triblock copolymer prepared by combination of atom transfer radical polymerization and ring opening polymerization

AbstractA glasses‐shaped triblock copolymer of poly(ε‐caprolactone)‐b‐polystyrene‐b‐poly(ε‐caprolactone) (PCL‐b‐PS‐b‐PCL) is prepared by combining atom transfer radical polymerization (ATRP) and ring opening polymerization (ROP). Polystyrene (PS) star polymers are prepared via ATRP using a tetra‐functional initiator, followed by azidation to yield azide end‐functionalized star polymers. An alkyne‐functionalized coupling agent, 2,2‐bis[(2‐propyn‐1‐yloxy)methyl]‐1‐propanol is employed to produce hydroxy 8‐shaped PS via copper(I)‐catalyzed alkyne‐azide cycloaddition. Herein, hydroxy 8‐shaped PS with high purity is obtained through preparative size exclusion chromatography (Prep SEC) and high‐performance liquid chromatography, followed by the characterizations using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS), size exclusion chromatography (SEC), infrared, and proton nuclear magnetic resonance (1H NMR) spectroscopy. The hydroxy groups of the 8‐shaped PS are utilized as initiators for the ROP of ε‐caprolactone to obtain linear chains attached to the 8‐shaped architecture. After SEC fractionation, the glasses‐shaped triblock copolymer is characterized using 1H NMR and SEC. This unprecedented topology possesses two free chain‐ends and two cycles; thus, both the properties of linear and cyclic polymers may be expected to be observed.

Electrospray Ionization-Mass Spectrometry of Synthetic Polymers Functionalized with Carboxylic Acid End-Groups.

Electrospray ionization-mass spectrometry (ESI-MS) of low-charging synthetic polymers typically produces mass spectra exhibiting a bias toward the low-mass region of the polymer mass distribution. To examine the origin(s) of this ionization bias, narrow dispersity polystyrene polymers (Đ < 1.10) were prepared with ionizable carboxylic acid end-groups at one or both chain termini. The mixture complexity was further reduced through preparative size-exclusion chromatography (SEC), and these well-defined polymers were subjected to negative ion ESI-MS on a high-resolution instrument with a mass-to-charge (m/z) range up to 8000. Incorporation of one carboxylic acid end-group facilitated the generation of singly charged [M - H]- ions across the entire range of the mass analyzer. The comparison of mass spectra with size-exclusion chromatograms of the same polymer revealed an ionization bias toward lower masses, which was partially overcome through fractionation, modification of electrospray solvent, and increased declustering potentials. Incorporation of a second ionizable moiety within polymers of equivalent size facilitated multiply charged [M - 2H]2- ion formation with significantly improved ionization efficiency, spectral coverage of the molar mass distribution, and minimal cluster ion formation. These findings indicate that increased charging of polymers through multiple, well-defined sites of ionization can enhance volatilization and ionization of higher-mass polymers. Generation of higher-molecular-weight polymers in low-charge states-while possible under ideal conditions-competes ineffectively with either nonspecific, multiple-charging of similar sized polymers or ionization of the smaller polymers in the distribution.

Sustainable valorization of meat processing wastewater with synergetic eutectic mixture based purification of R-Phycoerythrin from porphyrium cruentium

Wastewater effluent from meat processing industries are enriched with nutrients but remain underutilized. Therefore, an efficient method was administered by employing this resource for the cultivation of Porphyreum cruentium. The cultured biomass was exposed to a one-step disruption and selective purification method using a protic deep eutectic solvent to obtain a commercially significant pigment, R-Phycoerythrin (R-PE). Six protic deep eutectic solvents (pDES) were synthesized and their thermophysical activity determined. A synergy between microwave and cavitation (MACE-DLPME) has been achieved for the effective recovery of these membrane proteins. The addition of pDES provides the selective medium for the concentration of R-PE. Optimization of the MACE-DLPME method yields 95.9% (w/w) of R-PE. Preparative size exclusion chromatography resulted in (28 µg/g) of R-PE. Further ultra-purification by anion exchange chromatography enhances the purity fold of R-PE to 125. The resulting ultrapure fraction exhibits enhanced anti-platelet activity (1.56 mg/g ascorbic acid equivalent).

Macromolecular Model of the Pectic Polysaccharides Isolated from the Bark of Norway Spruce (Picea abies).

The bark of Norway spruce (Picea abies) contains up to 13% pectins that can be extracted by pressurized hot water, which constitute a valuable renewable resource in second-generation lignocellulosic biorefineries. This article proposes, for the first time, structural molecular models for the pectins present in spruce bark. Pectin fractions of tailored molar masses were obtained by fractionation of the pressurized hot water extract of the inner bark using preparative size-exclusion chromatography. The monosaccharide composition, average molar mass distribution, and the glycosidic linkage patterns were analyzed for each fraction. The pectin fraction with high molecular weight (Mw of 59,000 Da) contained a highly branched RG-I domain, which accounted for 80% of the fraction and was mainly substituted with arabinan and arabinogalactan (type I and II) side chains. On the other hand, the fractions with lower molar masses (Mw = 15,000 and 9000 Da) were enriched with linear homogalacturonan domains, and also branched arabinan populations. The integration of the analytical information from the macromolecular size distributions, domain composition, and branch lengths of each pectin fraction, results in a comprehensive understanding of the macromolecular architecture of the pectins extracted from the bark of Norway spruce. This paves the way for the valorization of spruce bark pectic polymers in targeted applications based on their distinct polymeric structures and properties.