Stereochemical Heterogeneity Analysis of Polylactides by Multidimensional Liquid Chromatography.

A new and robust high-performance liquid chromatography (HPLC) method that separates poly(lactic acid) (PLA) according to its stereochemical composition is presented. Using this method, poly(l-lactide) incorporating trace amounts of meso-lactide resulting from the racemization is separated from the pristine polymer. To prove this aspect in more detail, a representative poly(l-lactic acid) standard, assumed to be highly homogeneous, was separated using this method. The result showed that this was not the case as a fraction incorporating meso-lactide due to racemization occurring during the synthesis is separated. Employing two-dimensional liquid chromatography (2D-LC), the molar mass differences of the separated species were investigated, and fractions with similar molecular sizes were detected, confirming that the LC separation is solely based on stereochemical heterogeneity. The sample was further fractionated by preparative HPLC, followed by an in-depth analysis of the fractions using homonuclear decoupling in proton nuclear magnetic resonance (1H NMR). Convincing results that unveiled significant differences in the stereochemistry of the isolated PLA fractions were obtained. Subsequent analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) also confirmed oligomer series with different end group structures, indicating that the applied HPLC method is very sensitive to minor variations in stereochemistry and end groups. This integrated approach offers detailed insight into the structural characteristics of PLA polymers, contributing to a better understanding of their composition and potential applications.

Advanced tools for molecular characterization of bio-based and biodegradable polymers.

Bio-based and biodegradable materials play a vital role in a sustainable and green economy. These materials must exhibit properties that are similar to or better than the properties of oil- or coal-based materials and require sophisticated synthesis technologies and detailed knowledge of structure-property correlations. For comprehensive molecular structure elucidation, advanced analytical methods, including coupled and hyphenated techniques that combine advanced fractionation and information-rich spectroscopic detectors, are an indispensable tool. One important tool for fractionating complex polymers regarding molecular size is size exclusion chromatography. For fractionating polymers with regard to chemical composition, solvent (or temperature) gradient HPLC has been developed. The combination of different liquid chromatography methods in comprehensive two-dimensional HPLC setups is another important tool. Today, a toolbox of HPLC methods is in place that enables the fractionation of complex bio-based and biodegradable polymers according to the most important molecular parameters including molecular size, composition, functionality, and branching. Here, an overview of the different techniques and some major applications is presented. Some representative developments in the field are discussed, and different techniques, experimental protocols, and applications are highlighted.

Advanced Liquid Chromatography of Polyolefins Using Simultaneous Solvent and Temperature Gradients.

Olefin copolymers are complex polymer materials that exhibit multiple distributions in molecular properties such as molar mass, chemical composition, and branching. To address the multivariate molecular compositions, chromatographic protocols have been developed that synergistically combine solvent and temperature gradients. As representative examples, blends of olefin copolymers have been fractionated on porous graphitic carbon stationary phases. This is the first study that makes complementary use of solvent and temperature gradient interaction chromatography (SGIC and TGIC, respectively) to capitalize on the advantages of both techniques. In a first experimental setup, solvent and temperature gradients were used simultaneously and complex blends of low molar mass polyethylene and ethylene-co-1-octene copolymers were separated with high efficiency. The separation of oligomers was observed to be significantly better in SGIC as compared to TGIC, while comonomer blends could be separated in either TGIC or SGIC mode. In another innovation, a two-column setup was employed where the columns were placed in different temperature zones. It was demonstrated that the separation of both low and high comonomer content blends was improved significantly when the temperatures of the two zones were manipulated reasonably.

Characterization of Complex Branched Polymers by Multidetector Thermal Field-Flow Fractionation.

Size exclusion chromatography (SEC) for the molar mass analysis of polymers is of limited use for the analysis of branched polymers due to the co-elution of linear and branched molecules with similar hydrodynamic sizes but different molar masses. Thermal field-flow fractionation (ThFFF) in combination with multiple detection methods is a versatile alternative to SEC due to the fact that fractionation is not based entirely on molecular size but the interplay of thermal and translational diffusion that depend on molecular topology and molecular size. Multidetector ThFFF is used to investigate the correlation of branching and molar mass by determining polymer conformations from Mark-Houwink plots and the degrees of branching using functionality plots. The suitability of this approach is demonstrated for a set of 3-, 4-, and 6-arm star polystyrenes as well as a more complex hyperbranched polybutadiene-polystyrene copolymer. It is shown that ThFFF is a powerful tool for the analysis of the radius of gyration and the hydrodynamic radius when coupled online to static light scattering, viscometry, and dynamic light scattering. Shape factors are evaluated as influenced by branching where narrow dispersed star polystyrenes are used as model systems for the analysis of the more complex hyperbranched polybutadiene-polystyrene copolymer.

Thermal Field-Flow Fractionation with Quintuple Detection for the Comprehensive Analysis of Complex Polymers.

With a constantly increasing complexity of macromolecular structures, advanced polymer analysis faces new challenges with regard to the comprehensive analysis of these structures. Today it goes without saying that comprehensive polymer analysis requires selective and robust fractionation methods in combination with a set of information-rich detectors. Thermal field-flow fractionation (ThFFF) has proven to be a powerful technique for the fractionation of complex polymers as well as polymer assemblies. In the present study, ThFFF is coupled to a set of five detectors to simultaneously provide quantitative information on a number of important molecular parameters, including molar mass, molecular size, chemical composition, molecular topology, intrinsic viscosity, and normal and thermal diffusion coefficients. The five-detector setup includes a triple detector device (multiangle light scattering (MALS), differential refractive index (dRI), and differential viscometer (dVis)) that is coupled to an ultraviolet (UV) detector for dual concentration detection and an online dynamic light scattering (DLS) detector. Triple detection consisting of MALS, dRI, and dVis provides information on molar mass, molecular size, and molecular topology. Dual concentration detection offers compositional analysis from a combination of UV and dRI detectors, whereas DLS provides information on diffusion coefficients and hydrodynamic radii. The power of this novel quintuple detector ThFFF (ThFFF-QD) is documented for three important fields of application, namely, the comprehensive analysis of (1) linear and star-shaped polymers, (2) hydrogenated and deuterated polymers, and (3) block copolymer self-assemblies. These applications highlight the novel approach of determining the most relevant molecular parameters, including Mark-Houwink and conformation plots, simultaneously in a single experiment.

Comprehensive branching analysis of star-shaped polystyrenes using a liquid chromatography-based approach.

The comprehensive branching analysis of complex polymers is still a challenge in advanced polymer analysis. Average branching information (average number and length of branches) can be obtained by spectroscopic methods, mainly NMR spectroscopy. The determination of the branching distribution, i.e., the concentration of macromolecules with a given number of branches, however, requires fractionation. Typically, size exclusion chromatography is used that separates the complex mixture with regard to molecular size in solution and not strictly with regard to the number of branches. In the present approach, model star-shaped polystyrenes were synthesized with a pre-determined architecture to give theoretical three-arm, four-arm, and six-arm structures. The branched samples were compared with a linear analogue of comparable molar mass known not to contain branching. Triple detector size exclusion chromatography with refractive index, multiangle light scattering, and online viscometer detection was used to determine absolute molar masses, radii of gyration, and branching distributions of the star-shaped polymers. 1H-NMR was used to calculate the average functionality and a reasonable agreement between the results of the two methods was obtained. Thermal gradient interaction chromatography and solvent gradient interaction chromatography were employed to separate the complex reaction products according to chemical composition (number of branches) and to resolve by-products. The separation capabilities of the two chromatographic techniques were compared and evaluated. Comprehensive two-dimensional liquid chromatography was used to separate the polydisperse star-shaped polystyrenes with regard to both branching and molar mass. Graphical abstract.

Comprehensive analysis of tara tannins by reversed-phase and hydrophilic interaction chromatography coupled to ion mobility and high-resolution mass spectrometry.

Reversed-phase liquid chromatography (RP-LC) and hydrophilic interaction chromatography (HILIC) methods hyphenated to diode array detection and ion mobility (IM) high-resolution mass spectrometry (HR-MS) were used for the analysis of gallic acid derivatives and gallotannins in a commercial tara extract. UV spectra combined with low and high-collision energy mass spectral data and known RP-LC elution orders allowed the identification of 45 isomeric gallic acid derivatives and gallotannins. The synergy between IM and UV data was found to provide a simple means to determine the number of depsidic bonds and thus to distinguish between positional isomers. IM also facilitated the assignment of individual isomeric species between HILIC and RP-LC separations. For the hydrolysable tannins present in tara, RP-LC provided superior resolution and specificity compared to HILIC. The results reported in this paper highlight the utility of IM in combination with optimised complementary chromatographic separations and HR-MS for the detailed qualitative analysis of hydrolysable tannins in complex mixtures of these compounds. Graphical abstract.

Comprehensive two-dimensional liquid chromatography for the characterization of acrylate-modified hyaluronic acid.

Hyaluronic acid and its acrylate derivatives are important intermediates for various pharmaceutical, biomedical, and cosmetic applications due to their biocompatibility and viscoelasticity properties. However, these polymers are inherently difficult to characterize due to their significant heterogeneity regarding molar mass and chemical composition (degree of substitution, DS). The present study describes the development of a comprehensive online two-dimensional liquid chromatography (2D-LC) approach to characterize hyaluronic acid and its acrylate derivatives (DS ranging from 0.4 to 3.1) in terms of molar mass and degree of substitution. In the first dimension of the 2D-LC method, separation according to chemical composition/DS was achieved by using a stepwise solvent gradient and a reversed phase C8 column. Fractions from the first dimension were automatically transferred to the second dimension comprising size exclusion chromatographic separation of the fractions according to molar mass. It was found that the hyaluronic acid derivatives were broadly distributed with regard to both chemical composition and molar mass. Fractions with different degrees of substitution were identified, and their molar mass distributions were determined. The study proved that comprehensive 2D-LC is a powerful approach to reveal the complex nature of hyaluronic acid and its derivatives. Graphical abstract.

Comprehensive Analysis of Oxidized Waxes by Solvent and Thermal Gradient Interaction Chromatography and Two-Dimensional Liquid Chromatography.

This report addresses the comprehensive analysis of oxidized/functionalized polyethylene waxes according to chemical composition and molar mass by selective chromatographic methods. For the first time, tailored high-temperature interaction chromatography in solvent gradient (HT-SGIC) and thermal gradient (HT-TGIC) modes are used for the chemical composition separation of these materials. Separation protocols are developed using three model wax samples with different degrees of oxidation. For the chromatographic separations polar silica gel is used as the stationary phase. Solvent gradients of decane and cyclohexanone are used in HT-SGIC at 110 °C to separate the bulk waxes into several heterogeneous fractions according to polarity and the type of functionality. Column temperature and gradient manipulation are shown to influence chromatographic resolution and retention. The HT-SGIC investigations are complemented by HT-TGIC separations where a solvent mixture of decane and cyclohexanone is used as the mobile phase in isocratic mode. It is shown that HT-SGIC and HT-TGIC provide different types of separation, however, both are predominantly based on differences in functionality. To provide comprehensive information on chemical composition (functionality) and molar mass, HT-SGIC and HT-TGIC are coupled to HT-SEC, using ortho-dichlorobenzene as the second dimension mobile phase. Clear differences between oxidized and nonoxidized waxes are detected in HT-2D-LC providing comprehensive information on the molecular heterogeneity of these materials.

Stereocomplexation of Polymers in Micelle Nanoreactors As Studied by Multiple Detection Thermal Field-Flow Fractionation.

In this study the thermally induced in situ stereocomplexation (SC) of binary blends of symmetrical isotactic and syndiotactic polymethymethacrylates (i- and s-PMMAs) inside micellar nanoreactors (MNR) with polystyrene (PS) shells is investigated using thermal field-flow fractionation (ThFFF) as a separation technique. The MNRs are prepared from three systematic binary blending ratios of pure micelles of i-PMMA-PS and s-PMMA-PS in a nonsolvent for PMMAs in order to produce mixed micelles with a binary microstructural composition of the interior PMMA cores. The SC of these stereoregular PMMA cores inside the MNRs is shown to be thermally induced as a function of annealing temperatures from room temperature up to 150 °C. This SC is initially confirmed by Fourier transform infrared spectroscopy (FTIR), whereby signal shifts are observed in the carbonyl absorption regions. These signal shifts are as a result of SC interactions between adjacent ester and alpha-methyl groups. Furthermore, temperature-dependent dynamic light scattering (DLS) results show that MNRs with more SC domains have a much more drastic reduction in size. Additional corroboration is provided by multiple detection ThFFF results which show significant differences in retentions and sizes between MNRs with stereocomplexed cores and those without. Ratios of i-PMMA:s-PMMA in the order of 1:1, 1:2, and 2:1 were investigated with all ratios exhibiting thermal annealing induced SC, of which the 1:2 ratio is shown to have the highest predisposition to SC.

Comprehensive Three-Dimensional LC × LC × Ion Mobility Spectrometry Separation Combined with High-Resolution MS for the Analysis of Complex Samples.

Comprehensive two-dimensional liquid chromatography (LC × LC) and ion mobility spectrometry-mass spectrometry (IMS-MS) are increasingly being used to address challenges associated with the analysis of highly complex samples. In this work, we evaluate the potential of the combination of these techniques in the form of a comprehensive three-dimensional LC × LC × IMS separation system. As application, hydrophilic interaction chromatography (HILIC) × reversed phase LC (RP-LC) × IMS-high-resolution MS (HR-MS) was used to analyze a range of phenolic compounds, including hydrolyzable and condensed tannins, flavonoids, and phenolic acids in several natural products. A protocol for the extraction and visualization of the four-dimensional data obtained using this approach was developed. We show that the combination of HILIC, RP-LC, and IMS offers excellent separation of complex phenolic samples in three dimensions. Benefits associated with the incorporation of IMS include improved MS sensitivity and mass-spectral data quality. IMS also provided separation of trimeric procyanidin isomeric species that could not be differentiated by HILIC × RP-LC or HR-MS. On the traveling wave IMS (TWIMS) system used here, both IMS separation performance and the extent of second dimension (2D) undersampling depend on the upper mass scan limit, which might present a limitation for the analysis of larger molecular ions. The performance of the LC × LC × IMS system was characterized in terms of practical peak capacity and separation power, using established theory and taking undersampling and orthogonality into account. An average increase in separation performance by a factor of 13 was found for the samples analyzed here when IMS was incorporated into the HILIC × RP-LC-MS workflow.

Separation of hydrophobically modified hyaluronic acid according to the degree of substitution by gradient elution high performance liquid chromatography.

Amphiphilic hyaluronic acid (HA), synthesised by modifying HA to varying extents with acrylate groups, was successfully separated according to degree of substitution (DS) using solvent gradient high performance liquid chromatography (HPLC). Two HPLC methods based on the amphiphilic structure of the HA were developed. In the first approach, normal phase gradient HPLC was explored, and separation was based on the interactions of HA's polar hydroxyl groups with a polar cyano stationary phase. In the second approach, separation was based on the interaction of the hydrophobic acrylate moieties with a non-polar C8 stationary phase (reversed phase gradient HPLC). The separation was optimised by using an electrolyte in the sample solvent to suppress non-covalent interactions and improve the selectivity of the developed method. The photolytic stability of the modified and unmodified HA was also investigated in order to optimise the sample preparation procedure. Furthermore, an alternative method to NMR spectroscopy was developed for determining the DS of HA. Graphical abstract ᅟ.

Chemical Composition Fractionation of Olefin Plastomers/Elastomers by Solvent and Thermal Gradient Interaction Chromatography.

Olefin plastomers/elastomers are typically copolymers with high comonomer contents and low crystallinities. Therefore, the fractionation of these materials with crystallization-based methods is not feasible. On the other hand, solvent and temperature gradient interaction chromatography (SGIC and TGIC, respectively) are suitable techniques for the separation of olefin copolymers with regard to their chemical composition. In this study, the application ranges of both techniques are investigated and compared for ethylene-propylene (EP) copolymers. A linear dependency of ethylene content versus elution volume is obtained with SGIC in practically the whole ethylene range. In the case of TGIC, a linear dependency is obtained within certain ethylene content limits. The accessible ethylene content separation range for TGIC is 50-100 mol% ethylene, and a broader 26-100 mol% ethylene range is accessible for SGIC, the latter being the technique of choice in the analysis of EP rubbers.

Characterization of Complex Polymer Self-Assemblies and Large Aggregates by Multidetector Thermal Field-Flow Fractionation.

Micelles prepared from amphiphilic block copolymers (ABCs) have found numerous applications in pharmaceutical, electronics, environmental, cosmetics, and hygiene industries. These micelles, whether in the pure or mixed micelle form, often exist as multiple morphologies (spherical, cylindrical, worm, or vesicular) in equilibrium with each other. However, none of the current column-based fractionation techniques or any microscopic technique are capable of a successful separation, identification, and quantitation of these complex self-assemblies with regards to morphology, size, molar mass, and chemical composition in one experiment. Multidetector thermal field-flow fractionation (ThFFF) is shown to be capable of separating and characterizing not only pure micelles but also mixed micelles prepared from polystyrene-poly(ethylene oxide) ABCs. In addition, multidetector ThFFF is demonstrated to be capable of successfully characterizing multiple micellar morphological evolutions (induced by the addition of an electrolyte) and thus showcasing the potential of this novel approach to monitor the formation of polymer self-assemblies with multiple and complex morphological distributions.

Tacticity separation of poly(methyl methacrylate) by multidetector thermal field-flow fractionation.

For the first time, thermal field-flow fractionation (ThFFF) has been used for the separation of poly(methyl methacrylate) (PMMA) with regard to molecular microstructure. PMMA exists in three different isomeric forms, namely, isotactic, syndiotactic, and atactic. ThFFF analysis of the different PMMA isomers in tetrahydrofuran, acetonitrile (ACN), and dioxane reveals that isomers with similar molecular weights exhibit different Soret coefficients, and thus different retention times, under identical experimental conditions. Of the three solvents, ACN shows the greatest influence on fractionation of the isomers. The separation according to molecular microstructure is found to be based on the cooperative effects of the normal and thermal diffusion coefficients. Furthermore, it is found that blends of different PMMA isomers with similar molecular weights can be fractionated into their respective isomeric components. The distribution of the isomeric content in an atactic PMMA sample is determined quantitatively by fractionating the sample with ThFFF and subsequently analyzing the fractions by (1)H NMR. The isomeric distributions determined from NMR data correlate well with ThFFF retention data of the samples and thus further highlight the unique fractionating capabilities of ThFFF.

On the multimodality of preparative TREF fractionation as detected by advanced analytical methods.

Preparative temperature rising elution fractionation (prepTREF) is the standard technique for the preparative fractionation of polyolefins according to crystallisability. For olefin copolymers such as linear low-density polyethylene (LLDPE), it was believed that the TREF elution temperature correlates directly with the copolymer composition. For copolymers having different bulk comonomer contents, the prepTREF fractions of different samples collected at a given temperature were assumed to have the same chemical composition. It was acknowledged quite early that co-crystallisation effects may disturb TREF fractionation and fractions are obtained that are not completely homogeneous. This, however, has not been investigated quantitatively so far. The fundamental statement of prepTREF is challenged for the first time quantitatively using advanced analytical techniques including high-temperature high-performance liquid chromatography (HPLC). Ethylene-1-octene copolymers having bulk comonomer contents ranging from 0.3 to 6.4 mol% were fractionated by prepTREF, and the fractions were analysed by high-temperature size exclusion chromatography, crystallisation analysis fractionation, differential scanning calorimetry and high-temperature HPLC. All analytical results prove that the TREF fractions collected from different samples at the same elution temperature have different chemical compositions. The chemical compositions of the fractions correlate with the compositions of the bulk samples in that the comonomer contents of similar TREF fractions increase with an increase of the comonomer content of the bulk samples. These results are in clear contrast to the previous assumption that the TREF fraction composition is independent of the bulk copolymer composition for a given copolymer type. Graphical abstract Advanced analysis of LLDPE by combination of HT-HPLC, DSC and CRYSTAF.

Characterization of complex phthalic acid/propylene glycol based polyesters by the combination of 2D chromatography and MALDI-TOF mass spectrometry.

The combination of gradient HPLC, 2D chromatography, and MALDI-TOF MS facilitated the analysis of the various distributions of phthalic acid/propylene glycol-based model polyesters. Investigations of kinetic samples taken at various reaction times highlighted the subsequent differences at various stages of the polyesterification reaction in terms of molecular weight, chemical composition, and endgroups. Normal-phase gradient-HPLC analysis successfully enabled an oligomeric separation of the respective samples. Peak-splitting behavior in early eluting peaks suggested that the separation was affected by a combination of factors and not solely based on chemical composition, functionality type or degree of polycondensation. Two-dimensional chromatography provided the link between chemical composition and molecular weight distribution, confirming that the first dimension gradient HPLC separation was based on chemical composition with increasing degree of oligomerization in the second dimension. The off-line coupling of LAC with MALDI-TOF MS provided structural details in combination with improved molecular weight determination of the more homogeneous LC fractions. The study indicated that all aspects related to the model saturated anhydride system should be considered in the case of copolyester synthesis to produce industrial type polyester resins. It was shown that the present multidimensional approach provided most comprehensive structural information on the polyester system.

Comprehensive Microstructure and Molar Mass Analysis of Polybutadiene by Multidimensional Liquid Chromatography.

For the first time, polybutadiene is separated according to microstructure using solvent gradient interaction chromatography (SGIC). Superior separation of polybutadienes having different microstructures is obtained on a silica-based reversed stationary phase and a mobile phase of acetone-hexane. This SGIC system enables the baseline separation of 1,2-polybutadienes and 1,4-polybutadienes even in cases where the samples have similar molar masses. 2D liquid chromatography is performed with the SGIC method separating according to microstructure in the first dimension coupled to size exclusion chromatography separating according to molar mass in the second dimension, thus providing comprehensive information on both microstructure and molar mass.

Fractionation of Poly(butyl methacrylate) by Molecular Topology Using Multidetector Thermal Field-Flow Fractionation.

Thermal field-flow fractionation (ThFFF) is an interesting alternative to column-based fractionation being able to address different molecular parameters including size and composition. Until today it has not been shown to be able to fractionate polymers of similar molar masses and chemical compositions by molecular topology. The present study demonstrates that poly(butyl methacrylates) with identical molar masses can be fractionated by ThFFF according to the topology of the butyl group. The influence of the solvent polarity on the thermal diffusion behavior of these polymers is presented and it is shown to have a significant influence on the fractionation of poly(n-butyl methacrylate) and poly(t-butyl methacrylate). Fractionation improves with increasing solvent polarity and solvent polarity may have a greater influence on fractionation than solvent viscosity. It is found that the thermal diffusion coefficient, D(T), as well as the hydrodynamic diameter, D(h), exhibit increasing trends with increasing solvent polarity. The solvent quality has a significant influence on the fractionation. It is found that cyclohexane, being a theta solvent for poly(t-butyl methacrylate) but not for poly(n-butyl methacrylate), significantly improves the fractionation of the samples by decreasing the diffusion rate of the former but not the latter.

Preparative solution crystallization fractionation: a simple and rapid fractionation method for the chemical composition separation of complex ethylene-propylene copolymers.

The molecular structure elucidation of complex ethylene-propylene copolymers (EPCs) has benefited tremendously from the ability to combine preparative temperature rising elution fractionation (prep TREF) with various conventional analytical techniques. Recently reported, prep TREF-high-temperature solvent gradient interaction chromatography (HT-SGIC) (Cheruthazhekatt et. al, Macromolecules 45:2025-2034, 2012) is one of the most effective and highly useful coupled methods that allow for the exact measurement of the chemical composition distribution (CCD) present in various components of EPCs. The major drawback of prep TREF involving slow crystallization and elution steps is the long time per experiment. Here, we present a new and by far the simplest and fastest preparative fractionation method for complex polyolefins-preparative solution crystallization fractionation (prep SCF). The scope of the present study was to achieve a fast fractionation of complex bulk samples into an amorphous, semicrystalline and highly crystalline fraction, in sufficient amounts for the subsequent detailed compositional analysis. The effects of two different solvents, xylene and trichlorobenzene (TCB), and their influence on the solution crystallization of chemically different components of EPC were systematically investigated by combining prep SCF with crystallization analysis fractionation (CRYSTAF), FTIR, differential scanning calorimetry (DSC) and HT-SGIC analyses. Significant differences in the chemical composition of similar SCF fractions obtained from xylene and TCB were observed indicating the strong influence of the solvent on solution crystallization. Prep SCF-HT-SGIC results showed that, under similar experimental conditions, TCB as the fractionation solvent provides superior separation of complex semicrystalline ethylene-propylene (EP) components. Very interestingly, for the first time, separation of soluble fractions (30 °C) of iPP, EPC and PE homopolymer components in complex EPC was achieved by prep SCF in TCB. On the other hand, SCF fractionation in xylene provides a soluble fraction that is perfectly amorphous as has been shown by DSC and CRYSTAF. Based on these results, the present SCF approach and an updated method of the combination of prep SCF-HT-SGIC hold significant promise for the fractionation and characterization of similar complex EPCs in a simple way within a short analysis time, by using significantly smaller amounts of solvent compared to the previously reported, rather time-consuming, prep TREF-HT-SGIC combination. No similarly selective solution crystallization fractionations in preparative scale have been reported before.