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.

Conformation and persistence length of chitosan in aqueous solutions of different ionic strengths via asymmetric flow field-flow fractionation.

Conformation of chitosan in acidic aqueous solutions is strongly influenced by ionic strength, but the conventional employed size exclusion chromatography is limited to high ionic strength. Here we show that conformation of chitosan in acetate buffer down to millimolar ionic strength can be studied via asymmetric flow field-flow fractionation (AF4), where the separation is governed by the diffusion properties of the chitosan molecules and assisted by the electrostatic repulsion of the polyelectrolyte from the channel membrane. The size of chitosan decreases with ionic strength due to increasing screening of the polyelectrolyte effect. The persistence length of chitosan in the solutions, obtained by fitting the conformation plot by the wormlike chain model, decreases linearly with the Debye screening length from 44.5 nm at a salt concentration of 1.25 mM dominated by the electrostatic contribution to 8.6 nm in 800 mM acetate buffer close to its intrinsic persistence length of 7.7 nm.

Improving temperature gradient interaction chromatography of polyolefins by simultaneous use of different stationary phases.

Temperature gradient interaction chromatography (TGIC) at high temperatures is a powerful method for the chemical composition separation of polyolefins. TGIC is a two-step process where the sample is crystallized on the stationary phase at low temperature followed by the elution of the sample components using a temperature gradient towards high temperatures. For TGIC typically a porous graphitic carbon (PGC) stationary phase is used. The separation mechanism is based on crystallization and adsorption/desorption phenomena and it has been shown that co-crystallization and co-adsorption may affect the separation. The present study reports on the simultaneous use of a non-adsorptive and an adsorptive stationary phase (column) in series to utilize both crystallization and adsorption for improved separation in TGIC. A silica column is used as the non-adsorptive support to allow for the crystallization of the polyolefin sample in the absence of an adsorptive force followed by the typical PGC column for adsorption/desorption. Accordingly, the loci of crystallization and adsorption/desorption are well separated from each other and can be adjusted independently. This novel column setup allows the sample to be introduced slowly onto the second (adsorptive) column eliminating possible co-adsorption and poor selectivity. Low molar mass polyethylene comprising of oligomers with approximately C30C130 was used to illustrate the importance of a non-adsorptive column for improved separation. Utilizing a non-adsorptive silica column allows for higher dynamic flow rates during crystallization, which improves separation. Shorter adsorptive columns are found to be more efficient in this experimental protocol as compared to standard TGIC experiments. Smaller PGC column sizes result in reduced longitudinal and Eddy diffusion and, hence, higher resolution of low and high molar mass polyolefins.

Variable temperature asymmetric flow field-flow fractionation for the topology separation of poly(methyl methacrylate).

Size exclusion chromatography is the method of choice for the molar mass analysis of polymers, however, it is not selective towards polymer microstructure. Counterintuitively, asymmetric flow field-flow fractionation (AF4), a channel-based molar mass analysis technique, has been shown to be able to fractionate poly(methyl methacrylate (PMMA) according to tacticity. This was revealed by investigating the solution behaviour of syndiotactic- (s-PMMA), atactic- (a-PMMA) and isotactic (i-PMMA) poly(methyl methacrylate) of similar molecular masses in solvents with different thermodynamic properties. In addition to developing a selective fractionation method, the effects of solvent quality and channel temperature on the fractionation were investigated. It was concluded that the thermodynamic quality of the carrier liquid has a significant influence on the retention behaviour of polymers in AF4. It was shown that the separation of s-PMMA and i-PMMA can be improved by using a theta solvent (acetonitrile) as the carrier liquid instead of thermodynamically good solvents such as tetrahydrofuran and chloroform. In addition, it was found that by using a non-stereocomplexing solvent for PMMA, such as chloroform, blends of s-PMMA and i-PMMA can be separated. Lastly, it was established that the AF4 channel temperature has an influence on the retention behaviour of the PMMA samples. For THF as carrier liquid it was found that the maximum difference in retention between s-PMMA and i-PMMA can be achieved at a channel temperature of 35 °C. In the case of ACN being the carrier liquid, it was observed that the retention behaviour of i-PMMA was significantly more influenced by a change in temperature as compared to s-PMMA and a-PMMA.

Connecting the complex microstructure of LDPE to its rheology and processing properties via a combined fractionation and modelling approach.

Well-defined mini-plant low density polyethylene samples were fractionated preparatively according to their crystallizability via preparative temperature rising elution fractionation and according to molecular weight via preparative solvent gradient fractionation (pSGF). Rheology of the fractions was measured in both the small amplitude oscillatory shear (SAOS) and the non-linear extension regimes. The linear and non-linear rheology of the pTREF fractions were dominated by molecular weight effects, while the impact of the higher degree of long chain branching for the pSGF fractions with higher molecular weights was observed in van Gurp-Palmen plots and in strain hardening behavior in the extensional rheology measurements. Additionally, the experimental fractionation process was mimicked via modelling. The branching topologies of the bulk samples were obtained by coupled kinetic and Monte Carlo calculations. These topologies were fractionated computationally and the result were used to predict the rheological behavior of the individual fractions by applying the BoB algorithm with no parameter adjustment. The experimental observed trends were predicted by the model and the overall agreement was acceptable. This study demonstrates, that polymer fractionation is possible on a preparative scale and allows for the polymer flow properties characterization of the individual fractions, a method that is highly relevant during processing. Moreover, the fractionation process is followed and understood from the modelling point of view.

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.

Two-dimensional fractionation of complex polymers by comprehensive online-coupled thermal field-flow fractionation and size exclusion chromatography.

Thermal field-flow fractionation (ThFFF) was successfully coupled online to size exclusion chromatography (SEC) in a comprehensive two-dimensional configuration. In the first dimension, fractionation according to chemical composition based on the interplay of thermal and translational diffusion took place in the ThFFF channel. Fractions from the first dimension were comprehensively transferred to the second dimension, SEC, and separated according to hydrodynamic volume which is a function of molar mass. To illustrate the capabilities of this novel two-dimensional fractionation approach, polystyrene-poly (methyl methacrylate) block copolymers were comprehensively fractionated and characterized. Blends of homopolymers, homo- and copolymers, and copolymers with different compositions were fractionated and the effects of experimental conditions, the components' molar masses and compositions were investigated. The results illustrated the molar mass independence of the thermal diffusion coefficient in ThFFF. Translational diffusion coefficients were quantitatively determined via dynamic light scattering. The study aimed at proving the versatility of the comprehensive online coupling of ThFFF and SEC for the analysis of complex polymers having the ability to provide detailed molecular information (chemical composition and molar mass distribution) as well thermal and translational diffusion information in a single analysis. Finally, the merits of using information-rich detectors are highlighted.

Chromatographic mode transition from size exclusion to slalom chromatography as observed for chitosan.

Elution behavior of chitosan was studied using narrow-bore size exclusion chromatography (SEC) columns packed with 8 µm porous particles at different eluent flow rates. Delayed elution of the largest chitosan species from the columns was observed at high flow rates, which was attributable to strong chain deformation rather than chain degradation. In particular, a chromatographic mode transition from SEC to slalom chromatography (SC) was observed for high molar mass chitosan samples with abnormal elution behaviors, which originates from the coil-stretch transition of chitosan chains in elongational flow through the columns. By properly reducing the elongational rate in the columns via tuning the flow rate, particle size and column dimension, the chromatographic mode transition from SEC to SC for chitosan can be effectively suppressed.

Retention of polypropylene stereoisomers in solvent gradient interaction chromatography on porous graphitic carbon as influenced by temperature and mobile phase composition.

The behavior of isotactic, syndiotactic and atactic polypropylene stereoisomers on porous graphitic carbon (PGC) at different column temperatures is investigated with 1-decanol, decalin and decane as the adsorption promoting solvents using gradient interaction chromatography (SGIC). Column temperatures between 120 - 180 °C are investigated for the three adsorption promoting solvents with 1,2,4-trichlorobenzene (TCB) as the desorption promoting solvent. Owing to the different stereochemistry of the isomers, their interaction with the atomic level flat surface (ALFS) of porous graphitic carbon is observed to be different when injected from the three different adsorption promoting solvents. Atactic and isotactic polypropylene are not separable when 1-decanol is used as the adsorption solvent but can be separated from syndiotactic PP at column temperatures of between 120 - 180 °C. The three stereoisomers have almost similar elution volumes when decalin is used as the adsorption promoting solvent between 120 - 170 °C. Decane allows for both adsorption and desorption of the stereoisomers at distinct peak elution volumes at the studied column temperatures of 120 - 160 °C. Furthermore, it is shown that increasing the column temperature while maintaining other chromatographic conditions can either decrease or increase retention depending on the adsorption promoting solvent. More importantly, retention is influenced by adsorption conditions (temperature and adsorption promoting solvent) which may affect macromolecular conformations upon injection onto the PGC stationary phase. This is the first study on PP stereoisomers highlighting this behaviour.

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.

Improving chromatographic separation of polyolefins on porous graphitic carbon stationary phases: effects of adsorption promoting solvent and column length.

The chromatographic separation of complex polyolefins on porous graphitic carbon stationary phases is strongly influenced by the composition of the mobile phase. Of particular interest is the effect of the chemical structure of the adsorption promoting solvent as this component of the mobile phase determines the adsorption-desorption behavior of the polyolefin molecules. In a systematic study, alkyl alcohols and linear alkanes are used as adsorption promoting solvents and the effect of the molecules' carbon chain length on chromatographic resolution is investigated. As representative examples, solvent gradient interaction chromatography experiments on polypropylene stereoisomers and ethylene-co-1-octene copolymers are presented. In a further study, the effect of increasing chromatographic column length on the solvent gradient separation of ethylene-co-1-octene copolymers is investigated. In summary, it is shown that the polypropylene stereoisomers are retained in 1-octanol as well as in n-decane and n-dodecane, allowing for identification of the individual stereoisomers in complex blends. For ethylene-co-1-octene copolymers it is shown that separation improves with increasing carbon chain length of the adsorption promoting solvent. Maximum resolution is obtained when a column length of 300 mm is used with 1-dodecanol as the adsorption promoting solvent.

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

In this study, we report a methodology based on reversed phase LC (RP-LC) and hydrophilic interaction chromatography (HILIC) separations coupled to ion mobility (IM) and high resolution mass spectrometry (HR-MS) for the detailed analysis of hydrolysable tannins. The application of this approach to the analysis of an industrial chestnut (Castanea sativa, wood chips) tannin extract is demonstrated. A total of 38 molecular species, including a large number or isomers, were identified in this sample based on HR-MS(E) and UV absorption spectral information as well as retention behaviour in both separation modes. In total, 128 and 90 isomeric species were resolved by RP- and HILIC-LC-IM-TOF-MS, respectively. The combination of low- and high collision energy mass spectral data with complementary chromatographic separations allowed tentative and putative identification of twenty molecular species, comprising 78 isomers, in chestnut for the first time. Ion mobility resolved six new dimeric and trimeric vescalagin conformers with unique arrival (drift) times, including new conformers of roburin A-D which were not separated using either RP-LC or HILIC. HILIC was found to be the preferred separation mode for the analysis of vescalagin derivatives, while RP-LC is preferred for the analysis of ellagitannins with a cyclic glucose core. For the complete separation of the galloyl glucose species, comprehensive HILIC × RP-LC separation would be required.

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 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.

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 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.

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.

A multidimensional fractionation protocol for the oligomer analysis of oxidized waxes.

Oxidized waxes possess far superior properties as compared to the alkanes they are derived from. The separation of alkane oligomers via gas chromatography (GC) becomes a challenge when polar oxygen-containing functional groups are introduced or when higher molar masses are targeted. In the present study, the separation and analysis of oligomers in oxidized and non-oxidized waxes using different liquid chromatographic techniques are investigated. Oligomers in two oxidized waxes and a non-oxidized wax from which they are derived, are separated using high-temperature solvent gradient interaction chromatography (HT-SGIC) and high-temperature two-dimensional liquid chromatography (HT-2D-LC). Evaporative light scattering detector conditions are tailored to provide the best detection with the solvent system at use. It is shown that oligomers in oxidized and non-oxidized waxes can be separated and identified using the mentioned techniques. It has been found that the ELSD detector response systematically decreases as the oxidation levels of the waxes increase. Coupling of HT-HPLC and high-temperature size exclusion chromatography (HT-SEC) in a comprehensive 2D-LC setup shows a broadening of the molar mass distributions of the lower oligomer fractions as a consequence of the modification indicating changes in the oligomer chain microstructures. A preparative fractionation technique is utilized to collect specific oligomer fractions from the bulk waxes followed by hyphenation to HT-HPLC and other techniques. HPLC is shown to provide more detailed information on the oligomer composition of waxes when coupled to a pre-fractionation technique.