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

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

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

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

Size-Exclusion Chromatography: A Twenty-First Century Perspective.

Now in its sixth decade, size-exclusion chromatography (SEC) remains the premier method by which to determine the molar mass averages and distributions of natural and synthetic macromolecules. Aided by its coupling to a variety and multiplicity of detectors, it has also shown its ability to characterize a host of other physicochemical properties, such as branching, chemical, and sequence length heterogeneity size distribution; chain rigidity; fractal dimension and its change as a function of molar mass; etc. SEC is also an integral part of most macromolecular two-dimensional separations, providing a second-dimension size-based technique for determining the molar mass of the components separated in the first dimension according to chemical composition, thus yielding the combined chemical composition and molar mass distributions of a sample. While the potential of SEC remains strong, our awareness of the pitfalls and challenges inherent to it and to its practice must also be ever-present. This Perspective aims to highlight some of the advantages and applications of SEC, to bring to the fore these caveats with regard to its practice, and to provide an outlook as to potential areas for expansion and growth.

Multi-detector hydrodynamic chromatography of colloids: following in Hamish Small's footsteps.

Hamish Small, scientist extraordinaire, is best known as the inventor of both ion chromatography and hydrodynamic chromatography (HDC). The latter has experienced a renaissance during the last decade-plus, thanks principally to its coupling to a multiplicity of physicochemical detection methods and to the structural and compositional information this provides. Detection methods such as light scattering (both multi-angle static and dynamic), viscometry, and refractometry can combine to yield insight into macromolecular or colloidal size, structure, shape, and molar mass, all as a function of one another and continuously across a sample's chromatogram. It was the author's great fortune to have known Hamish during the last decade of his life, before his passing in 2019. Here, a brief personal recollection is followed by an introduction to HDC and its application, in quadruple-detector packed-column mode, to the analysis of a commercial colloidal silica with an elongated shape.

Method development in interaction polymer chromatography.

Interaction polymer chromatography (IPC) is an umbrella term covering a large variety of primarily enthalpically-dominated macromolecular separation methods. These include temperature-gradient interaction chromatography, interactive gradient polymer elution chromatography (GPEC), barrier methods, etc. Also included are methods such as liquid chromatography at the critical conditions and GPEC in traditional precipitation-redissolution mode. IPC techniques are employed to determine the chemical composition distribution of copolymers, to separate multicomponent polymeric samples according to their chemical constituents, to determine the tacticity and end-group distribution of polymers, and to determine the chemical composition and molar mass distributions of select blocks in block copolymers. These are all properties which greatly affect the processing and end-use behavior of macromolecules. While extremely powerful, IPC methods are rarely employed outside academic and select industrial laboratories. This is generally because most published methods are "bespoke" ones, applicable only to the particular polymer being examined; as such, potential practitioners are faced with a lack of inductive information regarding how to develop IPC separations in non-empirical fashion. The aim of the present review is to distill from the literature and the author's experience the necessary fundamental macromolecular and chromatographic information so that those interested in doing so may develop IPC methods for their particular analytes of interest, regardless of what these analytes may be, with as little trial-and-error as possible. While much remains to be determined in this area, especially, for most techniques, as regards the role of temperature and how to fine-tune this critical parameter, and while a need for IPC columns designed specifically for large-molecule separations remains apparent, it is hoped that the present review will help place IPC methods in the hands of a more general, yet simultaneously more applied audience.

Absolute molar mass determination in mixed solvents. 1. Solving for the SEC/MALS/DRI "trivial" case.

Size-exclusion chromatography (SEC) with on-line static light scattering, specifically multi-angle static light scattering (MALS), and differential refractometry (DRI) detection remains the premier method by which to determine absolute, calibrant-independent molar masses of polymers. The method is restricted to the use of either neat solvents or solvents with a small amount of additive. In mixed solvents, preferential solvation (i.e., the enrichment, within the solvated volume of the polymer in solution, of one solvent over the other as compared to the solvent ratio outside said volume) leads to errors in the areas of the MALS and DRI chromatograms, as the solvent baseline does not accurately represent the solvent contribution to these detectors' peaks. A seemingly trivial way by which to overcome this problem is through the use of an isorefractive solvent pair. This "trivial" solution is complicated by the fact that the solvents in the pair must be miscible with each other in all proportions; the individual solvents as well as the mix must be able to fully dissolve the analyte; the solvents must possess sufficient optical contrast with the solution so as to generate an adequate detector signal; the solvent mix must be compatible with the chromatographic stationary phase, such that enthalpic contributions to the separation are minimal and analyte recovery from the columns is quantitative; and the difference in the Rayleigh factors of the solvents can be ignored. Herein, we present the analysis of narrow dispersity polystyrene (PS) and poly(methyl methacrylate) (PMMA) samples, across a four-fold range in molar mass, using SEC/MALS/DRI in a mix of tetrahydrofuran (THF) and methyl isoamyl ketone (MIAK), solvents which are shown to be isorefractive with each other at the temperature and wavelength of the experiments. Molar mass averages and dispersities are demonstrated to be statistically independent of solvent composition and to correspond well to the values in neat THF. The experiments were augmented by the use of on- and off-line quasi-elastic light scattering and of off-line MALS and DRI, to study the effect of solvent composition on polymer size in solution and on dilute solution thermodynamics. Additionally, 1H nuclear magnetic resonance spectroscopy was used to study the effect of tacticity on the insolubility of PMMA100 in 100% MIAK. We believe this constitutes the first example of obtaining accurate molar masses of polymers by SEC/MALS/DRI employing mixed solvents. The value of these experiments to other forms of macromolecular liquid chromatographic separations is also noted.

Detection Orthogonality in Macromolecular Separations. 2: Exploring Wavelength Orthogonality and Spectroscopic Invisibility Using SEC/DRI/UV/FL.

We continue herein the exploration of detector orthogonality in size-based macromolecular separations. Previously [5], the sensitivity of viscometric detection was juxtaposed to that of differential refractometry (DRI) and light scattering (LS, both static and dynamic), and it was shown that viscometry is a truly orthogonal detection method to both DRI and LS. Here, via the size-exclusion chromatography (SEC) analysis of blends of polystyrene and poly(methyl methacrylate), we demonstrate the orthogonality of DRI to UV detection and, within the UV region of the electromagnetic spectrum, we also explore the phenomenon of "wavelength orthogonality:" Analytes observable by one detection method are shown to be spectroscopically invisible to another method, or even to the same detection method when operating at a different wavelength. While generally focusing on blends of analytes of different molar masses (different sizes in solution), we also investigate the less-explored case of blends of coeluting analytes (same sizes in solution) where detector orthogonality can inform one's knowledge of whether or not coelution has occurred. Finally, by incorporating a fluorescence (FL) detector into the experimental set-up, we demonstrate not only its orthogonality to DRI detection but also its sensitivity to the presence of even minor (≈ 1%) fluorescent components in a sample. We hope the present experiments assist in understanding the complementarity of different spectroscopic detection methods and also help highlight the potential role of FL detection, a method which has been largely overlooked in macromolecular separation science.

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

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

Aqueous size-exclusion chromatography of polyelectrolytes on reversed-phase and hydrophilic interaction chromatography columns.

The size-exclusion separation of a water-soluble polyelectrolyte polymer, sodium polystyrene sulfonate (NaPSS), was demonstrated on common reversed-phase (C18, C4, phenyl, and cyano) and hydrophilic interaction chromatography (HILIC) columns. The effect of common solvents - acetonitrile (ACN), tetrahydrofuran (THF), and methanol (MeOH), used as mobile phase modifiers - on the elution of NaPSS and the effect of column temperature (within a relatively narrow range corresponding to typical chromatographic conditions, i.e., 10 °C-60 °C) on the partition coefficient, KSEC, were also investigated. Non-size-exclusion chromatography (non-SEC) effects can be minimized by the addition of an electrolyte and an organic modifier to the mobile phase, and by increasing the column temperature (e.g., to 50 °C or 60 °C). Strong solvents such as THF and ACN are more successful in the reduction of such effects than is the weaker solvent MeOH. The best performance is seen on medium polarity and polar stationary phases, such as cyanopropyl- and diol-modified silica (HILIC), where the elution of the NaPSS polyelectrolyte is by a near-ideal SEC mechanism. Hydrophobic stationary phases, such as C18, C4, and phenyl, require a higher concentration of a strong solvent modifier (THF) in the mobile phase to reduce non-SEC interactions of the solute with the stationary phase.

Organic solvent modifier and temperature effects in non-aqueous size-exclusion chromatography on reversed-phase columns.

Common reversed-phase columns (C18, C4, phenyl, and cyano) offer inert surfaces suitable for the analysis of polymers by size-exclusion chromatography (SEC). The effect of tetrahydrofuran (THF) solvent and the mixtures of THF with a variety of common solvents used in high performance liquid chromatography (acetonitrile, methanol, dimethylformamide, 2-propanol, ethanol, acetone and chloroform) on reversed-phase stationary phase characteristics relevant to size exclusion were studied. The effect of solvent on the elution of polystyrene (PS) and poly(methyl methacrylate) (PMMA) and the effect of column temperature (within a relatively narrow range corresponding to typical chromatographic conditions, i.e., 10°C-60°C) on the SEC partition coefficients KSEC of PS and PMMA polymers, were also investigated. The bonded phases show remarkable differences in size separations when binary mixtures of THF with other solvents are used as the mobile phase. The solvent impact can be two-fold: (i) change of the polymeric coil size, and possible shape, and (ii) change of the stationary phase pore volume. If the effect of this impact is properly moderated, then the greatest benefit of optimized solute resolution can be achieved. Additionally, this work provides an insight on solvent-stationary phase interactions and their effects on column pore volume. The only effect of temperature observed in our studies was a decreased elution volume of the polymers with increasing temperature. SEC partition coefficients were temperature-independent in the range of 10°C-60°C and therefore, over this temperature range elution of PS and PMMA polymers is by near-ideal SEC on reversed-phase columns. Non-ideal SEC appears to occur for high molar mass PMMA polymers on a cyano column when alcohols are used as mobile phase modifiers.

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

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

Quantitative characterization of gold nanoparticles by size-exclusion and hydrodynamic chromatography, coupled to inductively coupled plasma mass spectrometry and quasi-elastic light scattering.

The physicochemical characterization of nanoparticles (NPs) is of paramount importance for tailoring and optimizing the properties of these materials as well as for evaluating the environmental fate and impact of the NPs. Characterizing the size and chemical identity of disperse NP sample populations can be accomplished by coupling size-based separation methods to physical and chemical detection methods. Informed decisions regarding the NPs can only be made, however, if the separations themselves are quantitative, i.e., if all or most of the analyte elutes from the column within the course of the experiment. We undertake here the size-exclusion chromatographic characterization of Au NPs spanning a six-fold range in mean size. The main problem which has plagued the size-exclusion chromatography (SEC) analysis of Au NPs, namely lack of quantitation accountability due to generally poor NP recovery from the columns, is overcome by carefully matching eluent formulation with the appropriate stationary phase chemistry, and by the use of on-line inductively coupled plasma mass spectrometry (ICP-MS) detection. Here, for the first time, we demonstrate the quantitative analysis of Au NPs by SEC/ICP-MS, including the analysis of a ternary NP blend. The SEC separations are contrasted to HDC/ICP-MS (HDC: hydrodynamic chromatography) separations employing the same stationary phase chemistry. Additionally, analysis of Au NPs by HDC with on-line quasi-elastic light scattering (QELS) allowed for continuous determination of NP size across the chromatographic profiles, circumventing issues related to the shedding of fines from the SEC columns. The use of chemically homogeneous reference materials with well-defined size range allowed for better assessment of the accuracy and precision of the analyses, and for a more direct interpretation of results, than would be possible employing less rigorously characterized analytes.

Size-exclusion chromatography of metal nanoparticles and quantum dots.

This review presents an overview of size-exclusion chromatographic separation and characterization of noble metal nanoparticles (NPs) and quantum dots (QDs) over the past 25 years. The properties of NPs and QDs that originate from quantum and surface effects are size dependent; to investigate these properties, a separation technique such as size-exclusion chromatography (SEC) is often needed to obtain narrow distribution NP populations that are also separated from the unreacted starting materials. Information on the size distributions and optical properties of NPs have been obtained by coupling SEC to detection methods such as ultraviolet-visible and/or fluorescence spectroscopy. Problems associated with the sorption of NPs and QDs onto various SEC stationary phases, employing both aqueous and organic eluents, are also discussed here.

Determining the core, corona, and total size of CdSeS/ZnS quantum dots by SEC/QELS and TEM.

The size (hydrodynamic or Stokes radius, R H) of non-functionalized CdSeS/ZnS (core/shell) quantum dots (QDs) was characterized by size-exclusion chromatography with on-line quasi-elastic light scattering (SEC/QELS). Accurate determination of the size of QDs is important, because many of the optical properties of these materials are size dependent. A clear advantage of SEC/QELS over many batch techniques (e.g., QELS without separation) is the capability of the hyphenated technique to characterize the entire size range of a disperse sample, rather than merely providing a statistical average of the sizes present. Here, the SEC/QELS-determined R H values of CdSeS/ZnS QDs are compared to those determined by a traditional SEC experiment employing a calibration curve based on polystyrene standards, providing for the first reported study on SEC/QELS of non-functionalized QDs while also demonstrating the shortcomings of the widely-employed calibration curve approach. Furthermore, combining the R H of the QDs obtained by SEC/QELS with core size measurements derived from transmission electron microscopy allowed further calculation of the size of the QDs' coronas. The latter result was found to be in close agreement to the previously measured dimension of the main corona constituent, as well as with the calculated size of this constituent.

Polysaccharide characterization by hollow-fiber flow field-flow fractionation with on-line multi-angle static light scattering and differential refractometry.

Accurate characterization of the molar mass and size of polysaccharides is an ongoing challenge, oftentimes due to architectural diversity but also to the broad molar mass (M) range over which a single polysaccharide can exist and to the ultra-high M of many polysaccharides. Because of the latter, many of these biomacromolecules experience on-column, flow-induced degradation during analysis by size-exclusion and, even, hydrodynamic chromatography (SEC and HDC, respectively). The necessity for gentler fractionation methods has, to date, been addressed employing asymmetric flow field-flow fractionation (AF4). Here, we introduce the coupling of hollow-fiber flow field-flow fractionation (HF5) to multi-angle static light scattering (MALS) and differential refractometry (DRI) detection for the analysis of polysaccharides. In HF5, less stresses are placed on the macromolecules during separation than in SEC or HDC, and HF5 can offer a higher sensitivity, with less propensity for system overloading and analyte aggregation, than generally found in AF4. The coupling to MALS and DRI affords the determination of absolute, calibration-curve-independent molar mass averages and dispersities. Results from the present HF5/MALS/DRI experiments with dextrans, pullulans, and larch arabinogalactan were augmented with hydrodynamic radius (RH) measurements from off-line quasi-elastic light scattering (QELS) and by RH distribution calculations and fractogram simulations obtained via a finite element analysis implementation of field-flow fractionation theory by commercially available software. As part of this study, we have investigated analyte recovery in HF5 and also possible reasons for discrepancies between calculated and simulated results vis-à-vis experimentally determined data.

HISTORY OF CHROMATOGRAPHY Hamish Small: Experimenter Extraordinaire.

Hamish Small spoke to André Striegel about the secrets of his success in separation science, including the development of ion chromatography, and the value of vague thoughts in scientific progress.

AF4/MALS/QELS/DRI characterization of regular star polymers and their "span analogs".

Asymmetric flow field-flow fractionation (AF4) coupled with multi-angle static and quasi-elastic light scattering and differential refractive index detectors, was employed for the separation and characterization of regular star-shaped polystyrenes and their linear and span analogs in tetrahydrofuran. Stars with different arm lengths were separated from each other by employing a binary slope cross-flow gradient. Cross-flow optimization enabled fast separation of polystyrenes in two- and three-component blends. Macromolecular parameters were obtained by using light-scattering and refractive index detection, and properties of polystyrenes with different molecular architectures were compared. To our knowledge, this is the first report on the separation of star polymers by AF4. Novel characterization approaches for stars are important from both applied and fundamental standpoints, as these macromolecules are valued for their tribological, drug delivery, catalytic and coating capabilities, and also serve as model compounds for the structured study of long-chain branching and its effects in polymers.

Influence of glycosidic linkage on the solution conformational entropy of gluco- and mannobioses.

Employing size-exclusion chromatography, an entropically-controlled separation technique, we have determined the solution conformational entropy (-ΔS) of (1→2)-, (1→3)-, (1→4)-, and (1→6)-linked gluco- and mannobioses with an α anomeric configuration, at quasi-physiological conditions of solvent, temperature, and pH. The experiments allowed for comparison both among and between each series of disaccharides. Results included quantitative information on how the additional degrees of freedom of the (1→6) linkage influence -ΔS, as well as on the influence on solution conformational entropy of a single axial hydroxyl (OH) group and of the relative positioning of the glycosidic linkage and the anomeric hydroxyl group. We also contrasted the (1→4)-α-D-linked gluco- and mannobioses to their counterparts with a ß anomeric configuration. Comparison between (1→4)-ß-D-linked glucobiose (cellobiose) and (1→4)-ß-D-linked mannobiose showed that the restrictive effect on solution flexibility of the axial OH in the latter disaccharide is offset by the combined effect of hydroxyl group orientation and anomeric configuration on intramolecular hydrogen bonding.