Publications by authors named "André M Striegel"

47 Publications

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

Heliyon 2021 Apr 27;7(4):e06691. Epub 2021 Apr 27.

Chemical Sciences Division, National Institute of Standards and Technology (NIST), 100 Bureau Drive, MS 8392, Gaithersburg, MD 20899-8392 USA.

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.
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http://dx.doi.org/10.1016/j.heliyon.2021.e06691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8102424PMC
April 2021

Method development in interaction polymer chromatography.

Trends Analyt Chem 2020 ;130

Chemical Sciences Division, National Institute of Standards & Technology (NIST), 100 Bureau Drive, MS 8390, Gaithersburg, MD, 20899-8390, USA.

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.
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http://dx.doi.org/10.1016/j.trac.2020.115990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919746PMC
January 2020

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

Anal Chim Acta 2019 Apr 5;1053:186-195. Epub 2018 Dec 5.

Chemical Sciences Division, National Institute of Standards and Technology (NIST), 100 Bureau Drive, MS 8392, Gaithersburg, MD, 20899-8392, USA.

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, H 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.
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http://dx.doi.org/10.1016/j.aca.2018.11.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6604610PMC
April 2019

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

Chromatographia 2019 ;83(1)

Chemical Sciences Division, National Institute of Standards and Technology (NIST), 100 Bureau Drive MS 8392, Gaithersburg, MD 20899, USA.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7539643PMC
January 2019

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

Chromatographia 2018 ;81

Process Research and Development, Merck and Co., Inc., Rahway, NJ 07065, USA.

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 (∂/∂) from the other component, chemical heterogeneity translates into ∂/∂ heterogeneity. The latter, in turn, affects the accuracy of the molar mass () 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---butyl methacrylate) copolymer. Also determined were the chemical-heterogeneity-corrected 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 calculations is seen to reach as high as 53,000 g mol at the high end of the MMD. This error could be much higher, however, in copolymers with higher or with larger difference among component ∂/∂ values, as compared to the current analyte.
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http://dx.doi.org/10.1007/s10337-018-3512-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605080PMC
January 2018

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

J Chromatogr A 2018 Jan 6;1532:161-174. Epub 2017 Dec 6.

Chemical Sciences Division, National Institute of Standards and Technology (NIST), 100 Bureau Drive, MS 8392, Gaithersburg, MD, 20899‑8392, USA.

The size-exclusion separation of a water-soluble polyelectrolyte polymer, sodium polystyrene sulfonate (NaPSS), was demonstrated on common reversed-phase (C C, 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, K, 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 C, C, 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.
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http://dx.doi.org/10.1016/j.chroma.2017.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605059PMC
January 2018

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

J Chromatogr A 2018 Jan 24;1531:83-103. Epub 2017 Nov 24.

Chemical Sciences Division, National Institute of Standards and Technology (NIST), 100 Bureau Drive, MS 8392, Gaithersburg, MD 20899-8392, USA.

Common reversed-phase columns (C, C, 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 K 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.
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http://dx.doi.org/10.1016/j.chroma.2017.11.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6604611PMC
January 2018

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

Chromatographia 2017 Jun 23;80(6):989-996. Epub 2017 Mar 23.

Chemical Sciences Division, National Institute of Standards and Technology (NIST), 100 Bureau Drive, MS 8392, Gaithersburg, MD USA 20899-8392.

The specific refractive index increment (∂/∂) is an essential datum for the accurate quantitation of molar mass averages and distributions () 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 ∂/∂ 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 ∂/∂ 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 ∂/∂ 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.
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http://dx.doi.org/10.1007/s10337-017-3294-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5572220PMC
June 2017

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

J Chromatogr A 2017 Aug 27;1511:59-67. Epub 2017 Jun 27.

Chemical Sciences Division, National Institute of Standards and Technology (NIST), 100 Bureau Drive, Gaithersburg, MD 20899, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.chroma.2017.06.064DOI Listing
August 2017

Rebooting the Franchise: The 2015 International Symposium on GPC/SEC and Related Techniques.

Chromatographia 2016 Aug 27;79(15):941-943. Epub 2016 May 27.

Chemical Sciences Division, National Institute of Standards & Technology (NIST), 100 Bureau Drive, MS 8392, Gaithersburg, Maryland 20899-8392 USA.

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http://dx.doi.org/10.1007/s10337-016-3085-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5117652PMC
August 2016

Size-exclusion chromatography of metal nanoparticles and quantum dots.

Trends Analyt Chem 2016 Jun 15;80:311-320. Epub 2015 Dec 15.

National Institute of Standards and Technology, Chemical Sciences Division, 100 Bureau Drive, MS 8392, Gaithersburg, MD 20899, USA.

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.
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http://dx.doi.org/10.1016/j.trac.2015.06.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4911637PMC
June 2016

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

Anal Bioanal Chem 2016 Jun 21;408(15):4003-10. Epub 2016 Mar 21.

Chemical Sciences Division, National Institute of Standards and Technology, 100 Bureau Drive, MS8392, Gaithersburg, MD, 20899, USA.

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.
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http://dx.doi.org/10.1007/s00216-016-9487-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4940127PMC
June 2016

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

J Chromatogr A 2015 Feb 3;1380:146-55. Epub 2015 Jan 3.

Chemical Sciences Division, National Institute of Standards and Technology (NIST), 100 Bureau Drive, MS 8392, Gaithersburg, MD 20899, United States. Electronic address:

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.
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http://dx.doi.org/10.1016/j.chroma.2014.12.070DOI Listing
February 2015

HISTORY OF CHROMATOGRAPHY Hamish Small: Experimenter Extraordinaire.

LC GC Eur 2015 ;33:776-781

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605081PMC
January 2015

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

Carbohydr Res 2014 Oct 30;398:31-5. Epub 2014 Jun 30.

Chemical Sciences Division, National Institute of Standards & Technology (NIST), 100 Bureau Drive, Mail Stop 8392, Gaithersburg, MD 20899, USA. Electronic address:

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 β anomeric configuration. Comparison between (1→4)-β-D-linked glucobiose (cellobiose) and (1→4)-β-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.
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http://dx.doi.org/10.1016/j.carres.2014.06.022DOI Listing
October 2014

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

Analyst 2014 Nov;139(22):5843-51

National Institute of Standards and Technology, Chemical Sciences Division, 100 Bureau Drive, MS 8392, Gaithersburg, MD 20899, USA.

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.
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http://dx.doi.org/10.1039/c4an01105hDOI Listing
November 2014

Do column frits contribute to the on-column, flow-induced degradation of macromolecules?

J Chromatogr A 2014 Sep 19;1359:147-55. Epub 2014 Jul 19.

Chemical Sciences Division, National Institute of Standards & Technology (NIST), 100 Bureau Drive, MS 8392, Gaithersburg, MD 20899, USA. Electronic address:

Flow-induced, on-column degradation is a major hindrance to the accurate characterization of ultra-high molar mass macromolecules and colloids. This degradation is a direct result of the large shear rates which are generated within the column, which cause chain scission to occur both in the interstitial medium and, it has been postulated, at the packing particle pore boundary. An additional putative source of degradation has been the column frits, though little experimental evidence exists to either support or refute this claim. To this effect, the present experiments examine the role of the frits in the degradation of high molar mass macromolecules. Two narrow dispersity polystyrene standards, the molar mass of which differs by a factor of two, were analyzed on three different size-exclusion chromatography (SEC) columns, each with frits of different pore size, at various flow rates. In the smallest pore size column, which also contained the smallest frits and which was packed with the smallest diameter particles, the larger standard was forced to degrade by increasing the flow rate of the mobile phase. During the course of the latter portion of the study, the inlet and the outlet frits were removed from the column, in stepwise fashion. It was concluded that neither frit played any appreciable role in the degradation. Results of our studies were applied to explain previously observed degradation in ultra-high pressure liquid chromatography of polymers. The general conclusion arrived at herein is that the column frits are likely to have a secondary role (as compared to interstitial and pore boundary stresses), or no role at all, in polymer degradation for cases where the frit radius is larger than or equal to the hydraulic radius rcof the column.
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http://dx.doi.org/10.1016/j.chroma.2014.07.033DOI Listing
September 2014

Determining the solution conformational entropy of oligosaccharides by SEC with on-line viscometry detection.

Carbohydr Polym 2014 Jun 16;106:230-7. Epub 2014 Feb 16.

Chemical Sciences Division, National Institute of Standards & Technology (NIST), 100 Bureau Drive, Mail Stop 8392, Gaithersburg, MD 20899, USA. Electronic address:

Introduced here is a method for determining the solution conformational entropy of oligosaccharides (-ΔS) that relies on the on-line coupling of size-exclusion chromatography (SEC), an entropically-controlled separation technique, and differential viscometry (VISC). Results from this SEC/VISC method were compared, for the same injections of the same sample dissolutions and under identical solvent/temperature conditions, to results from a benchmark SEC/differential refractometry (SEC/DRI) method which has been applied successfully over the last decade to determining -ΔS of a variety of mono-, di-, and oligosaccharides. The accuracy (as compared to SEC/DRI) and precision of SEC/VISC were found to be excellent, as was the sensitivity of the viscometer in the oligomeric region. The experiments presented here contrast three sets of (1→4)-β-d-oligosaccharides, namely manno-, cello-, and N-acetylchitooligosaccharides of degree of polymerization (DP) 2 through 6. For each series, the dependence of -ΔS on DP was found to be monotonic while, between series, differences at each DP could be ascribed to either the additional degrees of freedom imparted by large, multi-atomic substituent groups, or to the presence or absence of additional intramolecular hydrogen bonds, depending on the axial versus equatorial arrangement of particular hydroxyl groups. An hypothesis is advanced to explain the unexpectedly high sensitivity of viscometric detection for low-molar-mass analytes. The method presented can be extended to the analysis of oligosaccharides other than those studied here.
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http://dx.doi.org/10.1016/j.carbpol.2014.02.027DOI Listing
June 2014

Does the "C₃ effect" offset the Δ2 effect, as regards the solution flexibility of aldoses?

Biopolymers 2014 Jul;101(7):703-11

Chemical Sciences Division, National Institute of Standards & Technology (NIST), 100 Bureau Drive, Mail Stop 8392, Gaithersburg, MD, 20899; Department of Chemistry & Biochemistry, Florida State University, Tallahassee, FL, 32306-4390.

The solution flexibility of carbohydrates influences a variety of molecular recognition and regulatory processes. For aldoses and other monosaccharides, this flexibility is dictated by the orientations of the various hydroxyl (OH) groups present, which influences conformer and anomer ratios, interactions among these OH groups, and interactions between them and the surrounding solvent. Depending on the number and position of axial OH groups, a variety of structures can coexist in solutions at equilibrium. In 1950, as part of his pioneering studies on the shape of pyranoside rings, Reeves described the Δ2 effect, the greater destabilization of the pyranose ring conformation when the OH group on carbon 2 (C2 ) is in the axial position. It was later proposed by Angyal that the Δ2 effect could be cancelled by the presence of an axial OH on C3 , termed here the "C3 effect." Employing size-exclusion chromatography, an entropically-controlled separation technique, we have investigated whether or not the C3 and Δ2 effects indeed do compensate for one another with respect to their influence on the solution flexibility of select aldohexoses and aldopentoses. As will be seen, while qualitatively and semiquantitatively this mutual cancellation of effects does occur in aldohexoses, it does not appear to do so in aldopentoses. An explanation for the latter appears to lie in the variety of anomers and conformers present in equilibrium solutions of those aldopentoses studied and in the relative entropic contribution of individual conformers or anomers to the total solution flexibility.
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http://dx.doi.org/10.1002/bip.22446DOI Listing
July 2014

There's plenty of gloom at the bottom: the many challenges of accurate quantitation in size-based oligomeric separations.

Anal Bioanal Chem 2013 Nov 26;405(28):8959-67. Epub 2013 Jul 26.

Chemical Sciences Division, National Institute of Standards and Technology (NIST), 100 Bureau Drive, Mail Stop 8392, Gaithersburg, MD, 20899, USA,

There is a variety of small-molecule species (e.g., tackifiers, plasticizers, oligosaccharides) the size-based characterization of which is of considerable scientific and industrial importance. Likewise, quantitation of the amount of oligomers in a polymer sample is crucial for the import and export of substances into the USA and European Union (EU). While the characterization of ultra-high molar mass macromolecules by size-based separation techniques is generally considered a challenge, it is this author's contention that a greater challenge is encountered when trying to perform, for quantitation purposes, separations in and of the oligomeric region. The latter thesis is expounded herein, by detailing the various obstacles encountered en route to accurate, quantitative oligomeric separations by entropically dominated techniques such as size-exclusion chromatography, hydrodynamic chromatography, and asymmetric flow field-flow fractionation, as well as by methods which are, principally, enthalpically driven such as liquid adsorption and temperature gradient interaction chromatography. These obstacles include, among others, the diminished sensitivity of static light scattering (SLS) detection at low molar masses, the non-constancy of the response of SLS and of commonly employed concentration-sensitive detectors across the oligomeric region, and the loss of oligomers through the accumulation wall membrane in asymmetric flow field-flow fractionation. The battle is not lost, however, because, with some care and given a sufficient supply of sample, the quantitation of both individual oligomeric species and of the total oligomeric region is often possible.
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http://dx.doi.org/10.1007/s00216-013-7198-1DOI Listing
November 2013

Hydrodynamic chromatography.

Annu Rev Anal Chem (Palo Alto Calif) 2012 ;5:15-34

Analytical Chemistry Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA.

Hydrodynamic chromatography (HDC) has experienced a resurgence in recent years for particle and polymer characterization, principally because of its coupling to a multiplicity of physical detection methods. When coupled to light scattering (both multiangle static and quasi-elastic), viscometric, and refractometric detectors, HDC can determine the molar mass, size, shape, and structure of colloidal analytes continuously and as a function of one another, all in a single analysis. In so doing, it exposes the analytes to less shear force (and, hence, less potential for flow-induced degradation) than in, for instance, size-exclusion chromatography. In this review, we discuss the fundamental chromatographic underpinnings of this technique in terms of retention, band broadening, and resolution, and we describe the power of multidetector HDC with examples from the recent literature.
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http://dx.doi.org/10.1146/annurev-anchem-062011-143107DOI Listing
October 2012

Characterization of copolymers and blends by quintuple-detector size-exclusion chromatography.

Anal Chem 2012 Jun 16;84(11):4812-20. Epub 2012 May 16.

Department of Chemistry & Biochemistry, Florida State University, Tallahassee, Florida 32306-4390, United States.

The properties imparted, oftentimes synergistically, by the different components of copolymers and blends account for the widespread use of these in a variety of industrial products. Most often, however, processing and end-use of these materials (especially copolymers) is optimized empirically, due to a lack of understanding of the physicochemical phase-space occupied by the macromolecules. Here, this shortcoming is addressed via a quintuple-detector size-exclusion chromatography (SEC) method consisting of multiangle static light scattering (MALS), quasi-elastic light scattering (QELS), differential viscometry (VISC), ultraviolet absorption spectroscopy (UV), and differential refractometry (DRI) coupled online to the separation method. Applying the SEC/MALS/QELS/VISC/UV/DRI method to the study of a poly(acrylamide-co-N,N-dimethylacrylamide) copolymer in which both monomer functionalities absorb in the same region of the UV spectrum, we demonstrate how to determine the chemical heterogeneity, molar mass averages and distribution, and solution conformation of the copolymer all in a single analysis. Additionally, through the various mutually independent conformational and architectural metrics provided by combining the five detectors, including the fractal dimension (derived from two different detector combinations), two different dimensionless size parameters, the chemical heterogeneity, and the persistence length, it is shown that the monomeric arrangement is more alternating than random at lower molar masses, thus causing the copolymer to adopt a more extended conformation in solution in this molar mass (M) regime. At high M, however, the copolymer is shown to be and to behave more like a random coil homopolymer, after passing through a 250 kg mol(-1)-broad region of intermediate chain flexibility. Thus, the combination of five detectors provides a unique means by which to determine absolute properties of the copolymer, solution-specific physical behavior, and the underlying chemical basis of the latter. The quintuple-detector method was also extended to the study of blends of polyacrylamide and poly(N,N-dimethylacrylamide) homopolymers to quantitate their molar mass, solution conformation, and chemical heterogeneity and to shed light on the breadth of the distributions of the component species. The method presented should be applicable to the study of copolymers and blends in which either one or both component moieties or polymers absorb in the UV region and can be implemented using not only SEC but other size-based separation methods as well.
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http://dx.doi.org/10.1021/ac3003775DOI Listing
June 2012

The obstruction factor in size-exclusion chromatography. 2. The interparticle, intraparticle, and total obstruction factors.

J Chromatogr A 2012 Jun 17;1241:69-75. Epub 2012 Apr 17.

Department of Chemistry & Biochemistry, Florida State University, Tallahassee, FL 32306, USA.

"Obstruction factor" is a generic rubric under which are usually gathered the interparticle, intraparticle, stationary phase, and total obstruction factors, γ(e), γ(p), γ(s), and γ(t), respectively. These, in turn, affect longitudinal diffusion and stationary, mobile phase, and stagnant mobile phase mass transfer. We conclude here our investigation into the various obstruction factors operative in size-exclusion chromatography (SEC). Stop-flow experiments were employed to determine either the interparticle (for analytes with K(SEC)=0) or the total (for analytes with K(SEC)>0) obstruction factor, and these results were combined with those from variable-flow-rate experiments which provided the intraparticle obstruction factor. Because of minimal enthalpic interactions between the analytes and stationary phase, in SEC γ(s)≈0, which allows for isolation of the other obstruction factors. A relationship between γ(t), γ(e), and γ(p) was proposed for SEC, based on previous independent work and dependent upon the various column porosities. This relationship was extended to hydrodynamic chromatography, a technique in which, ideally, both γ(s) and γ(p) are equal to zero.
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http://dx.doi.org/10.1016/j.chroma.2012.04.024DOI Listing
June 2012

Hydrodynamic chromatography: packed columns, multiple detectors, and microcapillaries.

Anal Bioanal Chem 2012 Jan 7;402(1):77-81. Epub 2011 Sep 7.

Department of Chemistry & Biochemistry, Florida State University, Tallahassee, FL 32306-4390, USA.

Hydrodynamic chromatography (HDC) is a liquid chromatographic technique that separates analytes on the basis of their size in solution. Separation can be conducted either in an open tube or in a column packed with inert, nonporous beads. In HDC, larger analytes elute first and smaller ones later, due to preferential sampling of the streamlines of flow in the open tube or in the interstitial medium of the packed column. Because of the low shear rates experienced in HDC, coupled with the wealth of information obtained when employing a multiplicity of detection methods, the technique has experienced a resurgence in recent years in both the particle sizing and macromolecular arenas, where it can provide information on the mutual interdependence of molar mass, size, shape, and compactness. Additionally, microcapillary HDC is also gaining popularity amongst the bioanalytical community, who have employed the technique, inter alia, to separate DNA fragments over a base pair range spanning four orders in magnitude. Here, examples from the literature are used to show how HDC has been applied in each of the aforementioned areas, explaining the information that can be obtained from various detector combinations, and opining on the future of the technique.
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http://dx.doi.org/10.1007/s00216-011-5334-3DOI Listing
January 2012

Characterizing string-of-pearls colloidal silica by multidetector hydrodynamic chromatography and comparison to multidetector size-exclusion chromatography, off-line multiangle static light scattering, and transmission electron microscopy.

Anal Chem 2011 Apr 23;83(8):3068-75. Epub 2011 Mar 23.

Department of Chemistry & Biochemistry, Florida State University, Tallahassee, Florida 32306-4390, USA.

The string-of-pearls-type morphology is ubiquitous, manifesting itself variously in proteins, vesicles, bacteria, synthetic polymers, and biopolymers. Characterizing the size and shape of analytes with such morphology, however, presents a challenge, due chiefly to the ease with which the "strings" can be broken during chromatographic analysis or to the paucity of information obtained from the benchmark microscopy and off-line light scattering methods. Here, we address this challenge with multidetector hydrodynamic chromatography (HDC), which has the ability to determine, simultaneously, the size, shape, and compactness and their distributions of string-of-pearls samples. We present the quadruple-detector HDC analysis of colloidal string-of-pearls silica, employing static multiangle and quasielastic light scattering, differential viscometry, and differential refractometry as detection methods. The multidetector approach shows a sample that is broadly polydisperse in both molar mass and size, with strings ranging from two to five particles, but which also contains a high concentration of single, unattached "pearls". Synergistic combination of the various size parameters obtained from the multiplicity of detectors employed shows that the strings with higher degrees of polymerization have a shape similar to the theory-predicted shape of a Gaussian random coil chain of nonoverlapping beads, while the strings with lower degrees of polymerization have a prolate ellipsoidal shape. The HDC technique is contrasted experimentally with multidetector size-exclusion chromatography, where, even under extremely gentle conditions, the strings still degraded during analysis. Such degradation is shown to be absent in HDC, as evidenced by the fact that the molar mass and radius of gyration obtained by HDC with multiangle static light scattering detection (HDC/MALS) compare quite favorably to those determined by off-line MALS analysis under otherwise identical conditions. The multidetector HDC results were also comparable to those obtained by transmission electron microscopy (TEM). Unlike off-line MALS or TEM, however, multidetector HDC is able to provide complete particle analysis based on the molar mass, size, shape, and compactness and their distributions for the entire sample population in less than 20 min.
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http://dx.doi.org/10.1021/ac103314cDOI Listing
April 2011

Influence of glycosidic linkage on solution conformational entropy of oligosaccharides: Malto- vs. isomalto- and cello- vs. laminarioligosaccharides.

Biopolymers 2011 Apr 17;95(4):228-33. Epub 2010 Nov 17.

Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306-3290, USA.

Carbohydrate flexibility can influence a variety of recognition, processing, and end-use properties, at both the polymeric and oligomeric levels. The influence of glycosidic linkage, in particular, on carbohydrate flexibility is manifested in properties such as bacterial selectivity, solution viscosity, and the ability to regulate the spread of disease. Here, we apply size-exclusion chromatography, an entropically controlled technique, to determine the solution conformational entropy (ΔS) of various oligosaccharide series. The aim of the present study is to highlight how, for a given anomeric configuration, glycosidic linkage affects ΔS, and to do so quantitatively as a function of degree of polymerization (DP). To this end, we compare ΔS values for DP 1-7 for malto- and isomaltooligosaccharides, and for DP 1-5 for cello- and laminarioligosaccharides. To do so, we realize previously unattainable separations of disaccharides via a strict size-exclusion mechanism. Also given here are the requirements for extending our method to other oligomers, as well as to biopolymers
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http://dx.doi.org/10.1002/bip.21567DOI Listing
April 2011

Separation science of macromolecules.

Anal Bioanal Chem 2011 Feb;399(4):1399-400

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http://dx.doi.org/10.1007/s00216-010-4480-3DOI Listing
February 2011

Characterizing the size, shape, and compactness of a polydisperse prolate ellipsoidal particle via quadruple-detector hydrodynamic chromatography.

Analyst 2011 Feb 26;136(3):515-9. Epub 2010 Nov 26.

Department of Chemistry & Biochemistry, Florida State University, Tallahassee, Florida 32306-4390, USA.

A detailed quantitative description of particle size, shape, and their distributions is essential for understanding and optimization of the solid-, solution-, and melt-state properties of materials. Here, we employ quadruple-detector hydrodynamic chromatography (HDC) with multi-angle static light scattering, quasi-elastic light scattering, differential viscometry, and differential refractometry detection as a method for characterizing three important physical properties of materials, namely the molar mass, size, and shape of a polydisperse, non-spherical colloidal silica sample. These properties and their distributions were measured continuously across the HDC elution profile of the sample. By combining information from the various parameters determined, we were also able to obtain quantitative knowledge regarding the compactness or denseness of the sample. The applicability of multi-detector HDC to characterize polydisperse, non-spherical analytes was shown to be rapid, accurate, and precise. An advantage over traditional characterization methods is the ability of multi-detector HDC to determine particle size, shape, compactness, and their distributions simultaneously in a single analysis.
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http://dx.doi.org/10.1039/c0an00738bDOI Listing
February 2011

The obstruction factor in size-exclusion chromatography. 1. The intraparticle obstruction factor.

J Chromatogr A 2010 Nov 17;1217(45):7131-7. Epub 2010 Sep 17.

Department of Chemistry & Biochemistry, Florida State University, Tallahassee, FL 32306-4390, USA.

We report the first of a series of studies on the obstruction factor γ in size-exclusion chromatography (SEC). Here, using narrow dispersity polymer standards we examine how the intraparticle obstruction factor γ(p) depends individually on a number of analyte properties, column characteristics, and user-defined parameters. Far from being constant, γ(p) is seen to vary with analyte molar mass and solvent, as well as with the pore size and particle size of the column packing material, sometimes in seemingly counterintuitive manner. Over the limited temperature range accessible to our equipment, however, no statistically significant change in γ(p) with temperature was discovered. The results presented should be applicable to forms of packed column chromatography other than SEC. The latter technique, however, presents a convenient test bed for quantitative determination of the obstruction factor, due to minimized sorptive mass transfer and longitudinal diffusion contributions to band broadening in most forms of SEC.
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http://dx.doi.org/10.1016/j.chroma.2010.09.021DOI Listing
November 2010
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