The size exclusion chromatography calibration of ‘Mixed-A’ and ‘Mixed-D’ columns using various polymers and compounds: An application to coal-derived materials

This work attempts to obtain the calibration curves of two different size exclusion chromatography (SEC) columns operating with 1-methyl-2-pyrrolidinone (NMP) as eluent by using various standards. Polystyrene (PS) and polymethylmethacrylate (PMMA) standards were used for obtaining calibration curves, and checked against polysaccharide (PSAC) standards, some small aromatic polycyclic standards and miscellaneous polymers. Polystyrenes and polymethylmethacrylates gave identical calibrations while polysaccharides and miscellaneous polymers lay within 1 or 2 min of the polystyrene calibration. Small molecules of mass less than 1000 units lay on or near to the polystyrene calibration lines, with a shift to late elution for the smallest molecules. This shift may be caused by the interaction with the column packing. A sample has been examined by analytical size exclusion chromatography, which was calibrated using polystyrene and polymethylmethacrylate standards. Molecular mass (MM) distributions of the sample have been examined in terms of these calibrations.     

Molecular weight determination of hyaluronic acid by gel filtration chromatography coupled to matrix-assisted laser desorption ionization mass spectrometry.

An analytical approach has been described for the molecular weight characterization of enzymatically degraded hyaluronic acid (HA). The approach involved the combined use of aqueous gel filtration chromatography (GFC) with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Microfractions were collected across an eluting peak from the chromatography system, followed by mass spectrometric analysis of these narrow fractions. The molecular mass determined by MALDI-MS and the signal obtained from the chromatography established a calibration curve for other hyaluronic acid samples analyzed by this GFC system. Results of one HA sample were obtained from both the calibration curve and direct fraction-by-fraction MALDI-MS analysis, and comparison of these results showed reasonable agreement. In contrast, molecular weights resulted from external calibration using dextran and pullullan standards showed drastically different numbers. Therefore, the GFC-MALDI-MS approach is a reliable method for the molecular weight characterization of polydisperse polysaccharides for which suitable calibration standards are unavailable for conventional GFC analysis.     

Gel permeation chromatography: calibration procedures for chains containing p-phenylene units

Experimental gel permeation chromatography calibrations have been obtained for polystyrene standards, polysulphone fractions, and polycarbonate fractions in chloroform at 30°. Chloroform is a good solvent for all three polymers which have similar polymer solvent interactions. The fractions have narrow molecular weight distributions, so that viscosity average molecular weight can be taken as the peak molecular weight of a chromatogram. The experimental polysulphone and polycarbonate calibrations are compared with curves calculated from the polystyrene calibration using equations which assume that the unperturbed mean-square end-to-end distance and hydrodynamic volume are universal calibration parameters. For molecular weights between 20,000 and 100,000 both universal calibration procedures were found to be acceptable. For polycarbonate extended chain length was also found to be satisfactory for universal calibration. For polycarbonate molecular weights below 20,000, the predicted molecular weight calibration deviated from the experimental data. Possible reasons for this difference are discussed.     

Contactless conductivity detection of synthetic polymers in non-aqueous size-exclusion electrokinetic chromatography

A capacitively coupled contactless conductivity detection (CCD) system has been applied for the detection of neutral synthetic polymers in capillary size-exclusion electrokinetic chromatography (SEEC). Polystyrene standards, that were used as a model compounds, were separated on a capillary column packed with porous 10 microm silica particles with an electrokinetically driven mobile phase, and detected by CCD and UV detection simultaneously. Mass-calibration curves for polystyrene were constructed. Satisfactory results were obtained for the linearity, the run-to-run repeatability (<0.2% for the relative retention and <4% for the peak area) and the robustness of the detector. One of the major issues in this preliminary study was to investigate the origin of the peaks observed for the polystyrene standards. The effect of the molar mass of the polystyrenes on the sensitivity was small. Therefore, the signals obtained could not be explained as the result of an increased viscosity and a decreased solution conductivity of the solute zone. An alternative hypothesis is suggested, and recommendations for further research are given.     

Two-dimensional liquid chromatography of PDMS-PS block copolymers

In this study, liquid chromatography at critical conditions of polystyrene (PS) and polydimethylsiloxane (PDMS) is used as the first dimension for the two-dimensional analysis of polydimethylsiloxane-block-polystyrene copolymers. Comprehensive two-dimensional liquid chromatography with size exclusion chromatography as the second dimension reveals information about the molar mass distributions of all separated fractions from the first dimension. Furthermore, fractions eluting at the critical conditions were collected and subjected to analysis in the second dimension at the critical adsorption point of the other block. These fractions were analyzed by Fourier transform infrared spectroscopy to determine their chemical compositions. The combination of the above approaches and the calibration of the evaporative light scattering detector for the first-dimension analysis yield deep insights into the molecular heterogeneity of the block copolymer samples. The composition of the samples and the chemical composition of the real block copolymer are also calculated by combining the results obtained at both critical conditions.     

Determining the molar mass of a plasma substitute succinylated gelatin by size exclusion chromatography-multi-angle laser light scattering,sedimentation equilibrium and conventional size exclusion chromatography

The clinical effectiveness of succinylated gelatin as a plasma substitute depends strongly on its molar mass, determined conventionally by size exclusion chromatography (SEC). This study evaluates different SEC calibration standards in comparison with two independent "absolute" methods for determining the weight average molar mass (M(w)) of a succinylated gelatin sample. SEC calibrated using succinylated gelatin fractions correlated well with size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS) and sedimentation equilibrium whereas SEC calibrated with unmodified gelatin, sodium polystyrene sulfonates or pullulans overestimated M(w) by over 20%. Universal calibration was equivocal. The problems associated with the preparation of succinylated gelatin fractions suggest that an absolute method such as SEC-MALLS may be a more suitable choice for determining the M(w) in succinylated gelatins.     

Analysis of polyamides by size exclusion chromatography and laser light scattering

The molar mass analysis of polyamides is complicated due to the fact that only a limited range of solvents can be used and association and aggregation phenomena have to be screened by adding electrolytes to the mobile phase. Optimum SEC behaviour is obtained when hexafluoroisopropanol + 0.05 mol/L potassium trifluoroacetate is used as the mobile phase. The calibration of the SEC system can be conducted in different ways. While a calibration with narrow disperse polymethyl methacrylate standards does not yield accurate molar mass information, the quantification can be done using an “artificial” calibration curve. This calibration curve is obtained by correcting the PMMA calibration curve with polyamide molar mass data from light scattering. The resulting molar mass distributions for different types of polyamides are compared with molar masses that are determined by size exclusion chromatography with a light scattering detector and an excellent correlation is obtained.     

Generation of average mass values and end group information of polymers by means of a combination of matrix-assisted laser desorption/ionization-mass spectrometry and liquid secondary ion-tandem mass spectrometry

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and liquid secondary ion-tandem mass spectrometry (LSI-MS/MS) have been applied to the analysis of synthetic polymers to generate values for the average mass and the mass of the end groups. The average mass values were calculated for polymethylmethacrylate and polystyrene standards from the MALDI-MS data. Abundant fragment ions of the polymers, generated by means of LSI-MS/MS, were consistent with the known structures of the end groups of the polymers. Furthermore, losses from the side chains of the polymers were also observed in the LSI-MS/MS spectra.     

Characterization of cross-linking structures in UV-cured acrylic ester resin by MALDI-MS combined with supercritical methanolysis.

The cross-linking structure of the ultra violet (UV)-cured resin prepared from dipentaerithritol hexacrylate (DPHA) was characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with supercritical methanolysis. The MALDI-mass spectrum of the decomposition products obtained by supercritical methanolysis contained a series of peaks of sodium-cationized methyl acrylate (MA) oligomers up to around m/z = 4000 formed through selective cleavage and methylation occurred at ester linkages in UV-cured DPHA. Furthermore, in order to observe widely distributed sequence lengths in the cross-linking junctions, the decomposed products of the cured resin were then fractionated using size exclusion chromatography followed by the MALDI-MS measurements of the individual fractions. The MALDI-mass spectra of the lower molar mass fractions mainly consisted of a series of peaks of MA oligomers around m/z values of several thousands, whereas those of higher molecular weight showed a broad peak up to m/z ca. 180000. The observed distributions of the supercritical methanolysis products suggested that the network junctions in the given UV-cured resin were composed of up to around 2000 acrylate units.     

Monodisperse lignin fractions as standards in size-exclusion analysis: Comparison with polystyrene standards

The difficulty of preparing monodisperse lignin fractions on a large scale is a limiting factor in many applications. The present paper addresses this problem by examining the properties and size-exclusion behavior of lignin isolated by the acetosolv pulping process from post-extraction crushed sugarcane bagasse. The isolated lignin was subjected to a solvent pretreatment, followed by preparative gel permeation chromatography fractionation. The fractions were analyzed by high-performance size-exclusion chromatography (HPSEC) and these samples showed a great decrease in polydispersity, compared to the original acetosolv lignin. Several fractions of very low polydispersity, close to unity, were employed as calibration curve standards in HPSEC analysis. This original analytical approach allowed calibration with these lignin fractions to be compared with the polystyrene standards that are universally employed for lignin molecular mass determination. This led to a noteworthy result, namely that the lignin fractions and polystyrene standards showed very similar behavior over a large range of molecular masses in a typical HPSEC analysis of acetosolv lignin.     

Comprehensive two-dimensional liquid chromatography for the characterization of functional acrylate polymers

Comprehensive two-dimensional liquid chromatography-size-exclusion chromatography (LC x SEC) was investigated as a tool for the characterization of functional poly(methyl methacrylate) (PMMA) polymers. Ultraviolet-absorbance and evaporative light-scattering detection (ELSD) were used. A simple method to quantify ELSD data is presented. Each data point from the ELSD chromatogram can be converted into a mass concentration using experimental calibration curves. The qualitative and quantitative information obtained on two representative samples is used to demonstrate the applicability of LC x SEC for determining the mutually dependent molar-mass distributions (MMD) and functionality-type distributions (FTD) of functional polymers. The influence of the molar mass on the retention behavior in LC was investigated using LC x SEC for hydroxyl-functional PMMA polymers. The critical conditions, at which retention is--by definition--independent of molar mass, were not exactly the same for PMMA series with different end-groups. Our observations are in close agreement with theoretical curves reported in the literature. However, for practical applications of LC x SEC it is not strictly necessary to work at the exact critical solvent composition. Near-critical conditions are often sufficient to determine the mutually dependent distributions (MMD and FTD) of functional polymers.     

Probing the molecular weight distributions of non-boiling petroleum fractions by Ag+ electrospray ionization mass spectrometry

This work explores the possibility of Ag+ electrospray ionization mass spectrometry (ESI-MS) to determine the molecular weight distributions of non-boiling petroleum fractions. Information about the molecular weight distributions is needed for fundamental studies on the nature of heavy crude oils and bitumens and for the development of novel recovery and processing methods. The method does not depend on thermal processes for the introduction of the fractions into the gas phase of the mass spectrometer, which is a considerable advantage over most other ionization methods. The Ag+ electrospray mass spectra of the fractions analyzed by using a toluene/methanol/cyclohexane (60:28:12%) solvent system display bimodal distributions in the ranges m/z approximately 300 to approximately 3000 and m/z 3000 to approximately 20,000. The abundances of the high molecular weight peak distributions can be reduced by in-source collisional activation experiments. Comparisons with the results obtained for model heteroatom-containing compounds (molecular weight < 600 Da) and high molecular weight polystyrene standards (up to one million Da) indicate that the majority of the structures in the saturate, naphthenoaromatic and polar aromatic fractions, and a significant portion of the asphaltenes, are small molecules. However, a considerable portion of the asphaltenes and some portion of the other fractions contain high molecular weight structures bound by covalent or strong non-covalent bonds. The results obtained by the Ag+ ESI method in this study for the saturate, aromatic, and polar fractions in a bitumen are in qualitative agreement with published molecular weight average results obtained for Cold Lake bitumen fractions analyzed by conventional gel permeation chromatography and field desorption mass spectrometry. Further work is needed to study the nature of the bonds and the interactions of the molecules in the asphaltene fractions by Ag+ ESI-MS.     

Evaluation of size-exclusion chromatography and size-exclusion electrochromatography calibration curves

Size-exclusion chromatography (SEC) and size-exclusion electrochromatography (SEEC) are chromatographic techniques used to determine molecular mass (weight) distributions (MWD) of polymers. One important step in the data treatment to derive MWD parameters is the modelling of the calibration curves. The calibration curves applied in SEC and SEEC are generally not linear. In this study the modelling of calibration curves is being examined. Different polynomial models have been evaluated and compared, not only for model fit but also for their predictive properties. It was found that sometimes a straight line and sometimes a third-order polynomial model were best. The best model across the effective range (also called linear range) is not always found to be a straight line. The SEEC curves were found to have considerably higher prediction errors than the SEC ones. Reduction of the number of calibration standards to five or six did not greatly affect the predictive properties of the calibration curves, neither in SEC nor in SEEC.     

Characterization of synthetic copolymers by interaction polymer chromatography: Separation by microstructure

Gradient elution of synthetic polymers has been studied both theoretically and experimentally using normal and reversed-phase HPLC systems. An accurate equation describing the gradient elution of polymer-homologous series in the context of continuous random-flight model of a flexible polymer chain interacting with attractive surface of the porous material has been derived and experimentally verified against a series of narrow polystyrene standards. Both the theory and the experiment predict the existence of molar mass-independent gradient elution at critical point of adsorption (CPA). The extension of the theory to synthetic copolymers predicts the existence of the CPA for statistical copolymers and describes its dependence on chemical composition and microstructure (blockiness) of the polymer chains. One of the important theoretical conclusions is that blockiness always increases the retention, so that blockier polymer chains elute later than their more random counterparts with the same chemical composition. This prediction has been confirmed experimentally using block and statistical styrene-methylmethacrylate copolymers. Block copolymers do not have CPA and always elute between critical points of the corresponding homopolymers. The retention depends on the polymer molar mass and increases with the length of the blocks from a stronger absorbing monomer. These findings provide theoretical and experimental bases for separation of statistical and block copolymers by chemical composition and microstructure of polymer chains.     

Calibration in High Temperature Gel Permeation Chromatography of Linear Polyethylenes

Abstract

The mathematical relationship between the gel permeation chromatography calibration curves of polystyrene and linear polyethylene has been determined in 1,2,4-trichlorobenzene at 130, 135 and 140°C. The experimentally determined relationship is in good agreement with that predicted from application of the principles of the universal calibration technique and published Mark-Houwink coefficients. Definition of this relationship enables the use of polystyrene as a secondary standard for gel permeation chromatographic determination of linear polyethylene molecular weight determinations.     

Optimisation of ambient and high temperature asymmetric flow field-flow fractionation with dual/multi-angle light scattering and infrared/refractive index detection

Asymmetric flow field-flow fractionation (AF4) enables to analyse polymers with very high molar masses under mild conditions in comparison to size exclusion chromatography (SEC). Conventionally, membranes for AF4 are made from cellulose. Recently, a novel ceramic membrane has been developed which can withstand high temperatures above 130 °C and chlorinated organic solvents, thus making it possible to characterise semicrystalline polyolefins by HT-AF4. Two ceramic membranes and one cellulose membrane were compared with regard to their quality of molar mass separation and the loss of the polymer material through the pores. Separating polystyrene standards as model compounds at different cross-flow gradients the complex relationship between cross-flow velocity, separation efficiency, the molar mass and peak broadening could be elucidated in detail. Moreover, the dependence of signal quality and reproducibility on sample concentration and mass loading was investigated because the evaluation of the obtained fractograms substantially depends on the signal intensities. Finally, the performance of the whole system was tested at high temperature by separating PE reference materials of high molar mass.     

Molar mass of main-chain bile acid-based oligo-esters measured by SEC, MALDI-TOF spectrometry and NMR spectroscopy: a comparative study

Bile acid-based polymers are promising new materials for biomedical applications. The determination of their molar mass, as for other novel polymers, has been difficult, due to the lack of suitable standards for size exclusion chromatography (SEC). In order to solve this problem, a family of main-chain bile acid-based oligo-esters has been synthesized by acyclic diene metathesis to be used as analogues in such analysis. These oligomers have been characterized by SEC, MALDI-TOF mass spectrometry and NMR spectroscopy. The results show that SEC with polystyrene standards tends to overestimate the molar mass of these materials and that a correction factor between 0.50 and 0.60 should be used for more accuracy.     

Studies on Simultaneous Molecular Weight and Spreading Calibration in Steric Exclusion Liquid Chromatography

Abstract

Two improved methods of molecular weight calibration are described where simultaneously parameters of a symmetrical spreading function are obtained through the use of polymolecular molecular weight standards and of average retention volumes. In the first method a linear molecular weight calibration is assumed and the second method is based on a universal molecular weight calibration curve obtained with narrow MWD polystyrene standards.

The proposed methods have been tested using two polymolecular polystyrene standards confirming good convergence of the applied iteration procedures and giving very promising results.     

Unusually high efficiency of the separation of polymers by hydrodynamic chromatography on hollow capillary columns

A series of polystyrene standards was separated by means of hydrodynamic chromatography on a open capillary with a length of 5 m (active zone is 4.5 m) and an inner diameter of 2 μm. Unusually high efficiency in the separation of polymers with masses of up to 500 kDa was noticed: 1.5 × 10⁶ theoretical plates at a retention time of ～70 min for a sample of polystyrene with a mass of 390 kDa. It was established that the column efficiency decreases rapidly for polymers with a mass above 500 kDa. A calibration curve for polystyrene standards was constructed. It was shown that, in contrast to the models described in the literature, it is described by a simple two-term expression without quadratic terms. It is concluded that hydrodynamic chromatography is a technique complementary to exclusion chromatography, especially in the separation of high-molecular polymers.     

Application of off-line size-exclusion chromatographic fractionation-matrix assisted laser desorption ionization time of flight mass spectrometry for proanthocyanidin characterization

In this work, we wanted to devise a reliable method to characterize polymerized forms of tannins, their structural information and mass distribution. Size-exclusion chromatography (SEC) is a chromatographic technique used to determine molecular mass (weight) distributions of polymers. One important step in the data treatment is the modeling of the calibration curves. Polystyrenes (PS) are standards usually used because no commercial procyanidin (PC) standards are available. An off-line coupling of SEC and MALDI was carried out to measure differences between polystyrenes and procyanidins. Thus, a new calibration curve was established; from 1000 to 8000 Da, there is a good correlation between the MALDI and PS calibration curves, in this field the PS calibration is correct and enables true mass determination. For masses above 8000 Da, PS calibration overestimates the real molecular weight of PC, overestimation of 53%. And for masses below 1000 Da, PS calibration underestimates their real molecular weight (10-15%). This means that to truly characterize PC, calibration based on PC standards is required.