Contribution of size exclusion chromatography and thermal analysis methods in the study of the radiochemical behavior of plastic materials

Sterilization by irradiation techniques is considered to be an improved method to treat polluted products of human consumption. Secondary effects, that may be generated in the components of packaging materials, could induce organoleptic damage and some physico-chemical modifications, observed especially in plastic materials. In order to demonstrate any such secondary changes, we have investigated the effects of beta-irradiation (a treatment which could induce either a reticulation or a cleavage in a polymeric sequence) on three polymers; namely, low density polyethylene, polystyrene and polyvinyl chloride. The techniques used to identify irradiation-induced modifications of these polymers were size exclusion chromatography and thermal analysis. Thermal analysis showed a decrease in the percentage of crystallinity of polyethylene after its radiosterilization. Exclusion chromatography highlighted a reticulation for irradiated polyvinyl chloride and polystyrene.     

Automated sample cleanup using on-line coupling of size exclusion chromatography to high resolution gas chromatography

A fully automated on-line sample cleanup system based on the coupling of size exclusion chromatography to high resolution gas chromatography is described. The transfer technique employed is based on fully concurrent solvent evaporation using a loop-type interface, early vapor exit and co-solvent trapping. Optimization of the LC-GC transfer was done visually via an all-glass oven door. To circumvent the problem of mixing within the injection loop, an adaptation was made to the standard loop-type interface. The determination of a series of additives in a polymer matrix is presented as one example of the vast range of applications opened up by this technique.     

Characterization of polymers by size exclusion chromatography using multiple detection. Determination of the Mark-Houwink constants for Pullulan 100

Summary A size exclusion chromatography system is outlined using multiple detection by a low angle laser light scattering detector (LALLS) and a concentration detector (RI) in conjunction with a differential viscosimeter. The combination of these three detectors parmits the easy and rapid determination of Mw, Mn, Mn and of the Mark-Houwink constants k and α. This system is based on commercially available components and we describe its use with an aqeous solvent system. A similar configuration described in the literature employs a homemade differential viscosimeter constructed by the authors and uses an organic solvent for the SEC. Working with aqeous solvents requires high performance of all equipment components. We will demonstrate the capability of this system with Pullalan as an example.     

Error introduced into determination of pore size distribution by size exclusion chromatography

Simultaneous use of large standard molecules and small particles of the product examined gives rise to errors in pore size determination by size exclusion chromatography. This error is calculated for packings of spherical particles, thus making corrections possible.     

Interlaced size exclusion liquid chromatography of monoclonal antibodies

Size exclusion chromatography is a widely performed analysis of monoclonal antibodies, primarily used to monitor the levels of higher weight molecular species such as aggregates. Owing to the subtleties of these separation mechanisms and frequently observed partial resolutions of components in these separations, many common methods for increasing the method throughput are not practical as they trade off resolution for speed. Short columns, high flow rates and smaller particles are examples of these approaches. In this paper a practical method is demonstrated for injecting samples onto the column in rapid succession and gating the detection window to monitor the elution of each sample individually. At any given instant approximately two samples are eluting through the column. By co-ordinating the injection and detection time windows the samples can be kept discrete and significant throughput enhancements achieved, up to nearly 2-fold improvements are demonstrated. A rudimentary theory is development to show that the throughput improvements can be predicted to approximation by simple column characteristics. Experimental results for a series of monoclonal antibodies demonstrate the equivalency of the method to a conventional injection approach, the throughput increase, and the robustness of the method.     

Evaluation of silica gel-immobilized phosphorylcholine columns for size exclusion chromatography and their application in the analysis of the subunit structures of fish-egg lectins

Columns of phosphorylcholine (PC) immobilized on silica gel were shown to be useful for size exclusion chromatography (SEC) of proteins. The columns provided good separation of proteins in 50 mM sodium phosphate buffer (pH 6.9) containing 0.25 M NaCl, and there was a linear relationship between the retention times and the logarithmic values of the molecular weights with a correlation coefficient (R²) of 0.978–0.992. The columns were used in analyzing the subunit structures of the rhamnose-binding lectins CSL1, CSL2, and CSL3, isolated from chum salmon (Oncorhynchus keta) eggs. Although the lectins, which are a group of carbohydrate-binding and hydrophobic proteins, behaved anomalously in SEC with conventional matrices, they could be eluted from the immobilized PC columns without non-size-related retention, thereby allowing their molecular weights to be reliably estimated.     

Size exclusion chromatography – A blessing and a curse of science and technology of synthetic polymers

Size exclusion chromatography, SEC is one of the most popular methods for the separation of different kinds of macromolecules. This critical review gives concise information about macromolecules and their behavior in solution, basic understanding about principles, instrumentation, and application possibilities of SEC, and more in detail discusses drawbacks and pitfalls of the method with the emphasis on synthetic polymers. Selected practical advices are included to help enhance the quality of SEC results.     

Analysis of sodium polycarboxylates in detergents by size exclusion chromatography

Summary This paper describes the quantitative analysis and preparative isolation of sodium polycarboxylates in detergents by means of gel permeation chromatography. An analytical monitoring method separates the polymers from other low molecular detergent ingredients within 10 minutes. There is no separation of the various molecular weight polycarboxylate macromolecules themselves. They elute from the column as a single narrow peak at the exclusion volume. A second preparative gel filtration method allows isolation of polycarboxylates in amounts necessary for further characterization. Appropriate sample pretreatments and possible interferences are discussed.     

分子排阻色谱硅质填料的制备及其对蛋白质的分离机理

利用自制的四种不同粒径的硅溶胶,通过堆积法来制备分子排阻色谱多孔硅质填料,该填料在进行化学键合改性后,形成二醇固定相。利用二醇固定相对蛋白质进行分离分析方面的研究。此填料粒径小,有利于蛋白质生物大分子的高效快速分离分析。     

The fractal calibration method applied to the characterization of polymers in solvent mixtures and in mixed gel packings by SEC

The size-exclusion chromatographic (SEC) behaviour of different solvent/polymer systems in three packing sets has been analysed from fractal considerations. The three-column sets studied are specifically formed by: (i) 'pure' micro-styragel, (ii) 'mixed' TSK Gel H(HR + XL + HR) and (iii) mixed TSK Gel H(XL + HR + XL). The experimental data reveals that in most of the systems assayed the classical universal calibration (UC) is not fulfilled, denoting the existence of secondary effects accompanying the main SEC mechanism. In order to obtain an accurate characterization of different polymers eluted in solvent mixtures and/or mixed packings, the use of a reliable and trusted calibration curve is required. In this sense, two alternative procedures have been analysed: the specific (SC) and the fractal (FC) calibrations. The results have evidenced that the use of the FC instead of the classical universal method diminishes up to nine times (in the case of the micro-styragel set) the mean deviation on the calculated molar mass with respect to the value given by the supplier. In the case of TSK Gel-based sets, the mean deviation is reduced to the half. The SC curve made with standards of the sample under study also reduces the mean deviation values but needs a broad set of narrow standards, whereas the fractal approach only needs one polymeric sample to build up the calibration curve.     

Calibration of ultrafiltration membranes against size exclusion chromatography columns

Using the extension of the concept of universal calibration parameter, yielding a relation between the hydrodynamic volume of molecules and the elution volume in size exclusion chromatography (SEC), to retention coefficients in ultrafiltration (UF), we propose a direct calibration of UF membranes against chromatography columns. Plotting the retention coefficient by one given UF membrane of a series of probe molecules versus their elution volume in SEC chromatography provides a calibration curve for this membrane. For a wide range of retentions, such calibration can be directly used to predict the retention of any molecule: one only needs to measure its exclusion volume by the SEC column, and read the retention by the calibrated membrane on the calibration curves.     

Characterisation of polymers by size exclusion chromatography using multiple detection. Investigations on the determination of structural differences of hydroxyethyl starches

Summary An aqueous size exclusion chromatography system is outlined using dual detection by a multi-angel laser light scattering photometer (MALLS) and a concentration detector (RI). This system makes possible the determination of the molecular weight distribution of water soluble polymers in conjunction with the radius of gyration. The differences in the radii of gyration at the same molecular weights of two hydroxyethyl starches with different molecular structure are presented qualitatively. The determination of the Mark-Houwink relation for these polymers leads to a qualitatively similar result.     

Interactions of poly(2-methacryloyloxyethyl phosphorylcholine) with various salts studied by size exclusion chromatography

The objective of this research is to develop a relationship between salt type and concentration to poly(2-methacyloyloxyethyl phosphorylcholine) (PMPC) zwitterionic polymer solution behaviors. In particular, polyelectrolyte hydrodynamic volumes were analyzed through size exclusion chromatography in relation to the addition of various salts at various concentrations. The salt properties examined were salt concentration, ionic strength, solution pH, cation type/size, anion type/size, valency, and configuration. It was found that the effect of ion properties is related to mechanisms associated with the geometry of the polyelectrolyte. The negative charge group of the polyelectrolyte situated closer to the backbone (inside) is less important to the change in hydrodynamic volume than the positive charge group situated at the end of the side chain (outside). The extensive amount of data generated in this study provides a strong background for possible accurate formulation of a theory based on the salt effect on PMPC polyelectrolyte solution behavior.     

Effect of method of solution on observed molecular weight distribution of barley starch and starch derivatives in size exclusion chromatography

Summary Samples of native barley starch and six starch derivatives were suspended (0.1% sample concentration) in four different solvents: the eluent (pH 11 buffer), dimethylsulfoxide, 0.1 M NaOH or 0.5 M NaOH and kept in a boilling water bath for 5 to 60 minutes or shaken for 60 minutes. The average molecular weight values , and the polydispersity value were determined with a TSK PW-type column using narrow standard calibration. Only a small part of the samples dissolved in the eluent. The dissolution of sample in dimethylsulfoxide was dependent on sample type. Of the NaOH solutions, 0.5 M NaOH was the only one that dissolved all the samples. Therefore, 0.5 M NaOH appears to be the solvent of choice for starch molecules.     

Characterization of branched ultrahigh molar mass polymers by asymmetrical flow field-flow fractionation and size exclusion chromatography

The molar mass distribution (MMD) of synthetic polymers is frequently analyzed by size exclusion chromatography (SEC) coupled to multi angle light scattering (MALS) detection. For ultrahigh molar mass (UHM) or branched polymers this method is not sufficient, because shear degradation and abnormal elution effects falsify the calculated molar mass distribution and information on branching. High temperatures above 130 °C have to be applied for dissolution and separation of semi-crystalline materials like polyolefins which requires special hardware setups. Asymmetrical flow field-flow fractionation (AF4) offers the possibility to overcome some of the main problems of SEC due to the absence of an obstructing porous stationary phase. The SEC-separation mainly depends on the pore size distribution of the used column set. The analyte molecules can enter the pores of the stationary phase in dependence on their hydrodynamic volume. The archived separation is a result of the retention time of the analyte species inside SEC-column which depends on the accessibility of the pores, the residence time inside the pores and the diffusion ability of the analyte molecules. The elution order in SEC is typically from low to high hydrodynamic volume. On the contrary AF4 separates according to the diffusion coefficient of the analyte molecules as long as the chosen conditions support the normal FFF-separation mechanism. The separation takes place in an empty channel and is caused by a cross-flow field perpendicular to the solvent flow. The analyte molecules will arrange in different channel heights depending on the diffusion coefficients. The parabolic-shaped flow profile inside the channel leads to different elution velocities. The species with low hydrodynamic volume will elute first while the species with high hydrodynamic volume elute later. The AF4 can be performed at ambient or high temperature (AT-/HT-AF4). We have analyzed one low molar mass polyethylene sample and a number of narrow distributed polystyrene standards as reference materials with known structure by AT/HT-SEC and AT/HT-AF4. Low density polyethylenes as well as polypropylene and polybutadiene, containing high degrees of branching and high molar masses, have been analyzed with both methods. As in SEC the relationship between the radius of gyration (R(g)) or the molar mass and the elution volume is curved up towards high elution volumes, a correct calculation of the MMD and the molar mass average or branching ratio is not possible using the data from the SEC measurements. In contrast to SEC, AF4 allows the precise determination of the MMD, the molar mass averages as well as the degree of branching because the molar mass vs. elution volume curve and the conformation plot is not falsified in this technique. In addition, higher molar masses can be detected using HT-AF4 due to the absence of significant shear degradation in the channel. As a result the average molar masses obtained from AF4 are higher compared to SEC. The analysis time in AF4 is comparable to that of SEC but the adjustable cross-flow program allows the user to influence the separation efficiency which is not possible in SEC without a costly change of the whole column combination.     

Two-dimensional liquid chromatography analysis of synthetic polymers using fast size exclusion chromatography at high column temperature

In recent years, two-dimensional liquid chromatography (2D-LC) has been used increasingly for the analysis of synthetic polymers. A 2D-LC analysis provides richer information than a single chromatography analysis at the cost of longer analysis time. The time required for a comprehensive 2D-LC analysis is essentially proportional to the analysis time of the second dimension separation. Many of 2D-LC analyses of synthetic polymers have employed size exclusion chromatography (SEC) for the second-dimension analysis due to the relatively short analysis time in addition to the wide use in the polymer analysis. Nonetheless, short SEC columns are often used for 2D-LC analyses to reduce the separation time, which inevitably deteriorates the resolution. In this study, we demonstrated that high temperature SEC can be employed as an efficient second-LC in the 2D-LC separation of synthetic polymers. By virtue of high temperature operation (low solvent viscosity and high diffusivity of the polymer molecules), a normal length SEC column can be used at high flow rate with little loss in resolution.     

An analytical solution of a newly proposed peak broadening equation and extension of polydispersities to higher molecular weight averages in size exclusion chromatography (SEC)

Summary Herein is reported an analytical solution to the peak broadening or peak dispersion/flattening equation based on the recently proposed Instrumental Spreading Shape Function and its application to correction for imperfect resolution (inadequate peak separation and/or excessive peak broadening) for higher molecular weight averages. The relationship of these higher MW averages with the familiar Weight Average and number average molecular weights is also discussed. Criteria for perfect resolution are specified and a true molecular weight calibration curve is accordingly defined.     

A sub-two minutes method for monoclonal antibody-aggregate quantification using parallel interlaced size exclusion high performance liquid chromatography

In process development and during commercial production of monoclonal antibodies (mAb) the monitoring of aggregate levels is obligatory. The standard assay for mAb aggregate quantification is based on size exclusion chromatography (SEC) performed on a HPLC system. Advantages hereof are high precision and simplicity, however, standard SEC methodology is very time consuming. With an average throughput of usually two samples per hour, it neither fits to high throughput process development (HTPD), nor is it applicable for purification process monitoring. We present a comparison of three different SEC columns for mAb-aggregate quantification addressing throughput, resolution, and reproducibility. A short column (150 mm) with sub-two micron particles was shown to generate high resolution (~1.5) and precision (coefficient of variation (cv)<1) with an assay time below 6 min. This column type was then used to combine interlaced sample injections with parallelization of two columns aiming for an absolute minimal assay time. By doing so, both lag times before and after the peaks of interest were successfully eliminated resulting in an assay time below 2 min. It was demonstrated that determined aggregate levels and precision of the throughput optimized SEC assay were equal to those of a single injection based assay. Hence, the presented methodology of parallel interlaced SEC (PI-SEC) represents a valuable tool addressing HTPD and process monitoring.     

Application of size exclusion chromatography with surfactant eluent to the study of polymer–surfactant interactions: oligomeric and micellar chromatographic effects

Complexation of sodium dodecyl sulphate (SDS) with a wide range of molecular weights of poly(ethylene glycol) (PEG) and poly(ethylene oxide) (PEO) has been studied by size exclusion chromatography using aqueous SDS eluent. A multi-angle laser light scattering detector and a differential refractometer were applied to give direct measurement of the molecular weight of complexes without reference to elution volume, since the latter is not a reliable indicator of the complex size. Background light scattering from micellar eluents hampered quantitative size measurements, but was minimal in sub-micellar eluent, where saturated binding was observed for polymers larger than 1000 g mol⁻¹. Multiple peaks and voids were observed in the elution profiles of low molecular weight polymers (up to a mass of 600 g mol⁻¹) in eluent at micellar concentrations. Several sources contribute to this behavior, including micellar chromatographic separation of the PEG oligomers due to their different distribution coefficients between the micellar and water phases. Preliminary results are reported for distribution coefficients of individual oligomers in a 600 g mol⁻¹ PEG sample. Three distinct binding behaviors are observed with increasing degree of polymerization of PEG: no interaction for small glycols, equilibrium partitioning of intermediate oligomers in and out of micelles, and binding of micelles to the larger polymers.