Size Exclusion Chromatography Techniques Research Articles

Truths and myths about the determination of molar mass distribution of synthetic and natural polymers by size exclusion chromatography

ABSTRACTThis article discusses various aspects of the determination of molar mass distribution by means of size exclusion chromatography (SEC) in various application modes. The effects of erroneous specific refractive index increment (dn/dc), branching, column performance, and enthalpic interactions on the results obtained by different SEC techniques are discussed. Combination of SEC and a light scattering detector represents the most direct way to the molar mass distribution of all natural and synthetic polymers as it completely eliminates the need for column calibration and to a certain extent eliminates the dependence of the obtained results on some operational variables such as flow rate, temperature, or injected mass. A multiangle light scattering (MALS) photometer has become the most frequently used light scattering detector capable of determination of molecular size as another important polymer characteristic. This article contrasts SEC‐MALS method with other application modes of SEC from the viewpoint of some frequent confusions and misunderstandings. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40111.

On‐Line Monitoring of Molecular Weight Using NIR Spectroscopy in Reactive Extrusion Process

SummaryNear infrared spectroscopy (NIR) is a versatile tool for process control, scientific research and quality management. Consequently it is our aim to utilize NIR technique for the understanding and following of reactive extrusion processes. As preliminary results it was shown the molecular weight of degraded, non‐purified polyester can be measured and forecasted using purely NIR spectra analysis. In this paper we describe the close correlation of the received molecular weight values of partly glycolised Polyethylene terephthalate (PET) samples using reactive extrusion, measured using size exclusion chromatography (SEC) techniques and chemometric models basing on NIR measurements.

Synthesis and characterization of ether bridged polymers and their fluorescent, thermal, conductivity, optical and electrochemical properties

This paper describes the synthesis of novel ether bridged polymers containing carbazole units via HBr elimination. Also, these polymers include the ether bridged aliphatic chains at different lengths in their structures. The synthesized polymers were characterized by FT-IR, NMR and UV–Vis spectroscopy, and their thermal stabilities were determined by TGA measurements. Additionally, the oxidation peak potentials of compounds were demonstrated using cyclic voltammetry method. Fluorescence measurements were performed using dimethylformamide solution, and the optimization of concentrations to obtain the maximal emission intensity was also investigated in dimethylformamide solution. The morphologic properties of the polymers were obtained by using scanning electron microscopy at different amplifications. Moreover, the number average molecular weight, weight average molecular weight and polydispersity index values of polymers were determined by size exclusion chromatography technique. As a result, some remarkable properties related to the fluorescence and thermal measurements of the polymers were obtained. Because of the fluorescence and thermal properties of these polymers, they could be used in various application fields, such as fluorescence sensors and space crafts.

Intrinsically Disordered Enamel Matrix Protein Ameloblastin Forms Ribbon-like Supramolecular Structures via an N-terminal Segment Encoded by Exon 5

Tooth enamel, the hardest tissue in the body, is formed by the evolutionarily highly conserved biomineralization process that is controlled by extracellular matrix proteins. The intrinsically disordered matrix protein ameloblastin (AMBN) is the most abundant nonamelogenin protein of the developing enamel and a key element for correct enamel formation. AMBN was suggested to be a cell adhesion molecule that regulates proliferation and differentiation of ameloblasts. Nevertheless, detailed structural and functional studies on AMBN have been substantially limited by the paucity of the purified nondegraded protein. With this study, we have developed a procedure for production of a highly purified form of recombinant human AMBN in quantities that allowed its structural characterization. Using size exclusion chromatography, analytical ultracentrifugation, transmission electron, and atomic force microscopy techniques, we show that AMBN self-associates into ribbon-like supramolecular structures with average widths and thicknesses of 18 and 0.34 nm, respectively. The AMBN ribbons exhibited lengths ranging from tens to hundreds of nm. Deletion analysis and NMR spectroscopy revealed that an N-terminal segment encoded by exon 5 comprises two short independently structured regions and plays a key role in self-assembly of AMBN.

A Chemometric Approach to Assess the Frying Stability of Cottonseed Oil Blends During Deep‐Frying Process: I. Polar and Polymeric Compound Analyses

AbstractThe main goal of the present study was (i) to determine the formation of degradation products in cottonseed oil (CSO) blends during deep frying process by adsorption and high performance size exclusion chromatography techniques and (ii) to evaluate the impacts of food additives on total polar (TPC) and polymeric compound (PTAG) formation using a chemometric approach. In order to prepare the frying CSO blends; ascorbic palmitate, mixed tocopherols, dimethylpolysiloxane, lecithin and sesame oils were used as additives. To determine the real impacts of additives, a quarter‐fraction factorial experimental design with two levels and five factors was used. The changes in TPC and PTAG data were carefully evaluated during 10 h of frying at 170 ± 5 °C with normal distribution (ND) graphs and analyzed using a one‐way analysis of variance (ANOVA), followed by Tukey's Post‐hoc test (α = 0.05). The results indicated that the increasing values for TPC and PTAG during the frying processes for all blends, TPC and PTAG contents reached maximum levels of 16.37 and 6.01 % respectively, which are below the limit values stated by official authorities for the quality assessment of frying oils. The ANOVA test results were in good agreement with ND graphs and data indicated that the impact of mixed tocopherols was significant for TPC formation, meanwhile the impact of lecithin and ascorbic palmitate × dimethylpolysiloxane were significant for PTAG formation. Thus, the present study should be considered to be a very useful guide for developing new frying oil formulations based on CSO by using food additives.

Synthesis and characterization of aromatic and aliphatic ether bridged polymers containing carbazole moieties

Abstract In this study, the polymers were synthesized ether bridged containing carbazole moieties via HBr elimination. Also, these polymers include the aliphatic chains at the different lengths and aromatic groups in their structures. However, the polymers were characterized by FT-IR, NMR and UV–vis spectroscopy, and their thermal stabilities were determined via TGA measurements. Fluorescence measurements were performed using N,N′-dimethyl formamide solutions and also, the optimization of the concentrations to obtain maximal emission intensity was investigated in N,N′-dimethyl formamide. The morphologic properties of the polymers were investigated at different particle sizes by using Scanning Electron Microscopy (SEM). Additionally, the number average molecular weight (Mn), weight average molecular weight (Mw) and polydispersity index (PDI) values of the polymers were determined by size exclusion chromatography (SEC) technique. Resultantly, the remarkable properties related to the fluorescence and thermal measurements of the polymers were obtained. Therefore, these polymers could be used in various application fields because of the fluorescent and thermal properties.

Size exclusion chromatography as a universal method for the purification of quantum dots bioconjugates

The bioconjugation of fluorescent quantum dots (QDs) and purification of QDs bioconjugates are of vital importance in bioapplications. In this paper, we systematically investigated the purification efficiency of QDs bioconjugates and their stability during separation process by using size exclusion chromatography (SEC) technique, fluorescence spectroscopy, and fluorescence correlation spectroscopy (FCS). In this study, commercial QDs and fluorescein isothiocyanate (FITC) were used as labeling probes, and bovine serum albumin (BSA) and antibody (Erbitux) were used as mode samples. The covalently linkage and the electrostatic interaction were used in bioconjugation procedures. We systematically studied the effects of certain factors such as the scales of column, loading volume, elution buffer, and separation media on the purification efficiency of QDs bioconjugates by a home-built SEC device. And highly pure and monodisperse QDs bioconjugates could be obtained by SEC purification twice under the optimized conditions. Furthermore, we investigated the stability of QDs bioconjugates in different conjugation ways and purification conditions by FCS, and found that the stability of bioconjugates were poor when electrostatic binding mode was used. We also observed that Sephacryl S300 HR (separation range for globular proteins: 1 × 10(4) -1.5 × 10(6) Da) was the best choice for purifying the vast majority of QDs-bioconjugates. Our work described here will be constructive to popularization and applications of QDs in life science.

Development of 68Ga-labelled DTPA galactosyl human serum albumin for liver function imaging

The hepatic asialoglycoprotein receptor is responsible for degradation of desialylated glycoproteins through receptor-mediated endocytosis. It has been shown that imaging of the receptor density using [(99m)Tc]diethylenetriamine pentaacetic acid (DTPA) galactosyl human serum albumin ([(99m)Tc]GSA) allows non-invasive determination of functional hepatocellular mass. Here we present the synthesis and evaluation of [(68)Ga]GSA for the potential use with positron emission tomography (PET). Labelling of GSA with (68)Ga was carried out using a fractionated elution protocol. For quality control thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and size exclusion chromatography (SEC) techniques were evaluated. Stability of [(68)Ga]GSA was studied in phosphate-buffered saline (PBS) and human serum. For in vivo evaluation [(68)Ga]GSA distribution in Lewis rats was compared with [(99m)Tc]GSA by using a dual isotope protocol. PET and planar imaging studies were performed using the same scaled molar dose of [(68)Ga]GSA and [(99m)Tc]GSA. Time-activity curves (TAC) for heart and liver were generated and corresponding parameters calculated (t50, t90). [(68)Ga]GSA can be produced with high radiochemical purity. The best TLC methods for determining potential free (68)Ga include 0.1 M sodium citrate as eluent. None of the TLC methods tested were able to determine potential colloids. This can be achieved by SEC. HPLC confirmed high radiochemical purity (>98%). Stability after 120 min incubation at 37 °C was high in PBS (>95% intact tracer) and low in human serum (∼27% intact tracer). Biodistribution studies simultaneously injecting both tracers showed comparable liver uptake, whereas activity concentration in blood was higher for [(68)Ga]GSA compared to [(99m)Tc]GSA. The [(99m)Tc]GSA TACs exhibited a small degree of hepatic metabolism compared to the [(68)Ga]GSA curves. The mean [(68)Ga]GSA t90 was higher than the mean t90 for [(99m)Tc]GSA. The mean [(68)Ga]GSA t50 was not significantly different from the mean t50 for [(99m)Tc]GSA. This study provides a promising new (68)Ga-labelled compound based on a commercially used kit for imaging the functional hepatocellular mass.

The impact of optimised coagulation on membrane fouling for coagulation/ultrafiltration process

This work aimed to demonstrate the effect of optimised coagulation on minimise fouling for coagulation–ultrafiltration process. Coagulation as pretreatment has the potential to mitigate fouling and enhance flux. Operating parameters were tested by a matrix of experiments for various mixing conditions and coagulant doses. In coagulation experiments, varied shear forces were applied to generate different floc characteristics in order to assess the effect on membrane fouling. Floc properties were investigated with an optical monitoring technique to identify structure, size and growth of flocs. It was shown that stronger flocs are of advantage for fouling mitigation and that the coagulant dosage is crucial for the performance of filtration. The impact of water quality was assessed using general water quality parameters and organic characterisation techniques to investigate the performance of each treatment step. The treatment efficiency was further assessed based on comparing the molecular size fractions of the organic matter before and after coagulation using a size exclusion chromatography technique. The result confirmed the significance of organic character on treatment performance.

Aggregation Mechanism of an IgG2 and two IgG1 Monoclonal Antibodies at low pH: From Oligomers to Larger Aggregates

To identify the aggregation mechanism and the stability characteristics of three different monoclonal antibodies under acidic conditions. The aggregation kinetics is analyzed by a combination of light scattering, size exclusion chromatography and fluorescence techniques and the aggregation data are correlated to protein structure, hydrophobicity, charge and antibody subclass. In the investigated conditions, the antibody aggregation follows a mechanism consisting of two-steps: reversible monomer oligomerization followed by irreversible cluster-cluster aggregation. The kinetics of the two steps is differently affected by the operating conditions: mild destabilizing conditions induce formation of oligomers which are stable within weeks, while stronger denaturing conditions promote aggregation of oligomers to larger aggregates which eventually precipitate. For different antibodies significant differences in both oligomerization and growth rates are found, even for antibodies belonging to the same subclass. For all antibodies the aggregate formation is accompanied by a structure re-organization with an increase in the ordered β-sheet structures. At low pH the aggregation propensity of the investigated antibodies does not correlate with antibody subclass, surface net charge and hydrophobicity of the non-native state. The aggregation mechanism of three antibodies in acidic conditions as well as differences and analogies in their stability behavior has been characterized.

Application of HPLC, Gel electrophoresis and size exclusion chromatography for separation and characterization of storage proteins of soybean and peanut

Summary Certain practical aspects in the separation of large biomolecules have been discussed with the chosen examples of storage proteins from two important crops, viz, soybean and peanut. Importance of seeds and storage proteins along with classification and nomenclature of plant proteins is briefly mentioned. The present paper describes an updated knowledge and status on structural features of glycinin (major storage protein of soybean) and arachin (major storage protein of peanut) with respect to their molecular species, subunit structure, and their association-dissociation behavior by using techniques of high-performance liquid chromatography (HPLC), electrophoresis and size exclusion chromatography (SEC) which supplemented each other.

Filler effect on properties of “All‐Acrylic” copolymer/clay elastomeric materials synthesized by “in situ” nitroxide mediated polymerization

AbstractIn this work, we describe the “in situ” synthesis of “all‐acrylic” copolymer (n‐butyl acrylate‐co‐methyl methacrylate)/clay materials at different low contents of raw and modified Montmorillonite (1–4 wt % versus monomer). The cationic 2,2′ azobis‐(amidinopropane)dihydrochloride initiator was used to modified the clay by cation exchange in combination with the N‐tert‐butyl‐N‐[1‐diethylphosphono‐(2,2‐dimethylpropyl)] (SG1) nitroxide to synthesize the polymer/clay nanocomposite via nitroxide mediated controlled radical polymerization. All synthesized materials are characterized by proton nuclear magnetic resonance, size exclusion chromatography, thermogravimetric analysis and differential scanning calorimetry techniques. The thermo‐mechanical properties of the synthesized materials are also reported. The results show that a decrease in molar masses and/or slight changes in molar compositions of poly (n‐butyl acrylate‐ co‐methyl methacrylate)/clay systems can be balanced by clay loading in polymer matrix, and consequently compensated or masked clay effects on physical properties of obtained materials. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012

Synthesis of Tri- and Multiblock Polymers with Asymmetric Poly(ethylene oxide) End Blocks.

We report the anionic synthesis of poly(ethylene oxide-b-isoprene-b-ethylene oxide) (OIO') triblock copolymers and poly(ethylene oxide-b-styrene-b-butadiene-b-ethylene oxide) (OSBO') tetrablock terpolymers with asymmetrically sized poly(ethylene oxide) (PEO) end blocks using triisopropylsilyloxy-1-propyllithium (TIPSOPrLi) as a protected initiator and diphenylmethyl potassium (DPMK) as the base activator for EO polymerization. All final products and precursors were characterized by size exclusion chromatography (SEC) and nuclear magnetic resonance (NMR) techniques confirming monomodal and narrow molecular weight distributions and controlled molar masses. The growth of independently controlled PEO blocks was additionally demonstrated by the characterization of residual PEO blocks obtained from the ozonolysis of OIO'.

Molecular modelling of polyphthalamides thermal properties: Comparison between modelling and experimental results

The aim of this study was to investigate new polyphthalamides (PPA) through the use of molecular modelling techniques and comparison with experimental data. The objectives were to provide a computer simulation method that can be used to predict the properties of polyphthalamides with a good reliability with respect to the experimentally synthesized materials. Several PPA with a variable amount of aromatic units derived from condensation with hexamethylene diamine (HMDA) and terephthalic acid (TPA) or isophthalic acid (IPA) were synthesized. These polymers were characterized using thermal analysis, size-exclusion chromatography, NMR and viscosimetry techniques. The same PPA were modelled at the atomistic level and molecular dynamics simulations were run. The glass transition temperature (Tg) of the various materials was used to make comparisons between simulations and experiments. The results were found to be in good agreement and the simulation method can be used to predict thermal properties of this class of polyamides.

Anionic synthesis of binary random in-chain multi-functionalized poly(styrene/butadiene/isoprene and dimethyl [4-phenylvinyl)-phenyl]silane) (PS-DPESiH, PB-DPESiH, PI-DPESiH) copolymers

The binary random in-chain silyl-hydride multi-functionalized poly(styrene/butadiene/isoprene and dimethyl[4-(1-phenylvinyl) phenyl]silane) (PS-DPESiH, PB-DPESiH, PI-DPESiH) copolymers were successfully synthesized. These functionalized copolymers were prepared in hexane with n-BuLi as the initiator at 50 °C for 4 h. The silyl-hydride groups were introduced into polymer backbones quantitatively by living anionic polymerization. The copolymers were determined through 1H NMR, size exclusion chromatography (SEC) and differential scanning calorimetry (DSC) techniques, while the number of silyl-hydride groups was calculated and discussed.

Electrochemical syntheses and characterizations of poly(2-aminobenzothiazole)s

Poly(2-aminobenzothiazole)s were synthesized by electropolymerization method using 2-aminobenzothiazole (2ABT) and 6-ethoxy-2-aminobenzothiazole (EtO-2ABT) as the monomers. Cyclic voltammetry (CV) was used to determine the electrochemical oxidation–reduction characteristics. Electrochemical deposition of the polymers on indium tin oxide (ITO) coated glass surface was carried out by “bulk electrolysis” method. Spectroelectrochemistry was used to determine the spectral changes during the electropolymerization. All of the structural, optical and thermal characterizations were made using the polymers obtained on ITO by bulk electrolysis. The CV behaviours were investigated using glassy carbon (GCE) as the working electrode. Structural characterizations were made by FT-IR, NMR, and size exclusion chromatography (SEC) techniques. Optical properties were investigated by UV–vis and fluorescence analyses. Solid state electrical conductivities were measured on polymeric film surface by four point probe technique using an electrometer. Also, morphological characterizations were made by scanning electron microscopy (SEM) technique.

Syntheses and characterizations of oligo(azomethine ether)s derived from 2,2′-[1,4-enylenebis (methyleneoxy)]dibenzaldehyde and 2,2′-[1,2-phenylenebis (methyleneoxy)]dibenzaldehyde

The oligo(azomethine ether)s were synthesized via polycondensation of alkyl diamines with aromatic dialdehydes. Two series of dialdehydes, namely 2,2′-[1,2-phenylenebis(methyleneoxy)]dibenzaldehyde [2,2′-1,2-(PBMODB)] and 2,2′-[1,4-phenylenebis(methyleneoxy)]dibenzaldehyde [2,2′-1,4-(PBMODB)] were prepared from the condensation reactions of salicylaldehyde with o-xylenedibromide and p-xylenedibromide, respectively. The structures of dialdehydes and oligomers were determined by FT-IR, 1H-NMR and 13C-NMR. The thermal analyses of oligomers were conducted by DSC and TG/DTA techniques, respectively. Size exclusion chromatography (SEC) technique was also used to determine molecular weights and molecular weight distributions of oligomers. Electrochemical properties of the synthesized products were investigated.

Circulating MicroRNA in Digestive Tract Cancers

d t For many decades, cell-free nucleic acids have been known to be present in peripheral blood. Several tudies have identified tumor-specific and/or tumor-assoiated alterations in the circulating nucleic acids of paients with various cancers. In recent years, cell-free miroRNA (miRNA) have been stably detected in the plasma nd serum, like other molecules; their presence in the lood has attracted the attention of researchers due to heir potential use as valuable blood biomarkers.1 MiRNAs are short, noncoding RNAs that play important roles in various physiologic and developmental processes. The mature miRNAs are produced from long primary transcripts through 2 sequential cleavage steps. The long primary miRNA transcript is cleaved by the Drosha complex in the nucleus, generating intermediate precursor miRNA. Precursor miRNA is transported by exportin-5 from the nucleus into the cytoplasm, and then subjected to further cleavage by a Dicer RNAase III enzyme, generating a short double-strand miRNA. One strand (guided strand) of mature miRNA is then incorporated into the RNA-induced silencing complex and subsequently hybridize to the 3=-untranslated region of their target mRNAs to repress translation or degrade these mRNAs. Thus, a single miRNA can influence the expression of hundreds of genes and allow them to function in a coordinated manner. Therefore, miRNAs have been implicated as key molecules in all cellular processes. Numerous studies have shown that alterations in miRNA expression correlate with various diseases, including the development and progression of cancer, and some miRNAs can function as oncogenes or tumor suppressors. These findings have opened up a new and interesting field in the diagnosis of cancer and the treatments of cancer patients. Mitchell et al2 first demonstrated that circulating miRNAs had the potential to be new biomarkers in patients with solid cancers. In recent years, several papers have demonstrated that circulating miRNAs can also be detected in the peripheral blood of patients with digestive tract cancers. Although the origins and physiologic functions of cell-free miRNAs in the blood remain to be fully elucidated, a noninvasive assay for miRNAs should be developed to exploit these molecules as potential diagnostic and prognostic biomarkers. This assay undoubtedly contributes to an improvement in the clinical outcomes of cancer patients. In this article, we review the current state of biological and clinical research regarding circulating miRNAs of digestive tract cancer patients and discuss the future perspectives.

Estimating measurement uncertainty in high-performance liquid chromatography methods

In every measurement procedure, it is important to know the components of measurement uncertainty affecting the quality of measured result and reliability of quantified result. The procedure for recognizing measurement uncertainty is not universal but depends on the method and sample type. It has to be made according to good laboratory practice. This paper aims at showing the comparison of measurement uncertainty component estimations for three methods using the high-performance liquid chromatography techniques: determination of the type and content of aromatic hydrocarbons in diesel fuels and petroleum distillates by normal phase high-performance liquid chromatography, determination of nitrates in water samples by ion chromatography, and determination of molecular weights of polystyrene by size exclusion chromatography technique. Both similarity and differences were found during the measurement uncertainty component estimation, and conclusions about influences of certain components on the result uncertainty were made.

Hydrothermal Treatment (HTT) of Microalgae: Detailed Molecular Characterization of HTT Oil in View of HTT Mechanism Elucidation

The understanding of the chemical nature of the oil is important for both the optimization of the process and the design of upgrading strategies for further use as an energy carrier or toward transportation fuels. Hydrothermal treatment (HTT) oil is a complex matrix, whose composition is strongly affected by the feedstock type and by the HTT experimental conditions. In the present work, HTT oil from Desmodesmus sp. was subjected to a detailed chemical analysis. Various characterization techniques (silica gel chromatography, methanolysis, size exclusion chromatography, analytical pyrolysis, elemental analysis, and thermogravimetric techniques) were coupled to gather clearer information on the chemical nature of HTT oil obtained at different reaction times, temperatures, and slurry concentrations. Special attention was paid to the fate of N in the HTT process and the nature of the N-containing species in the oil. By cross-checking results from the chemical characterization of the oil with process data, it was finally possible to identify some different competitive reactions involved in the formation of HTT oil at different conditions. Results show that main compounds obtained at low temperature are still classifiable as lipids, which are extractable without the HTT, together with some short chain algaenan and some hydrophobic protein fragments that end up in the organic solvent phase. At higher temperature (300–375 °C), proteins and cellulose started to break down, giving cyclic dipeptides and amino acids side chains (by pyrolysis-like reactions), carbohydrates derivatives (e.g., furans) and products from the cross reaction of proteins and carbohydrates (e.g., formation of alkyl-pyrrolidinones, pyrazines, pyrroles and melanoidin-like materials). This phenomenon is responsible for the observed increase in oil mass yield with increasing processing temperature, as well as the increase in nitrogen content of the oil. Optimization of the production of fuels and fuel precursors by HTT should be done in conjunction with evaluation of downstream processing options and/or the possibility to recycle unconverted material to the algae cultivation.