Low-pressure Size Exclusion Chromatography Research Articles

Electron microscopy of stable electrophoretic fractions of natural humic acids – the key to the understanding of their structural organization

Transmission electron microscopy (TEM) with contrast staining by uranyl acetate solution was used to study morphological differences between soil humic acids (HAs) and their A, B and C + D fractions obtained using coupling preparative low-pressure size exclusion chromatography and analytical polyacrylamide gel electrophoresis. The electrophoretic mobility of fractions varied in order C + D B A. The distribution of various morphological elements between fractions showed that large structures such as vesicle-like formations 70–150 nm long and 30–80 nm wide with clear edges were found exclusively in fraction A and occupied ~55% of the TEM image area. On the other hand, long fibrils with a length of 60–100 nm, a width of 4–6 nm and a thickness of 2–3 nm, as well as their bundles with a length of 150 nm and a diameter of 30–70 nm were identified only in the C + D fraction and occupied ~59 % area of TEM images. Smaller morphological elements such as point particles with a diameter of 2–3 nm, ring particles with a diameter of 4–6 nm, worm-shaped short particles with a length of 20–30 nm, and spheroids with a diameter of 10– 30 nm were observed in all samples, but in varying quantities. Significant morphological differences between the fractions can be explained by their composition, previously established by using a few physico-chemical methods. The ratio Car(165–108 ppm)/Calk(108–0 ppm), or aromaticity index, calculated from 13C-NMR, could be one of the indicators of the various morphological structures formation. The obtained TEM results clearly confirm the supramolecular organization of soil HAs.

Transmission electron microscopy of electrophoretic humic acids fractions obtained by coupling size exclusion chromatography-polyacrylamide gel electrophoresis: The next step to understanding structural organization of soil humic matter

The transmission electron microscopy (TEM) with contrast staining by uranyl acetate solution was used to study morphological differences between soil humic acids (HAs) and their A, B and C + D fractions obtained using coupling preparative low-pressure size exclusion chromatography and analytical polyacrylamide gel electrophoresis. The electrophoretic mobility of fractions varied in order C + D > B > A. The distribution of various morphological elements between the bulk HAs and their fractions clearly showed that larger structures, such as smeared drops (70–150 nm long), were formed exclusively in fraction A, whereas elongated fibers (60–100 nm long) and their bundles (>150 nm long) were found only in fraction C + D. Smaller morphological elements such as point particles (2–3 nm in diameter), ring particles (4–6 nm in diameter) with hole in the center, worm-like short particles (20–30 nm long) and spheroids (10–30 nm in diameter) were observed in all samples, but in varying quantities. Significant morphological differences between the samples can be explained by their contrasting properties obtained earlier by different physical–chemical methods. The ratio Car(165–108 ppm)/Calk (108–0 ppm) or aromaticity derived from 13C NMR data appears to be one of the indicators for the formation of different morphological structures. This study provides the new information about morphological structures of soil organic matter that are consistent with the formation of different supramolecular aggregates, probably controlled by the association of smaller ring particles. The data obtained can clarify the role of humic acids in the environment and their involvement in physical–chemical processes in soils.

Isolation and Quantification of Polystyrene Nanoplastics in Tissues by Low Pressure Size Exclusion Chromatography.

Ecotoxicity investigations of plastic nanoparticles (NPs) should pay more attention to their ability to pass barriers, accumulate, and initiate toxicity in cells. The purpose of this study was to develop a simple size exclusion chromatography (SEC) methodology to measure plastic NPs in biological tissues. A SEC column was prepared using a high-resolution gel for large macromolecules to separate plastic NPs from the protein/lipid pools in tissues. It was necessary to prepare the samples in high salt and non-ionic detergent (0.5 M NaCl and 0.2% Tween-20) and apply 0.2% Tween-20 containing 14 mM NaCl for the elution buffer to limit proteins adsorption to NPs. This methodology was able to resolve 50 and 100 nm polystyrene NPs from the protein/lipid pools in tissue homogenates. The fluorescent dye neutral red (NR) was also used for transparent NPs. Moreover, a sample fractionation step was also proposed for plastic NPs concentration using a salting-out methodology with saturated NaCl (5 M) and acetonitrile. Polystyrene NPs partition in acetonitrile, which were further analyzed by SEC. This methodology was tested in two case studies with clams collected in a high boat traffic (harbor) area and with caged freshwater mussels downstream of a large urban area. Although the present methodology was developed with polystyrene NPs it should be amenable to other plastic polymers that react with the NR fluorescent probe.

Macromolecular Structure of a Commercial Humic Acid Sample

The molecular structure of a commercial sample of humic acids (HA) was investigated by membrane dialysis experiments (MD) and low-pressure size-exclusion chromatography (LP-SEC). MD showed that HA molecules were retained by dialysis membrane with a cut-off of 6–8 kDa, independently from HA concentration (15 or 150 mg L−1), NaHCO3 concentration (0.005–2.0 mol L−1), and from propan 2-ol (0–5 v/v %). SEC experiments at low pressure gave chromatograms with a broad peak, with an elution volume between those of the globular proteins bovine serum albumin (molecular weight = 66.5 kDa) and lysozyme from egg (molecular weight = 14.4 kDa). The pattern of the chromatogram did not vary with HA concentration, and second-run chromatograms of single eluted fractions showed relatively sharp peaks. From these data, we reveal that the commercial HA sample analysed has a macromolecular structure rather than being a supramolecular aggregate of relatively small molecules, as recently proposed for some samples of HA obtained from different sources.

Preparation, functionalization and characterization of engineered carbon nanodots.

Carbon-based dots (CDs) and their functionalized (nano)composites have recently attracted attention due to their seemingly easy preparation and numerous potential applications, ranging from those in the biomedical field (i.e., imaging and drug delivery) to those in (opto)electronics (i.e., solar cells and LEDs). This protocol details step-by-step procedures for synthesis, purification, functionalization and characterization of nitrogen-doped carbon nanodots (NCNDs), which we have been preparing for the past few years. First, we describe the bottom-up synthesis of NCNDs, starting with the use of molecular precursors (arginine (Arg) and ethylenediamine (EDA)) and making use of microwave-assisted hydrothermal heating. We also provide guidelines for the purification of these materials, through either dialysis or low-pressure size-exclusion chromatography (SEC). Second, we outline post-functionalization procedures for the surface modification of NCNDs, such as alkylation and amidation reactions. Third, we provide instructions for the preparation of NCNDs with different properties, such as color emission, electrochemistry and chirality. Given the fast evolution of preparations and applications of CDs, issues that might arise from artifacts, errors and impurities should be avoided. In this context, the present protocol aims to provide details and guidelines for the synthesis of high-quality nanomaterials with high reproducibility, for various applications. Furthermore, specific needs might require the CDs to be prepared by different synthetic procedures and/or from different molecular precursors, but such CDs can still benefit from the purification and characterization procedures outlined in this protocol. The sample preparation takes various time frames, ranging from 4 to 18 d, depending on the adopted synthesis and purification steps.

Appraisal and perceptivity of proteins purified from Zygogramma bicolorata (Coleoptera: Chrysomelidae) against pathogens engendering mycotic infection

The multidrug resistivity (MDR) of opportunistic microorganisms is still a major concern which requires the development of a new efficient drug with fewer side effects that can be added to the antifungal armamentarium. This study is the first to report the antifungal nature of proteins purified from the hemolymph of Zygogramma bicolorata, immunized with Micrococcus luteus (MTCC 106) and Escherichia coli (MTCC 443). Immunized hemolymph was assayed for antimicrobial sensitivity against pathogens, Candida albicans (ITCC 4718) and Aspergillus niger (ITCC 5479) by disc diffusion method. The crude extract of hemolymph was separated and purified into fractions by low-pressure size exclusion chromatography using a Sephadex G-25 column. Protein estimation for crude fraction was performed by Lowry's method followed by the assessment of antifungal activity. The study revealed that four fractions (fraction 6, 9, 11 and 15) out of 30 fractions exhibited activity against the test organisms and accommodated high and low molecular weight proteins/peptides of 120, 100, 90 and 66 kDa and two relatively low molecular weight polypeptides (36 and 18 kDa) respectively. Being MDR in dire matter, the efficacy of the drug is momentous.

The association of lipophilic phospholipids with native bovine casein micelles in skim milk: Effect of lactation stage and casein micelle size

A known biological role of casein micelles is to transport calcium from mother to young and provide amino acids for growth and development. Previous reports demonstrated that modified casein micelles can be used to transport and deliver hydrophobic probes. In this study, the distribution of lipid-soluble phospholipids, including sphingomyelins (SM) and phosphatidylcholines (PC), was quantified in whole raw milk, skim raw milk, and casein micelles of various sizes during early, mid, and late lactation stages. Low-pressure size exclusion chromatography was used to separate casein micelles by size, followed by hydrophobic extraction and liquid chromatography-mass spectrometry for the quantification of PC and SM. Results showed that the SM d18:1/23:0, d18:1/22:0, d18:1/16:0, d16:1/22:0, d16:1/23:0, and d18:1/24:0 and the PC 16:0/18:1, 18:0/18:2, and 16:0/16:0 were dominating candidates appearing in maximum concentration in whole raw milk obtained from late lactation, with 21 to 50% of total SM and 16 to 35% of total PC appearing in skim milk. Of the total SM and PC found in skim milk, 35 to 46% of SM and 22 to 29% of PC were associated with the casein micelle fraction. The highest concentrations of SM d18:1/22:0 (341 ± 17 µg/g of casein protein) and PC 16:0/18:1 (180 ± 20 µg/g of casein protein) were found to be associated with the largest casein micelles (diameter = 149 nm) isolated in milk from late lactation, followed by a decrease in concentration as the casein micelle size decreased.

Molecular Size Distribution of Fluorophores in Aquatic Natural Organic Matter: Application of HPSEC with Multi-Wavelength Absorption and Fluorescence Detection Following LPSEC-PAGE Fractionation.

Analytical high performance size exclusion liquid chromatography (HPSEC) with multiwavelength absorbance and fluorescence detections was used for the analysis of molecular size distribution and optical properties of dissolved natural organic matter. Experiments were conducted on Suwannee River organic matter (SRNOM) and its fractions A, B, C+D preliminary obtained by combination of preparative low pressure size exclusion chromatography and polyacrylamide gel electrophoresis (LPSEC-PAGE) and purified by dialysis on membrane with nominal cutoff 10 kDa, the fractions molecular size varied in order A > B > C + D > 10 kDa. The multistep fractionation of SRNOM enabled the size-separation of at least five types of humic-like fluorophores within NOM showing emission maxima at 465, 450, 435, 420, and 405 nm. The decrease of the humic-like emission maxima paralleled the decrease of the nominal molecular size of fluorescent SRNOM. The protein-like fluorescence was split into tyrosine-like and tryptophan-like fluorophores and only detected in fractions A and B. This work provides new data on the optical properties of size-fractionated NOM, which consistent with the formation of supramolecular NOM assemblies, likely controlled by association of low-molecular size components. It is clearly observed for the high molecular size fraction A, containing free amino acids or short peptides. The combination of several different fractionation procedures is very useful for obtaining less complex NOM compounds and understanding the NOM function in the environment.

Synthesis, Separation, and Characterization of Small and Highly Fluorescent Nitrogen-Doped Carbon NanoDots.

A facile bottom-up approach to carbon nanodots (CNDs) is reported, using a microwave-assisted procedure under controlled conditions. The as-prepared nitrogen-doped CNDs (NCNDs) show narrow size-distribution, abundant surface traps and functional groups, resulting in tunable fluorescent emission and excellent solubility in water. Moreover, we present a general method for the separation of NCNDs by low-pressure size-exclusion chromatography, leading to an even narrower size distribution, different surface composition, and optical properties. They display among the smallest size and the highest FLQYs reported so far. (13)C-enriched starting materials produced N(13) CNDs suitable for thorough NMR studies, which gave useful information on their molecular structure. Moreover, they can be easily functionalized and can be used as water-soluble carriers. This work provides an avenue to size- and surface-controllable and structurally defined NCNDs for applications in areas such as optoelectronics, biomedicine, and bioimaging.

Comparison of extractable soil carbon and dissolved organic carbon by their molecular characteristics

The aim of this study was to compare the molecular characteristics of extractable soil carbon from biotopes of the Rašeliník watershed and dissolved organic carbon (DOC) from Rašeliník creek, by using the 0.1M pyrophosphate, 0.01M CaCl2 and Britton-Robinson buffer as extraction agents. The molecular weight Mr and weight-average molecular weight Mw, determined by the low pressure size exclusion chromatography, increased in the following sequence: humic substances (HS) in CaCl2 < aquatic HS < HS in sodium pyrophosphate ≤ HS in a buffer. Elution curves of all humic substances were characteristic by two peaks with predominant low-molecular fraction. Mr and Mw values of aquatic humic substances were 5.9 and 7.9 kDa, respectively, and proportion of this low-molecular fraction reached 97%. This corresponds to the fact, that the main fraction of HS in surface waters constitute fulvic acids. Using soil extraction in CaCl2 we obtained the fraction of organic carbon similar to the humic substances contain in DOC. Differences in quantity of humic substances extracted from soils among CaCl2 (mean 0.42 ± 0.39), Britton-Robinson buffer (34.9 ± 11.2) and sodium pyrophosphate (293.2 ± 113.4) were statistically significant. The A465/A665 ratio negatively correlated with molecular weight of humic substances.

Preclinical Manufacture of an Anti-HER2 scFv-PEG-DSPE, Liposome-Inserting Conjugate. 1. Gram-Scale Production and Purification

A GMP-compliant process is described for producing F5cys-PEG-lipid conjugate. This material fuses with preformed, drug-loaded liposomes, to form "immunoliposomes" that bind to HER2/neu overexpressing carcinomas, stimulates drug internalization, and ideally improves the encapsulated drug's therapeutic index. The soluble, single-chain, variable region antibody fragment, designated F5cys, was produced in E. coli strain RV308 using high-density cultures. Affinity adsorption onto horizontally tumbled Streamline rProtein-A resin robustly recovered F5cys from high-pressure-disrupted, whole-cell homogenates. Two product-related impurity classes were identified: F5cys with mid-sequence discontinuities and F5cys with remnants of a pelB leader peptide. Low-pressure cation exchange chromatography, conducted at elevated pH under reducing conditions, enriched target F5cys relative to these impurities and prepared a C-terminal cysteine for conjugation. Site-directed conjugation, conducted at pH 5.9 +/- 0.1 with reaction monitoring and cysteine quenching, yielded F5cys-MP-PEG(2000)-DSPE. Low-pressure size exclusion chromatography separated spontaneously formed, high-molecular-weight conjugate micelles from low-molecular-weight impurities. When formulated at 1-2 mg/mL in 10 mM trisodium citrate, 10% sucrose (w/v), at pH 6.4 (HCl), the conjugate was stable when stored below -70 degrees C. Six scale-up lots were compared. The largest 40-L culture produced enough F5cys to manufacture 2,085 mg of conjugate, enough to support planned preclinical and future clinical trials. The conjugate was 93% pure, as measured by polyacrylamide gel electrophoresis. Impurities were primarily identified as product-related. Residual endotoxin, rProtein A, and genomic DNA, were at acceptable levels. This study successfully addressed a necessary step in the scale-up of immunoliposome-encapsulated therapeutics.

Efficient purification of small unsaturated oligoglucuronides by reversed-phase chromatography

Ion-exchange chromatography has been applied to purification of unsaturated oligoglucuronans. After an isocratic elution on a strong anion-exchange column, the collected fractions were desalted by low pressure size exclusion chromatography. However, this efficient separation was limited by the time required to desalt. So, we developed a reversed-phase chromatography method using back ionization of oligomers. Two C18 columns were tested with trifluoroacetic acid (TFA 0.7%) as eluent. Different selectivities and column stabilities were observed in this acidic condition. The scale up for semi-preparative applications enabled us to recover pure unsaturated oligoglucuronans without desalting step.

A complete set of hyaluronan fragments obtained from hydrolysis catalyzed by hyaluronidase: Application to studies of hyaluronan mass distribution by simple HPLC devices

Hyaluronan (HA) has different biological functions according to its molar mass; short HA fragments are involved in inflammation processes and angiogenesis, whereas native HA is not. Physicochemically, studies of native HA hydrolysis catalyzed by bovine testicular hyaluronidase (HAase) have suggested that kinetic parameters depend on HA chain length. To study the influence of HA chain length in more detail, and to try to correlate the physicochemical and biological properties of HA, HA hydrolysis catalyzed by HAase was used in a new procedure to obtain HA fragments of different molar masses. HA fragments (10-mg scale) with a molar mass from 800 to 300,000 g mol −1 were prepared, purified using low-pressure size exclusion chromatography (SEC), lyophilized, and characterized in molar mass by either mass spectrometry or HPLC–SEC–multiangle laser light scattering. The polydispersity index of the purified fractions was less than 1.25. The complete set of HA standards obtained was used to calibrate our routine HPLC–SEC device using only a refractive index (RI) detector. We showed that the N-acetyl- d-glucosamine reducing end assay and the calibrated HPLC–SEC–RI gave equivalent kinetic data. In addition, the HPLC–SEC–RI furnished the mass distribution of the polysaccharide during its hydrolysis.

Large-scale preparation of α-D-(14)-oligogalacturonic acids from pectic acid

An efficient and inexpensive method for large-scale preparation of α-D-(1[Formula: see text]4)-oligogalacturonic acids (oligo-GalA), up to DP 5, from pectic acid is described. Pectic acid was digested with a commercially available pectinase to yield a mixture of oligo-GalA, which was effectively separated by a combination of low-pressure – size-exclusion chromatography based on ion-exchange chromatography to obtain pure oligo-GalA of DP 2-5. Key words: pectic acid, galacturonic acid, galabiose, galatriose, pectinase.

Search for plant-induced technetium species with size-exclusion and reversed-phase ion-pair chromatography

Abstract In this work an attempt is made to have a closer look at technetium species (TcX) induced by spinach plants, grown on TcO − 4 containing nutrient solutions. To this end, size-exclusion chromatography (SEC) and reversed-phase ion-pair chromatography (RP-IPC) were used: The performance was established with model compounds, i.e., anionic Tc-DTPA and cationic Tc-cyclam, used as mimics for TcX. Under the conditions applied, the information retrieved from high performance RP-IPC was minimal due to strong interactions of the technetium species with the Nucleosil C18 stationary phase. However, in low-pressure SEC non-size effects allowed differentiation of TcX into two distinct classes of technetium compounds, i.e., TcX1 and TcX2. From retention behavior in SEC, it was possible to speculate on the chemical properties of these two technetium species.

Implementation of an HPLC polystyrene divinylbenzene column for separation of activated sludge exopolymers

A high-pressure size-exclusion chromatography procedure for separation of activated sludge exopolymers was investigated and implemented in order to achieve a documented and faster separation procedure than the conventional low-pressure size-exclusion chromatography methods previously suggested in studies of activated sludges from a traditional and an advanced from activated sludges from a traditional and an advanced activated sludge treatment plant performing biological nitrogen and phosphorus removal were used. For both types of exopolymers the separation was largely dependent on the mobile-phase. Using NaCl and ortho-phosphate in the molar proportion 10:1 it was shown that for a mobile-phase ionic strength of 0.011 and pH in the range 7.0–10.0 no irreversible column adsorption occurred. For a standard procedure a mobile-phase pH of 7.0 was selected in order to separate the exopolymers into the maximal number of peaks. Alterations in the mobile-phase, i.e. using a pH below 7.0 or a mobile-phase ionic strength above 0.011, changed the separation for both types of exopolymers and caused irreversible column adsorption. Similarly, using deionized water as the mobile-phase irreversible column adsorption was introduced and the separation was strongly affected. The method applicability for qualitative characterization of exopolymers was demonstrated. The method was found to be successful in showing differences and similarities between exopolymers from two different activated sludge treatment plants, showing degradation of exopolymer compounds due to exoenzymes in the exopolymers and showing that snow melting and subsequent high conductivity in the inlet to the waste-water treatment plant had an impact on the chromatographic fingerprint of the extracted exopolymers.

Characterization of genital tract growth factor‐like activity from testis sheaths and fat body of Heliothis virescens

AbstractTestis sheaths and fat body from developing male pupae of Heliothis virescens synthesize soluble growth factor‐like products when exposed to 20‐hydroxyecdysone (20HE). These factors promote growth and development of the genital tract. Saline extracts of modified Grace's medium and 20HE‐treated testis sheaths and fat body were subjected to heat, freeze‐thaw, organic solvents, and low pressure size exclusion chromatography. Although extracts were stable to repeated freeze‐thawing, activity was lost after exposure to organic solvents; activities of fractions heavier than 6.5 KDa were inhibited by heating to 100°C. Size exclusion chromatography yielded 10 active testis extract fractions and 9 active fat body fractions. Although the approximate molecular weights of most of the extract fractions were similar, enzyme studies using protease, lipase, and α‐amylase indicated differences in the chemistry of active fractions derived from the two tissues. Active factors were inhibited by protease or lipase or both enzymes. © 1994 Wiley‐Liss, Inc.

The application of the size exclusion chromatography/ viscometry technique to the determination of molar-mass and long-chain branching in polychloroprene

The coupling of a low-pressure size exclusion chromatography (SEC) with a modified Ubbelohde capillary viscometer is described. This SEC/viscometry system measures the flow time of aliquot fractions (5ml) of the SEC effluent along with the refractive index change. This dual detection leads to the determination of the intrinsic viscosity as a function of the elution volume, thus allowing a precise use of Benoit's universal calibration. Moreover information of the branching factors (degree of long-chain branching, long-chain branching frequency) can be calculated under certain assumptions. As an example the changes in molar-mass distribution and branching factors during mechanical shear degradation (mastication) of special polychloroprene samples were investigated. It is shown that the SEC/viscometry system is especially suitable for the characterization of polymers with broad molar-mass distribution and extremely high molecular tails. The data provided by this method are useful for the investigation of the viscoelastic behaviour of concentated polychloroprene solutions and for quality control of polymers in the rubber and adhesives industries.

Interactions of lens proteins. Concentration dependence of β-crystallin aggregation

The concentration-dependence of beta-crystallin aggregation was studied by both high- and low-pressure size exclusion chromatography of calf lens cortical extract (0.5-249 mg ml-1), nuclear extract (0.2-304 mg ml-1) and purified beta-crystallins (0.4-52 mg ml-1). A reversible equilibrium exists between beta H(igh)-crystallins (predominantly hexamers) and a portion of the beta L(ow)-crystallins (predominantly dimers). Association to beta H-crystallin is more extensive in the nucleus than in the cortex. Moreover, at physiological protein concentrations, the weight percentage of beta H-crystallins is greater than that of beta L-crystallins, in both the cortex and the nucleus. beta H-Crystallins can be fully dissociated to beta L-crystallin at low protein concentration. On the other hand, not all of the beta L species are competent to associate to beta H at high concentrations. This association appears to be directly dependent on the presence of beta B1 chains. We therefore propose that the concentration and spatial distribution of beta H-crystallin in vivo is actually regulated by differential synthesis of beta B1 polypeptides.