High Molecular Size Fraction Research Articles

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.

Inhibitory effect of NOM in photocatalysis process: Explanation and resolution

The present study explained the inhibitory effect of natural organic matter (NOM) on the photocatalytic degradation of clofibric acid (CA), and attempted to eliminate this inhibition by changing reaction conditions. The NOM degradation was followed by size-exclusion chromatography for dissolved organic carbon and ultraviolet absorption (SEC-DOC and SEC-UV254).The results demonstrated that the deactivation of NOM on catalyst was more dependent on the molecular weight of NOM than on its concentration. Higher molecular weight fractions were prone to adsorb onto the catalysts’ surface and acted as electron-hole scavengers and light filters to reduce the photocatalytic degradation rate of CA. pH adjustment changed the adsorption property of NOM, while, decreasing of its inhibition was not observed. However, the oxygen addition can significantly eliminate this inhibitory effect by increasing the degradation rate of high molecular size fractions of NOM.

High amounts of free aromatic amino acids in the protein-like fluorescence of water-dissolved organic matter

Fluorescence spectroscopy is widely used to study water pollution. The fluorescence of water natural organic matter can be classified into two groups: the protein-like fluorescence originating from aromatic amino acids and the humic fluorescence originating from humic substances. Actually, the precise molecular origin of the protein-like fluorescence is unknown because this fluorescence may be caused by either free amino acids, peptides or proteins. Therefore, we studied the molecular origin of the protein-like fluorescence of Suwannee River natural organic matter and fractions A, B and C + D obtained by size exclusion chromatography/polyacrylamide gel electrophoresis. Fractions were analyzed by reversed-phase high-performance liquid chromatography. The electrophoretic mobilities of fractions varied in the order C + D > B > A and the molecular size in the opposite order. Our results show that the protein-like fluorescence is almost exclusively located in high molecular size fraction A and medium molecular size fraction B. Retention times and fluorescence emission spectra of authentic free aromatic amino acids tyrosine and tryptophan were identical with the retention times and emission spectra of several chromatographic peaks of fractions A and B. More than 50 % of the protein-like fluorescence is due to free aromatic amino acids incorporated in water natural organic matter.

Almond protein hydrolysate fraction modulates the expression of proinflammatory cytokines and enzymes in activated macrophages

Simulated gastrointestinal treatment of almond proteins with pepsin and pancreatic proteases resulting in 16.6% degree of hydrolysis or 1.33 milliequivalent leucine per g protein yielded a hydrolysate that modulated excessive nitric oxide production in lipopolysaccharide-activated RAW264.7 macrophages. After fractionation, a resulting fraction of molecular size > 5 kDa retained the nitric oxide modulatory effect observed initially in the crude hydrolysate. The high molecular size fraction was found to modulate levels of proinflammatory cytokines, interleukin (IL)-6, IL-1β, and tumour necrosis factor (TNF)-α in the activated cells. Immunoblotting analysis indicated that the hydrolysate fraction decreased the expression levels of inflammatory enzyme indicators, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 in the activated cells. RT-PCR analysis showed that treatment of the activated cells with the hydrolysate fraction resulted in the inhibition of relative gene expressions of proinflammatory IL-6, IL-1β, TNF-α, iNOS and COX-2. These results indicate a potential application of almond protein hydrolysates against inflammatory conditions, and will contribute to delineating the possible contributions of proteins to health benefits attributed to almond consumption.

Analysis of electrophoretic soil humic acids fractions by reversed-phase high performance liquid chromatography with on-line absorbance and fluorescence detection

A combination of reversed-phase high performance liquid chromatography (RP HPLC) with on-line absorbance and fluorescence detection was used for analysis of chernozem soil humic acids (HAs) and their fractions A, B and C+D with different electrophoretic mobility (EM) and molecular size (MS). Samples were injected onto the column at the identical volume and absorbance. All chromatograms exhibit the resolution of seven peaks. The estimation of relative recovery of HAs and fractions from the reverse-phase column has been done. High MS fraction A, which possesses the low EM, is essentially more hydrophobic (73% of the fraction amount remained adsorbed on the column) and aliphatic than medium MS and EM fraction B (33% of the fraction amount remained adsorbed on the column). The most hydrophilic and aromatic properties belong to low MS fraction C+D, which possess the highest EM and practically was not adsorbed on the column. The hydrophobicity of the bulk HAs lies within the range of fractions hydrophobicity. The absorption spectra of bulk HAs, electrophoretic fractions A, B, C+D and corresponding RP HPLC peaks were featureless but had differences in the values of absorbance ratio at 300 and 400nm (A3/A4). For fractions A and B this ratio gradually decreased from peak 1 to 7 (from 3.05 to 2.80 and 3.00 to 2.40, respectively). This trend was less pronounced in HAs and practically absent in fraction C+D, where ratio A3/A4 varied within a small range. The strong relationship between fluorescence properties, EM, MS, polarity and aliphaticity/aromaticity of HAs fractions was found. Humic and protein-like fluorescence had different polarity nature. The protein-like fluorescence is located in humic material which irreversibly adsorbed on the reverse-phase column and not subjected to RP HPLC characterization. The humic-like fluorescence at Ex/Em 270/450nm is mostly located in the hydrophilic peak of low MS fraction C+D. Taking into account that high MS fraction A consisted mainly of aliphatic components it is reasonable to suggest that these associations are capable of organizing into micellar structures. These data could be of great environmental importance, because the different fractions might reflect different soil physical–chemical properties.

Antiaggregation potential of berry fractions against pairs of Streptococcus mutans with Fusobacterium nucleatum or Actinomyces naeslundii

Coaggregation is an interspecies adhesion process, which is essential to the development of dental plaque. This is an in vitro study of the composition of the soluble solids in the berry juice molecular size fractions (<10 kDa, FI; 10-100 kDa, FII; >100 kDa, FIII) derived from apple, bilberry, blackcurrant, cloudberry, crowberry and lingonberry and their ability to inhibit and reverse coaggregation of the pairs of common species in dental plaque: Streptococcus mutans with Fusobacterium nucleatum or Actinomyces naeslundii. Inhibitory and reversal activity was found in the molecular size fractions FII and FIII of bilberry, blackcurrant, crowberry and lingonberry. The active fractions contained higher amounts of polyphenols (5-12% of soluble solids) than those without activity (<2% of soluble solids). Proanthocyanidins dominated in the active lingonberry juice fractions FII and FIII and also small amounts of anthocyanins were detected. Anthocyanins, proanthocyanidins and flavonol glycosides were prevalent in FII and FIII fractions of bilberry, blackcurrant and crowberry juices. Comparable amounts of sugars and titratable acids were present in the latter three berry juice fractions of different size. The results indicate that the high molecular size fractions of lingonberry, bilberry, blackcurrant and crowberry juices have antiaggregation potential on common oral bacteria, the potential being associated with their polyphenolic content.

DIRECT EFFECTS OF HUMIC-LIKE SUBSTANCE ON GROWTH, WATER, AND MINERAL NUTRITION OF VARIOUS SPECIES

The influence of humic-like substances was studied on various crop plants (cucumber, maize, pelargonium, and wheat, for example) cultivated under hydroponic conditions. The humic-like product, called SHB®, was extracted from poplar sawdust by a thermical and mechanical process and applied diluted into nutrient solutions of soilless culture at concentrations ranging between 100 to 400 mg SHB® L−1. The application of the humic-like substance induced a biostimulating effect on plant growth of each species studied. Treated plants present a faster development and reach reproductive stage three to five days earlier than control. SHB® also provoked a better efficiency of plant water uptake and they improved the mineral nutrition. It is assumed that most of direct effects on plant metabolisms are related to the low molecular size fractions (LMS) because it is the only part of SHB® which can penetrate into root tissues. But, it is also suggested that high molecular size fractions (HMS) could participate in plant water saving by slowing down its passage in roots.

Electrofocusing the compost organic matter obtained by coupling SEC–PAGE

Humic acids (HA)-like extracted from compost at the beginning ( t 0) and after 130 days of composting ( t 130) were fractionated by coupling size exclusion chromatography to polyacrylamide gel electrophoresis (SEC–PAGE). HA-like fractions with the same molecular size (MS) and electrophoretic mobility were pooled and further characterised by analytical polyacrylamide gel electrofocusing (EF) and compared with HA separated from a Typic Chernozem soil. During the composting process all fractions were subjected to quantitative and qualitative modifications: the high MS fraction was degraded, the mid MS fractions were qualitatively changed, the content of low MS fractions increased and changed qualitatively. The main changes in EF pattern of the non fractionated HA-like t 130 were associated to low MS fractions. Such data seem to be reliable for explanation what mechanisms and monitoring of the evolution of the compost organic matter for their agricultural uses.

Spectroscopic approach for elucidation of structural peculiarities of Andisol soil humic acid fractionated by SEC-PAGE setup

A Andisol humic acid (HA) was fractioned by preparative size exclusion chromatography and polyacrylamide gel electrophoresis in the presence of denaturising agent (SEC-PAGE setup). Three fractions exhibiting high, medium and low molecular size (MS) and showing well-defined and distinct electrophoretic mobility were obtained. These fractions were analysed by UV–visible, FTIR and 3-D fluorescence spectroscopies. UV–visible absorbance and A 465/ A 665 ratio of fractions increased as the MS decreased. FTIR measurements coupled with HCl treatment of samples revealed that aliphatic moieties such as polysaccharides and peptides were mainly localized in high MS fractions while carboxylic functions in low MS fractions. Excitation-emission matrix spectra showed that long-wavelength emissions were almost exclusively observed upon excitation of low MS fraction. These data indicate that fractionation by SEC-PAGE setup yielded pools of soil HA macromolecules showing distinct structural characteristics.

Improvement of Defect Issues for Advanced 193nm Resist

The first generation 193nm resist for sub-90nm DRAM device has brought up defect issues in mass production. Improvement from several aspects has been applied along three different types of defect, 1) micro-bridge, 2) gel-like defect, 3) nozzle drying defect. Diversified attempts were considered from monomer design and polymerization to resist purification process via solvent selection as keys for defect-free solution. Polymerization and following purification process should be well considered to reduce high molecular size fraction. Co-solvent system for resist was also effective to avoid drying out of resist on coating nozzle tip. Also material design of monomers was essentially important to keep composition uniformity in polymer chain and not to generate defect precursor.

Fluorescence of soil humic acids and their fractions obtained by tandem size exclusion chromatography–polyacrylamide gel electrophoresis

Humic acids (HAs) extracted from soils of different origin (chernozem, ferralsol and ranker) and their fractions (A, B and C+D) obtained by tandem size exclusion chromatography–polyacrylamide gel electrophoresis were investigated by steady-state fluorescence spectroscopy in the emission mode. Independently of HA source, high molecular size fractions A and B are shown to be weakly fluorescent. The main fluorophores, especially those emitting at long wavelength (around 500–510 nm), are contained in the polar and low molecular size fractions C+D. As indicated by the observed pH effect, aromatic structures bearing carboxylate and OH substituents may be involved in these longer wavelength emissions.

Aquatic humus from an unpolluted Brazilian dark-brown stream: general characterization and size fractionation of bound heavy metals.

The main pool of dissolved organic carbon in tropical aquatic environments, notably in dark-coloured streams, is concentrated in humic substances (HS). Aquatic HS are large organic molecules formed by micro-biotic degradation of biopolymers and polymerization of smaller organic molecules. From an environmental point of view, the study of metal humic interactions is often aimed at predicting the effect of aquatic HS on the bioavailability of heavy metal ions in the environment. In the present work the aquatic humic substances (HS) isolated from a dark-brown stream (located in an environmental protection area near Cubatão city in São Paulo-State, Brazil) by means of the collector XAD-8 were investigated. FTIR studies showed that the carboxylic carbons are probably the most important binding sites for Hg(II) ions within humic molecules. 13C-NMR and 1H-NMR studies of aquatic HS showed the presence of constituents with a high degree of aromaticity (40% of carbons) and small substitution. A special five-stage tangential-flow ultrafiltration device (UF) was used for size fractionation of the aquatic HS under study and for their metal species in the molecular size range 1-100 kDa (six fractions). The fractionation patterns showed that metal traces remaining in aquatic HS after their XAD-8 isolation have different distributions. Generally, the major percentage of traces of Mn, Cd and Ni (determined by ICP-AES) was preferably complexed by molecules with relatively high molecular size. Cu was bound by fractions with low molecular size and Co showed no preferential binding site in the various humic fractions. Moreover, the species formed between aquatic HS and Hg(II), prepared by spiking (determined by CVAAS), appeared to be concentrated in the relatively high molecular size fraction F1 (> 100 kDa).

Amino acid distribution in soil humic acids fractionated by tandem size exclusion chromatography polyacrylamide gel electrophoresis

The objective of this work was to study humic acids (HAs), and their fractions obtained by coupling size exclusion chromatography-polyacrylamide gel electrophoresis, from arctic soil, podzolic soil, grey forest soil, chernozem and red soil, in respect to their amino acids composition and content. The fractions had exactly defined molecular size (MS) and electrophoretic mobilities (EM). While HAs and fractions had remarkably similar amino acids compositions, they differed greatly in amino acids contents. The highest quantity of amino acids was found in high MS fractions (11.5–21.7%). Amino acids content essentially decreased (to 2.1–5.5%) with the increasing of elution volumes and EM of HAs fractions, independently of HA sources. Separation of HA into less complex fractions with defined molecular and electrophoretic entities, which differ by their content of amino acids, could be of great value for studies on physical-chemical structures of HA and its genesis in soil.

Biodegradation of wheat straw lignocarbohydrate complexes (LCC)

Hot water extractions of ground wheat straw were performed as part of a study to assess digestibility and utility as a substrate for ligninolytic fungi. The treatments gave solutions containing phenolics and carbohydrates. After 48 h reflux percolation or 3 h autoclaving, little additional carbohydrates or phenolics were extracted into the liquors. During percolation, the pH of the wet straw in the reflux thimble fell only slightly and it is difficult to attribute the extraction in this case to an autohydrolytic process catalyzed by organic acids liberated from the straw. Gel permeation chromatography enabled the separation of the extracts into fractions, several of which were free of reducing sugars but contained phenolics and bound carbohydrates. Rechromatography of these fractions failed to separate these two components, indicating a tight linkage between them. The bound sugars were identified as glucose, arabinose, xylose and traces of galactose, but uronic acids were absent. The high molecular size fraction was rich in glucose whereas lower molecular size fractions were richer in xylose and arabinose and poor in glucose.