Size-exclusion Chromatography Polyacrylamide Gel Electrophoresis Research Articles

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.

Evaluation of Suwannee River NOM electrophoretic fractions by RP-HPLC with online absorbance and fluorescence detection.

Reversed-phase high-performance liquid chromatography (RP-HPLC) with online absorbance and fluorescence detections was used for the evaluation of Suwannee River natural organic matter (SRNOM) and its fractions A, B, and C+D, obtained by conventional size exclusion chromatography-polyacrylamide gel electrophoresis (SEC-PAGE) setup, for which the electrophoretic mobility (EM) and the absorptivity varied in the order C+D > B > A, and the molecular size (MS) in the opposite order. Analysis of SRNOM and its fractions in part of their relative irreversible adsorption on C18-column and relative distribution of eluted from the column matter on hydrophobic and hydrophilic peaks showed that hydrophobicity of fractions decreased in order: A > B > C+D. The online fluorescence detection showed that SRNOM and its fractions contained at least three groups of humic substances (HS)-like fluorophores with emission maxima at 435, 455-465, and 455/420nm and two protein-like fluorophores with emission maxima at around 300 and 340nm. The HS-like fluorophore with emission maximum at 435nm was located in the hydrophilic peak in all the samples. Those with maxima at 455-465nm were detected in hydrophobic peaks of fractions A and B. SEC-PAGE setup followed by RP-HPLC allowed us to develop new approach of SRNOM separation on less heterogeneous compounds mixture for their further study and structural identification.

1H-NMR and 13C-NMR spectroscopy of chernozem soil humic acid fractionated by combined size-exclusion chromatography and electrophoresis

Humic acid (HA) from chernozem soil was fractionated using combination size-exclusion chromatography–polyacrylamide gel electrophoresis, and three fractions showing distinct electrophoretic mobilities (EM) and molecular sizes (MS) were obtained. Unfractionated HA, all fractions and HA after 7 M urea treatment (HAU) were examined using both solid 13C and 1H in D2O and DMSO-d 6 NMR. The ratio C ar (165−108 ppm)/C alk (108−0 ppm) increased by a factor of 5 from the highest to the lowest MS fraction. This suggests that there is significant heterogeneity in terms of molecular structure and functionality among the various fractions, which differ in both EM and MS. A significant contrast in aromatic range was observed between 1H-NMR spectra of the original unfractionated HA and HAU in DMSO-d 6 solvent. Taking into account that the high MS fraction consists mainly of aliphatic components and the low MS fraction mainly of aromatic components, it is reasonable to suggest that these fractions have different chemical structures. These results are of great environmental importance because different MS fractions might be differently involved in chemical/biological processes in soils.

Tandem size-exclusion chromatography-polyacrylamide gel electrophoresis fractionation of aquatic humic substances: characterization of size-separates with TMAH treatment and pyrolysis-GC/MS

Aquatic humic (HA) and fulvic acids (FA) from two different origins were fractionated by the tandem size-exclusion chromatography and polyacrylamide gel electrophoresis (tandem SECPAGE). Three HA and two FA fractions exhibited different molecular sizes, electrophoretic mobilities and structural compositions based on elemental analyses and pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) after tetramethyl-ammonium hydroxide (TMAH) treatment. Based on comparison of the size-separates and unfractionated bulk samples, the highest molecular size fractions of aquatic humic substances (HS) appear to be enriched in fatty acids (mainly C16 and C18), which are strictly linked to the humic core. This and the presence of low chain fatty acids and unsaturated and di-tri acids may suggest a microbial rather than a vascular plant origin. Even if the majority of lignin derived components in aquatic HS represent relatively high state of biodegradation (oxidation), a certain variation between different sizefractions also exists, supporting a high heterogeneity and composite nature of aquatic HS. Although SEC-PAGE fractionation appears to produce fairly homogeneous HS fractions, the absorption of some urea into of the separates cannot be completely avoided in this method, which limits its suitability for evaluating, especially the presence of N-containing constituents.

Photodegrading properties of soil humic acids fractionated by SEC-PAGE set-up. Are they connected with absorbance?

Humic acids (HAs) extracted from soils of different origins were fractionated using size exclusion chromatography-polyacrylamide gel electrophoresis set-up. For each HAs, three fractions showing distinct electrophoretic mobilities and molecular sizes (MS) were obtained. Bulk HAs and their fractions (25 mg L −1) were then compared for their ability to photosensitize the degradation of 2,4,6-trimethylphenol upon irradiation at 365 nm. Rate coefficients were deduced from pseudo-first order kinetics. For five of the six series, the rate coefficient varied in the order: high MS < medium MS < low MS. Except for one HA, the general tendency was a linear increase of the rate coefficient with 1–10 − A , where A is the absorbance of HAs or fractions solution at 365 nm. It is an indication that the ratio of photosensitizing to non-photosensitizing chromophores is roughly constant among fractions.

Surface-Enhanced Raman Spectroscopy of Chernozem Humic Acid and Their Fractions Obtained by Coupled Size Exclusion Chromatography—Polyacrylamide Gel Electrophoresis (SEC-PAGE)

A humic acid extracted from a chernozem soil was fractionated combining size exclusion chromatography and polyacrylamide electrophoresis (SEC-PAGE). Three fractions named A, B, and C+D, with different electrophoretic mobilities and molecular sizes (MS), were obtained and subsequently characterized by thermochemolysis and surface-enhanced Raman spectroscopy (SERS). The data confirmed that fraction A, with the higher MS, was more aliphatic than fractions B and C+D and, in turn, fractions with lower MS (B and C+D) denoted an enrichment in lignin residues. These structural features explain conformational changes when varying the pH in the humic fraction A and indicated that combination of the two techniques is a good approach for characterizing humic substances.

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

Humic acids (HA) from soils of very different origin and bioclimatic conditions (podzoluvisol, phasozem, chernozem, and ferralsol) were fractionated by coupling size-exclusion chromatography and polyacrylamide gel electrophoresis (tandem SEC–PAGE). From each HA, three fractions—A, B, and C+D, with different molecular sizes and electrophoretic mobilities—were obtained and analysed by thermochemolysis, the products being identified subsequently by gas chromatography/mass spectrometry. Regardless of HA origin, fraction A contained considerably more n-fatty acids and n-alkanes derived from plant waxes than did fractions B and C+D. Lignins accumulated mainly in fractions B and C+D. These results show that tandem SEC–PAGE allowed the separation of soil HA into fractions enriched in different structural components whose distribution was independent of HA genesis.

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.

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

Humic acids (HAs) from three soils of different origin (Chernozem, Ferralsol and Ranker) have been fractionated by coupling size exclusion chromatography (SEC) and polyacrylamide gel electrophoresis (PAGE) on three fractions (fractions A, B, C + D) with different molecular sizes (MSs) and exactly defined electrophoretic mobility (EM). Fractions identically marked had similar EM and MS, independently of HA sources. The photoinductive properties of the whole HAs and their fractions were compared by studying the photoinduced transformation of fenuron at 365 nm. High MS fractions A and B appeared to exhibit poor photoinductive activities compared to the whole HAs, whereas low MS fraction C + D in Chernozem and Ranker were more efficient than the whole HAs. A fourth intermediary fraction containing a mixture of fractions B and C + D with small amount of D was shown to photoinduce poorly the transformation of fenuron. It was therefore concluded that the molecules capable of photoinducing the transformation of fenuron were mainly contained in fraction D. Fluorescence properties of Chernozem HA and its fractions have been tested. Fraction C + D exhibited a very similar fluorescence emission spectrum in comparison with the whole HA and in contrast, the fractions A and B emitted very weakly.

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.