Polyacrylamide gel electrophoresis of soil humic acid fractionated by size-exclusion chromatography and ultrafiltration

Trubetskoj, Oleg; Trubetskaya, O. E.; Afanas'eva, G. V.; Reznikova, O. I.; Sáiz‐Jiménez, Cesáreo

doi:10.1016/s0021-9673(97)00019-8

Cited by 73 publications

(35 citation statements)

References 18 publications

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“…Trubetskoj et al (1997) used size-exclusion chromatography in the presence of concentrated urea for collecting the HA fractions with different electrophoretic behavior. They have applied this method for the investigation of soil HAs (Trubetskoj et al 1997(Trubetskoj et al , 1999(Trubetskoj et al , 2010Trubetskaya et al 1997Trubetskaya et al , 2002Trubetskaya et al , 2007Trubetskaya et al , 2011Aguer et al 2001;Saiz-Jimenez et al 2006;Sanchez-Cortes et al 2006;Richard et al 2007;Francioso et al 2009). This method is advantageous because a sufficient amount of the fractions with different electrophoretic behavior could be collected for characterization.…”

Section: Infrared Spectroscopy Of the Fractionsmentioning

confidence: 99%

“…As mentioned above, concentrated urea is considered to dissociate the humic substances by disrupting hydrogen bonding and hydrophobic interactions. Trubetskoj et al (1997) developed a method for the fractionation of soil HAs based on the combined use of size-exclusion chromatography and PAGE in the presence of concentrated urea. They have been applying this method to the investigation of soil HAs (Trubetskaya et al 1998(Trubetskaya et al , 2002(Trubetskaya et al , 2011Trubetskoj et al 1999;Aguer et al 2001;Saiz-Jimenez et al 2006;Sanchez-Cortes et al 2006;Guyot et al 2007;Richard et al 2007;Francioso et al 2009).…”

Section: Introductionmentioning

confidence: 99%

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Fractionation of soil humic acids by preparative polyacrylamide gel electrophoresis in the presence of concentrated urea

Karim

Aoyama

2013

Soil Science and Plant Nutrition

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To separate soil humic acids (HAs) into their constituents and characterize them, polyacrylamide gel electrophoresis (PAGE) was carried out in the presence of 7 M urea using a preparative electrophoresis system. Two types of soil HAs were fractionated into nine fractions by PAGE. The dark-colored constituents were recovered from the electrophoretic fractions by precipitation on acidification, and the brown-colored constituents dissolved in the acidic solution of fast-moving fractions were recovered by adsorption onto DAX-8 resin. High-performance size-exclusion chromatography (HPSEC) confirmed that the constituents of the HAs were separated based on their molecular size by PAGE. The dark-colored constituents exhibited higher degrees of humification than did the corresponding unfractionated HAs, except for the constituents remaining in the electrophoretic gels at the end of electrophoresis. Diffuse reflectance infrared spectroscopy revealed that the chemical properties of the dark-colored constituents changed regularly: the content of carboxyl groups decreased and the proportions of proteinous, aliphatic and polysaccharide moieties increased with increasing molecular size. The humification degrees of the constituents adsorbed onto DAX-8 resin were considerably lower than those of the corresponding unfractionated HAs. The chemical properties of the DAX-8-adsorbed constituents were different from those of the dark-colored constituents. Observation of electrophoretic fractions under blue light (470 nm) and HPSEC with fluorescence detection at an excitation wavelength of 460 nm and an emission wavelength of 520 nm showed that green fluorescent substances were largely concentrated in the smallest molecular size fractions and were partitioned into both the dark-colored precipitates and DAX-8-adsorbed fractions. The proportion of organic carbon recovered by precipitation and adsorption onto DAX-8 resin was 45-63%, indicating that substantial parts of the HA constituents were missing. The unrecovered constituents were considered to be acid-soluble, nearly colorless substances. The dissociation of the acid-soluble constituents from the acid-insoluble dark-colored constituents during the preparative PAGE of soil HAs was ascribed to disruption of hydrogen bonding and hydrophobic interactions caused by concentrated urea.

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Section: Infrared Spectroscopy Of the Fractionsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Fractionation of soil humic acids by preparative polyacrylamide gel electrophoresis in the presence of concentrated urea

Karim

Aoyama

2013

Soil Science and Plant Nutrition

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“…There are a number of techniques that are used for measuring the molecular weight distribution of macromolecules such as size exclusion chromatography (SEC), field flow fractionation, ultrafiltration, ultracentrifugation or vapor pressure osmometry (Chin et al, 1991;Chin et al, 1994;Christl et al, 2000;Trubetskoj et al, 1997;Beckett et al, 1987;Perminova et al, 2003). We chose SEC not only because it is widely used but also because it is the most suited for measurements of high molecular weight compounds in atmospheric aerosols, because small amounts of sample can be analyzed, and because it is relatively fast.…”

Section: Sec Analysismentioning

confidence: 99%

Characterization of high molecular weight compounds in urban atmospheric particles

2005

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Abstract. The chemical nature of a large mass fraction of ambient organic aerosol particles is not known. High molecular weight compounds (often named humic-like substances) have recently been detected by several authors and these compounds seem to account for a significant fraction of the total organic aerosol mass. Due to the unknown chemical structure of these compounds quantification as well as a determination of their molecular weight is difficult. In this paper we investigate water soluble humic-like substances in ambient urban aerosol using size exclusion chromatography-UV spectroscopy and laser desorption/ionization mass spectrometry (LDI-MS). LDI-MS was used for the first time to investigate HULIS from atmospheric aerosols. A careful evaluation of the two method shows that both methods complement each other and that both are needed to learn more about the molecular weight distribution and the concentration of humic-like substances. An upper molecular weight limit of humic-like substances of about 700 Da and a concentration of 0.3-1.6 µg/m 3 air can be estimated, corresponding to 9-30% of the total organic carbon for an urban background site.

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“…Moreover, it was confirmed that the brown-colored acid-soluble constituents, as well as the nearly colorless acid-soluble constituents, were dissociated from HAs by concentrated urea treatment. Concentrated urea is considered to disrupt the hydrogen-bonding and hydrophobic interactions [1,12,19]. Therefore, the dissociation of HA constituents observed in this study can be attributed to the disruption of the hydrogen-bonding and hydrophobic interactions triggered by concentrated urea.…”

Section: Distribution Of Ha Constituents Among the Fractionsmentioning

confidence: 68%

Separation of acid-soluble constituents of soil humic acids by dissolution in alkaline urea solution and precipitation with acid

Aoyama

2015

Chem. Biol. Technol. Agric.

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Background: Humic substances are considered to be composed of relatively small, heterogeneous molecules bound by weak linkages. The dissociation of acid-soluble constituents from soil humic acids (HAs) during the preparative polyacrylamide gel electrophoresis in the presence of concentrated urea has been previously demonstrated. Moreover, the dissociation of acid-soluble constituents has been attributed to the action of concentrated urea. The aim of this study was to investigate the effects of concentrated urea on the dissociation of acid-soluble constituents of soil HAs. Results: Three types of soil HAs were solubilized in 0.1 M NaOH containing 7 M urea and precipitated after 16 h by acidifying the samples to pH 1.0. The acid-soluble constituents were separated from the dark-colored precipitates by concentrated urea treatment and accounted for 16-45 % of the total organic carbon in HAs. Approximately half of the acid-soluble constituents was recovered in the DAX-8-adsorbed fraction. The humification degree of the DAX-8-adsorbed fraction was considerably lower than that of the corresponding unfractionated HA. In contrast, the humification degree of the precipitated fraction increased due to the separation of acid-soluble constituents. The molecular sizes of the DAX-8-adsorbed and DAX-8-non-adsorbed fractions, estimated by high-performance size exclusion chromatography, were similar and smaller than the precipitated fraction. Three-dimensional excitationemission matrix fluorescence spectroscopy revealed that the acid-soluble constituents exhibited fluorescence similar to that of fulvic acid (FA), added to which the DAX-8-non-adsorbed fraction exhibited protein-like fluorescence. Diffuse reflectance infrared Fourier transform spectroscopy showed that the DAX-8-adsorbed fraction contained proteinous moieties and the DAX-8-non-adsorbed fraction was rich in proteinous and polysaccharide moieties.

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Polyacrylamide gel electrophoresis of soil humic acid fractionated by size-exclusion chromatography and ultrafiltration

Cited by 73 publications

References 18 publications

Fractionation of soil humic acids by preparative polyacrylamide gel electrophoresis in the presence of concentrated urea

Fractionation of soil humic acids by preparative polyacrylamide gel electrophoresis in the presence of concentrated urea

Characterization of high molecular weight compounds in urban atmospheric particles

Separation of acid-soluble constituents of soil humic acids by dissolution in alkaline urea solution and precipitation with acid

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