Combining electrophoresis with detection under ultraviolet light and multiple ultrafiltration for isolation of humic fluorescence fractions

Polyacrylamide gel electrophoresis of chernozem soil humic acids (HAs) followed by observation under UV (312 nm) excitation light reveals new low molecular weight (MW) fluorescent fractions. Ultrafiltration of HAs sample in 7 M urea on a membrane of low nominal MW retention (NMWR, 5 kDa) was repetitively used for separation of fluorescent and non-fluorescent species. Thirty ultrafiltrates and the final retentate R were obtained. Fluorescence maxima of separate ultrafiltrates were different and non-monotonously changed in the range of 475–505 nm. Fluorescence maxima of less than 490 nm were detected only in the four first utrafiltrates. For further physical–chemical analyses all utrafiltrates were combined into a fraction called UF < 5 (NMW < 5 kDa). Retentate R demonstrated very weak fluorescence under 270 nm excitation, while fluorescence intensity of UF < 5 was about six times higher than of the bulk HAs. Fraction UF < 5 was further ultrafiltrated on membranes of MNWR 3 kDa and 1 kDa, yielding three subfractions UF3-5, UF1-3 and UF < 1 with NMW 3–5 kDa, 1–3 kDa and <1 kDa, respectively. The validation of the UF procedure was performed by size exclusion chromatography on Sephadex G-25 column. The fluorescence maxima were found to be at 505, 488 and 465 nm for UF3-5, UF1-3 and UF < 1, respectively, with increasing of fluorescence intensity from UF3-5 to UF1-3 to UF < 1 fraction. EPR analysis showed that the amount of free radicals was the largest in retentate R and drastically decreased in fluorescent ultrafiltrates. The results demonstrate that more than one fluorophore is present in chernozem soil HAs complex.     

An analysis of dissolved organic matter from freshwater Karelian Lakes using reversed-phase high-performance liquid chromatography with online absorbance and fluorescence analysis

The spectral and optical properties of the fractionated components of dissolved organic matter (DOM) of three freshwater lakes in Karelia were studied using reversed-phase high-performance liquid chromatography (RP-HPLC) with online detection of fluorescence and absorption spectra. It is shown that the DOM fractions are qualitatively similar, but differ quantitatively in the ratio of components and consist of at least three types of fluorophores: (1) hydrophilic “humic-like” fluorophore(s) with the emission maximum in the region of 420 nm and an absorption band at 260–270 nm; (2) hydrophobic “humic-like” fluorophore(s) with the emission maximum at approximately 450 nm that has no characteristic absorption maxima in the region from 220 to 400 nm; and (3) a “protein-like” fluorophore with the emission maximum in the region of 340–350 nm, which is typical of proteins and peptides containing tryptophan.     

Capillary zone electrophoresis of soil humic acid fractions obtained by coupling size-exclusion chromatography and polyacrylamide gel electrophoresis

Capillary zone electrophoresis (CZE) was used for characterisation of soil humic acid (HA) fractions obtained by coupling size-exclusion chromatography with polyacrylamide gel electrophoresis, on the basis of their molecular size and electrophoretic mobility. CZE was conducted using several low alkaline buffers as background electrolyte (BGE): 50 mM carbonate, pH 9.0; 50 mM phosphate, pH 8.5; 50 mM borate, pH 8.3; 50 mM Tris-borate+1 mM EDTA+7 M urea+0.1% sodium dodecyl sulphate (SDS), pH 8.3. Independently of BGE conditions, the effective electrophoretic mobility of HA fractions were in good agreement with their molecular size. The better resolution of HA were obtained in Tris-borate-EDTA buffer with urea and SDS. This results indicated that CZE, mostly with BGE-contained disaggregating agents, is useful for separating HAs in fractions with different molecular sizes.     

Role of fractionation in studying the photochemical properties of humic substances

The purpose of the cooperative research between laboratories of Pushchino Biological Centre Russian Academy of Sciences and Laboratoire de Photochimie Moléculaire et Macromoléculaire, CNRS, France was the investigation of photochemical properties of humic substance fractions with different molecular sizes and electrophoretic mobilities. Humic acids from five different soils were used. The resulting data showed that a great part of photosensitizing fluorophores and chromophores are located in low-molecular fractions of humic acids, independently of their source.     

The Effects of a Strong Disaggregating Agent on Sec-Page of Aquatic and Soil Humic Matter

Abstract

Size-exclusion chromatographic (SEC) fractionation and electrophoretic separation of aquatic humic matter samples from a Finnish lake using Sephadex G-75 with 7 M urea solution as eluent and 10% polyacrylamide gel (PAGE) with urea and sodium dodecyl sulphate solution (SDS), respectively, were performed and compared to similar analyses performed on a Russian chernozem soil humic acid sample and Nordic reference fulvic and humic acid samples. The integrated whole of aquatic humic solutes and soil humic acids were found to exhibit similar SEC-PAGE behaviours. Humic matter was not excessively disaggregated by the 7 M urea and hence SEC-PAGE can with confidence be applied as a coarse, initial fractionation procedure or for certain predeterminations of the structural composition.     

Radiation gas dynamic processes in an explosion caused by high-velocity impact of a cosmic body in mars' atmosphere

Translated from Prikladnaya Mekhanika i Tekhnicheskaya Fizika, No. 6, pp. 35–45, November–December, 1993.