High-performance liquid chromatographic fractionation and characterization of fulvic acid

High-performance immobilized metal ion affinity chromatography (HP-IMAC) was used to fractionate humic substances (HS) based on their affinity for the immobilized copper(II) ion using acidic and glycine eluents. The work was carried out with two naturally occurring aqueous fulvic acids and commercially available Suwannee River fulvic acid. The IMAC-fractionated HS were then characterized by reversed-phase high-performance liquid chromatography (RP-HPLC) and size exclusion chromatography. The results showed that the affinity HS fraction eluted first using an acidic pH=2 eluent exhibited a relatively high hydrophilic character, whereas the fraction eluted later using a glycine eluent exhibited both a higher hydrophobic character and larger molecular size. On the other hand, the HS fraction with no affinity for the immobilized copper had low molecular size. The affinity of the HS fraction for copper(II) increased with increasing molecular weight. Based on the composite results of three different HS, it is evident that strong relationships exist between affinity, molecular weight, and hydrophilic/hydrophobic properties during the HP-IMAC fractionation. The results presented here have significance for understanding the nature of chemical interactions at the molecular level between dissolved organic matter and trace metals. IMAC, coupled with other liquid chromatographic strategies, is a promising tool for chemical fractionation and characterization of HS.     

Prediction of Properties of Soil Humic Substances from FTIR Spectra using Partial Least Squares Regression

Abstract

Humic substances (HS) play a key role in aquatic and terrestrial ecosystems. The understanding of the ecological functionalities of HS is based on the analysis of their properties, which is normally a very time-consuming procedure. Therefore we tested the possibility to apply the partial least squares regression (PLSR) method in connection with the mid infrared Fourier transform (FTIR) spectra of a series of soil humic substances for the prediction of different HS properties. The results with humic acid (HA) and fulvic acid (FA) fractions of soil HS from different environments show the possibility to predict several properties of an unknown soil HA with satisfying reliability above all the elemental composition.     

Determination of Organic Compounds,Fulvic Acid,Humic Acid,and Humin in Peat and Sapropel Alkaline Extracts

Black, brown, and light peat and sapropel were analyzed as natural sources of organic and humic substances. These specific substances are applicable in industry, agriculture, the environment, and biomedicine with well-known and novel approaches. Analysis of the organic compounds fulvic acid, humic acid, and humin in different peat and sapropel extracts from Lithuania was performed in this study. The dominant organic compound was bis(tert-butyldimethylsilyl) carbonate, which varied from 6.90% to 25.68% in peat extracts. The highest mass fraction of malonic acid amide was in the sapropel extract; it varied from 12.44% to 26.84%. Significant amounts of acetohydroxamic, lactic, and glycolic acid derivatives were identified in peat and sapropel extracts. Comparing the two extraction methods, it was concluded that active maceration was more efficient than ultrasound extraction in yielding higher amounts of organic compounds. The highest amounts of fulvic acid (1%) and humic acid and humin (15.3%) were determined in pure brown peat samples. This research on humic substances is useful to characterize the peat of different origins, to develop possible aspects of standardization, and to describe potential of the chemical constituents.     

High-performance liquid chromatography (HPLC)-size exclusion chromatography (SEC) for qualitative detection of humic substances and dissolved organic matter in mineral soils and peats in Lithuania

High-performance liquid chromatography (HPLC) with size exclusion (SEC) separation function was used to isolate and examine the molecular mass (MM) distributions and polydispersity of humic substances (HSs) and dissolved organic matter (DOM) from mineral soils and peats. The aim was to improve their detailed characterisation and to inform of their soil carbon (C) sequestration and environmental quality. This is the first study conducted in Lithuania in which HSs and DOM, separated from two soil types, have been used to characterise soil at the molecular level. The HPLC-SEC, as a separation method, was coupled with diode-array detection (DAD), thus enabling the separation of molecular fractions. Results showed that HPLC-SEC can be used to determine the MM of HSs in soil, provided that the relation between retention time and MM is known and a suitable method for fitting the HS peak is available. The UV-spectra analysis showed that DOM has a larger MM (M_w = 2439–3436 Da), which contains more aliphatic C. The HS fraction has a smaller MM (M_w = 2776 Da), with aromatic structures that reflect a higher aromaticity. Separated fractions had characteristic MMs of humic acid (HA) and fulvic acid (FA) and DOM. The HSs separated from peat samples were characterised by higher aromaticity, humification and stability. The HSs extracted from mineral soil samples showed a higher degradability level. The results also show the MM distribution and polydispersity of HS and DOM fractions (M_w/M_n = 1.009–1.252) are relatively homogenous in both soil types. Findings confirm that chromatographic and spectrometric parameters can be used for characterisation of both HSs and DOM, and for detecting changes in organic matter quality. Moreover, they can also be used for a further understanding the C-cycle and could be applied for enhancing soil C-sequestration and informing environmental quality management.     

Characterisation of humic substances by acid catalysed transesterification

The chemical composition of low molecular weight moieties linked to the core structures of humic substances (HS) are of substantial importance for the understanding of the chemical structures and mode of interactions of HS with other substances in the environment. In this study a novel approach to characterise certain low molecular weight compounds bound to HS is suggested. The method includes transesterification (TE) of ester and amide bound structures, and esterification (E) of free carboxylic groups using acid catalysed methanolysis followed by gas chromatography (GC)-mass spectrometry (MS) and GC-FID analysis. Methanolysis of five HS of different origin, demonstrated the presence of multifunctional hydroxy-substituted benzoic acids, hexoses, and long chain fatty acids. Based on GC-FID and addition of the internal standard before methanolysis, the total amounts of low molecular weight material could be estimated. In case of HS from the aqueous sources the yields were below 1%, whereas in case of the HS derived from lignite the yield was significantly higher. The hydrophobic long chain fatty acids constituted about one-third of this material. Principal component analysis (PCA), which was used for extended data evaluation, differentiated between the samples in terms of chemical composition.     

Analysis of aquatic‐phase natural organic matter by optimized LDI‐MS method

The composition and physiochemical properties of aquatic‐phase natural organic matter (NOM) are most important problems for both environmental studies and water industry. Laser desorption/ionization (LDI) mass spectrometry facilitated successful examinations of NOM, as humic and fulvic acids in NOM are readily ionized by the nitrogen laser. In this study, hydrophobic NOMs (HPO NOMs) from river, reservoir and waste water were characterized by this technique. The effect of analytical variables like concentration, solvent composition and laser energy was investigated. The exact masses of small molecular NOM moieties in the range of 200–1200 m/z were determined in reflectron mode. In addition, spectra of post‐source‐decay experiments in this range showed that some compounds from different natural NOMs had the same fragmental ions. In the large mass range of 1200–15 000 Da, macromolecules and their aggregates were found in HPO NOMs from natural waters. Highly humic HPO exhibited mass peaks larger than 8000 Da. On the other hand, the waste water and reservoir water mainly had relatively smaller molecules of about 2000 Da. The LDI‐MS measurements indicated that highly humic river waters were able to form large aggregates and membrane foulants, while the HPO NOMs from waste water and reservoir water were unlikely to form large aggregates. Copyright © 2014 John Wiley & Sons, Ltd.     

Chromatographic separation simulation of metal‐chelating peptides from surface plasmon resonance binding parameters

Some metal‐chelating peptides have antioxidant properties, with potential nutrition, health, and cosmetics applications. This study aimed to simulate their separation on immobilized metal ion affinity chromatography from their affinity constant for immobilized metal ion determined in surface plasmon resonance, both technics are based on peptide‐metal ion interactions. In our approach, first, the affinity constant of synthetic peptides was determined by surface plasmon resonance and used as input data to numerically simulate the chromatographic separation with a transport‐dispersive model based on Langmuir adsorption isotherm. Then, chromatographic separation was applied on the same peptides to determine their retention time and compare this experimental t_R with the simulated t_R obtained from simulation from surface plasmon resonance data. For the investigated peptides, the relative values of t_R were comparable. Hence, our study demonstrated the pertinence of such numerical simulation correlating immobilized metal ion affinity chromatography and surface plasmon resonance.     

Electrospray ionization mass spectrometry of terrestrial humic substances and their size fractions

Electrospray ionization mass spectrometry (ESI-MS) was used to evaluate the average molecular mass of terrestrial humic substances, such as humic (HA) and fulvic (FA) acids from a soil, and humic acid from a lignite (NDL). Their ESI mass spectra, by direct infusion, gave average molecular masses comparable to those previously obtained for aquatic humic materials. The soil HA and FA were further separated in size-fractions by preparative high performance size exclusion chromatography (HPSEC) and analyzed with ESI-MS by both direct infusion and a further on-line analytical HPSEC. Unexpectedly, their average molecular mass was only slightly less than for the bulk sample and, despite different nominal molecular size, did not substantially vary among size-fractions. The values increased significantly (up to around 1200 Da) after on-line analytical HPSEC for the HA bulk sample, at both pH 8 and 4, and for the HA size-fractions when pH was reduced from 8 to 4. It was noticed that HA size-fractions at pH 8 were separated by on-line HPSEC in further peaks showing average masses which progressively increased with elution volume. Furthermore, when the HA and NDL bulk samples were sequentially ultracentrifuged at increasing rotational speed, their supernatants showed mass values which were larger than bulk samples and increased with rotational speed. These variations in mass values indicate that the electrospray ionization is dependent on the composition of the humic molecular mixtures and increases when their heterogeneity is progressively reduced. It is suggested that the dominance of hydrophobic compounds in humic supramolecular associations may inhibit the electrospray ionization of hydrophilic components. Our results show that ESI-MS is reasonably applicable to humic substances only after an extensive reduction of their chemical complexity.     

土壤腐殖质的分析化学研究进展

对土壤腐殖质的分析化学研究进展作了综述。具体包含如下几个方面的内容；土壤腐殖质的组成和结构的研究，土壤腐殖质的起源和形成的研究，土壤腐殖质与土壤中有机，无机物质的相互作用的研究以及有机物料的分解，转化及其对腐殖质影响的研究，同时，对土壤腐殖质的研究中有待进一步用分析化学方法解决的几个问题进行了探讨。     

食品中有机酸的高效液相色谱分析法

对离子交换色谱法、离子排斥色谱法和反相高效液相色谱法分析食品中有机酸的特色和近几年的研究与应用状况作一综述。     

基于色谱法与质谱法分析唾液酸的衍生方法的研究进展

唾液酸是一类拥有9碳核心结构的单糖,广泛存在于脊椎动物体内,以及部分无脊椎动物、真菌和细菌中。唾液酸在生物体内可以以游离形式存在,也经常作为重要的组成部分连接于糖缀合物的末端,这使得其能够参与到多项生理活动中,且与炎症、癌症等疾病密切相关。以色谱法、质谱法为核心的分析方法,是表征生物样品中唾液酸的最主要研究方法。为了提高检测灵敏度或色谱分离度,在分析之前通常需要对唾液酸进行衍生化处理。经过几十年的发展,研究者们逐步建立了多种衍生化方法。该文从单糖、自由唾液酸、N/O-聚糖、糖脂的角度综述了用于色谱与质谱分析唾液酸的衍生方法,并展望了该领域的应用及发展趋势。     

A speciation methodology to study the contributions of humic-like and fulvic-like acids to the mobilization of metals from compost using size exclusion chromatography-ultraviolet absorption-inductively coupled plasma mass spectrometry and deconvolution analysis

High performance size-exclusion chromatography (HP-SEC) with UV absorption for organic matter detection and inductively coupled plasma mass spectrometry (ICP-MS) for elemental detection have been used to study the mobilization of metals from compost as a function of pH and the molecular mass of their complexes with dissolved organic matter (DOM). Due to its heterogeneous nature, organic matter mobilized from compost shows a continuous distribution of molecular masses in the range studied (up to 80 kDa). In order to differentiate between the contribution of humic and fulvic acids (FA) to the organic matter mobilized in the pH range 5-10, their UV absorption chromatographic profiles have been deconvoluted with respect to the adjusted gaussian profiles of the humic and fulvic acids isolated from compost. Results show a preponderant contribution of fulvic acids at low pH values and an increasing percentage of humic acids (HA) mobilized at basic pH (up to 49% of total DOM at pH 10). A similar deconvolution procedure has been applied to the ICP-MS chromatograms of selected metals (Co, Cu, Pb and Bi). In general, both fulvic and humic acids contribute to the mobilization of divalent transition metals, such as copper or cobalt, whereas bismuth or lead are preferably associated to humic acids. Non-humic substances (HS) also contribute to the mobilization of cations, especially at acidic pHs. These conclusions have been extended to different elements based on deconvolution analysis results at pH 7.     

The association between selenium and humic substances in forested ecosystems—laboratory evidence

In the soils and aquatic systems of coniferous forests, selenium is usually associated with humic substances. To clarify further some of the mechanisms involved, labelled and unlabelled selenite were added to two forest floors and to a brownwater lake. Sequential extraction procedures and chromatographic methods were used to evaluate the resulting association between selenium and humic substances. It was observed that the forest floors fixed most of the added selenite by means of microbial reductive incorporation and that selenium was preferentially incorporated into lowmolecular-weight fractions of the humic substances. By contrast, selenium reduction was much slower in the brown-water lake and instead, inorganic complexation of selenite to metal–humic complexes was important during the experiment, provided that the concentrations of competing ligands were low.     

The application of electrospray ionization coupled to ultrahigh resolution mass spectrometry for the molecular characterization of natural organic matter

Mass spectrometry has recently played a key role in the understanding of natural organic matter (NOM) by providing molecular-level details about its composition. NOM, a complex assemblage of organic molecules present in natural waters and soils/sediments, has the ability to bind and transport anthropogenic materials. An improved understanding of its composition is crucial in order to understand how pollutants interact with NOM and how NOM cycles through global carbon cycles. In the past, low-resolution (>3000) mass analyzers have offered some insights into the structure of NOM, but emerging ultrahigh resolution (>200,000) techniques such as electrospray ionization (ESI) coupled to Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) have significantly advanced our knowledge of NOM chemistry. Here, a review of the recent literature on the advancements of NOM characterization and the applications of mass spectrometry to this central task is presented. Various methods for the analysis and display of the extremely complex mass spectra, such as the van Krevelen diagram and Kendrick mass defect analysis, are discussed. We also review tandem mass spectrometry techniques employed to gain structural information about NOM components. Finally, we show how ESI-FT-ICR-MS has been applied to examine specific issues that are important to the NOM scientific community, such as NOM reactivity, transport and fate, degradation, and existence of components, which are indicators of NOM origin. In general, ultrahigh resolution provided by FT-ICR-MS is essential for the complete separation of the thousands of peaks present in the complex NOM mixture and will clearly lead to additional future advancements in the areas of aquatic, soil, and analytical chemistry.     

Development of an automated system for isolation and purification of humic substances

Characterization of humic substances (HS) in environmental samples generally involves labor-intensive and time-consuming isolation and purification procedures. In this paper, the development of an automated system for HS isolation and purification is described. The novelty of the developed system lies in the way the multiple liquids and columns used in the isolation/purification procedure are handled in both forward and back-elution mode by solenoid valves. The automated procedure significantly reduces the total throughput time needed, from 6–7 days to 48 h, and the amount of labor to obtain purified HS for further characterization. Chemical characterization of purified HS showed that results were in good agreement with previously published values for HS from a variety of sources, including the IHSS standard HS collection. It was also shown that the general properties of HS were consistent among the different source materials (soil, waste, aquatic) used in this study. The developed system greatly facilitates isolation and characterization of HS and reduces the risk of potential (time-dependent) alteration of HS properties in the manual procedure. Figure Photograph of the rear (left) and front (right) of the automated system for the isolation and purification of humic substances Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

泥炭土连续碱抽提腐殖酸的分子结构特征

从Pahokee泥炭土中连续碱抽提分离出8个不同的腐殖酸级分，并对每一级分进行了元素分析、傅里叶变换红外光谱（FTIR）、固态^13C核磁共振（^13CNMR)、高效排阻色谱（HPSEC）等一系列定性、定量研究。结果表明：所分离出的8个腐殖酸级分存在明显的结构性质差异，随提取和蔼的增加，O／C原子比由0．52减少到0．36，H／C原子比由1．05增加到1．52，相应于结构中含氧基团的减少和脂族基的增加，表观分子量也由7．7K增加到22．1K。同时，^13C NMR显示长链脂肪碳结构由无定型向晶型转变。此工作表明在所研究的腐殖酸中可能存在分别具有芳香或脂肪特性的两类腐殖酸结构，每种类型都有不同的分子量分布、元素组成、基团结构和母质来源。在特定的环境因素下，不同类型的腐殖酸会共存于同一体系中，增加了腐殖物质的非均匀性和复杂性。     

Quantification of Diaminomaleonitrile Using High Performance Liquid Chromatography with UV and Electrospray Ionization Mass Spectrometric Detection

Abstract

A method was developed to quantify diaminomaleonitrile (DAMN) in complex aqueous matrices using high performance liquid chromatography with UV and electrospray ionization mass spectrometric (ESI-MS) detection. The method is rapid (analytical times <10 min), able to quantify DAMN against complex backgrounds, and achieved with part per billion detection limits. Given the central role of DAMN in generating heterocycles of known biological significance, this study provides a rapid and sensitive method for quantifying DAMN in complex solutions.     

Characterization of synthetic nucleic acids by electrospray ionization quadrupole time-of-flight mass spectrometry

The potential of electrospray ionization quadrupole-quadrupole-time-of-flight mass spectrometry (ESI-QqTOF-MS) for the characterization of synthetic nucleic acids was evaluated. Oligonucleotides ranging in size from 12 up to 51 nucleotides were analyzed via direct infusion MS as well as via liquid chromatography (LC) online hyphenated to MS. These experiments proved the outstanding mass spectrometric performance of the TOF mass analyzer in regard of accuracy, reproducibility, resolution, and sensitivity. During a 1-min run, the monoisotopic mass of (dT)(24) was measured with a maximum relative mass deviation of 7.64 ppm proving the high mass accuracy of the TOF analyzer. Over a period of 1 h, mean deviations were determined in the range between -3.58 ppm and 3.06 ppm demonstrating the high stability of the applied external calibration. The molecular mass of a 51-mer was measured with a deviation smaller than 3.23 ppm from the theoretical value. The resolution exceeded a value of m/Deltam = 20 000 (m is the measured mass and Deltam the full peak width at half-maximum), which enabled the separation of the isotopic peaks of all investigated oligonucleotides. Because of the outstanding transmission and detection efficiency of the TOF mass analyzer, detection limits in the amol/microl to low fmol/microl range were reached. The usability of LC-ESI-QqTOF-MS for the qualitative and quantitative analysis of synthetic oligonucleotide mixtures was demonstrated.