Accepted Manuscript Title: Novel short-column anion-exchange chromatography for soil and peat humic substances profiling by step-wise gradient of high pH aqueous sodium ethylenediaminetetraacetate Author: Milan Hutta Janka R´aczov´a R´obert G´ora Juraj Pessl PII: DOI: Reference:

S0021-9673(15)00879-1 http://dx.doi.org/doi:10.1016/j.chroma.2015.06.032 CHROMA 356590

To appear in:

Journal of Chromatography A

Received date: Revised date: Accepted date:

17-2-2015 23-5-2015 15-6-2015

Please cite this article as: M. Hutta, J. R´aczov´a, R. G´ora, J. Pessl, Novel short-column anion-exchange chromatography for soil and peat humic substances profiling by stepwise gradient of high pH aqueous sodium ethylenediaminetetraacetate, Journal of Chromatography A (2015), http://dx.doi.org/10.1016/j.chroma.2015.06.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Highlights of article manuscript

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“Novel short-column anion-exchange chromatography for soil and peat humic substances profiling by step-wise gradient of high pH aqueous sodium ethylenediaminetetraacetate”

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by

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Milan Hutta*, Janka Ráczová, Róbert Góra, Juraj Pessl

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 DEAE anex chromatography for profiling humic substances in alkaline extract of soil.  Novel humic and fulvic acids characterization close to their operational definition.

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 Results show discrimination power, fast analysis, good sensitivity and reproducibility.

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Corresponding Author

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Milan Hutta

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Novel short-column anion-exchange chromatography for soil and peat humic substances profiling by step-wise gradient of high pH aqueous sodium ethylenediaminetetraacetate

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E-mail: [email protected]

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Abstract

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Novel anion-exchange liquid chromatographic method with step gradient of aqueous EDTA4- based mobile phase elution has been developed to profile available Slovak soil humic substances and alkaline extracts of various soils. The method utilize short glass column (30mmx3 mm) filled in with hydrolytically stable particles (60 μm diameter) Separon HEMABIO 1000 having (diethylamino)ethyl functional groups. Step gradient was programmed by mixing mobile phase composed of aqueous solution of sodium EDTA (pH 12.0; 5 mmol L-1) and mobile phase constituted of aqueous solution of sodium EDTA (pH 12.0, 500 mmol L-1). The FLD of HSs was set to excitation wavelength 480 nm and emission wavelength 530 nm (em). Separation mechanism was studied by use of selected aromatic acids related to humic acids with the aid of UV spectrophotometric detection at 280 nm. The proposed method benefits from high ionic strength (I = 5 mol L-1) of the end mobile phase buffer and provides high recovery of humic acids (98%). Accurate and reproducible profiling of studied humic substances, alkaline extracts of various types of soils enables straightforward characterization and differentiation of HSs in arable and forest soils. Selected model aromatic acids were used for separation mechanism elucidation.

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1. Introduction

Milan Hutta*, Janka Ráczová, Róbert Góra, Juraj Pessl

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Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynská dolina CH-2, 842 15 Bratislava, Slovak Republic

Keywords:

Anion-exchange chromatography HEMA DEAE, Na4EDTA humic substances soils aromatic acids

An ion-exchange mechanism is one of frequently considered explanation of interactions among humic acids, fulvic acids and clays in soil milieu [1]. However, its application in ionexchange chromatographic (IEC) separation of humic substances (HSs) is relatively rare. For instance, even Thurman and Malcolm [2] in their classical article on preparative isolation of aquatic HSs preferred combination of adsorption chromatography and size-exclusion chromatography, whereas ion-exchange was utilized only for hydrogen saturation before lyophilization to obtain low ash aqueous HSs. Later, Zhou and Banks [3] used three kinds of resin sorbents for separation, fractionation into 6 fractions and clean-up humic acid (HA) components in a process consisted of adsorption chromatography followed by cation IEC and

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anion IEC, rotary vacuum evaporation, desalting and crystallization. The procedure recovered 96% of the total mass of HA. Tebbe and Vahjen [4] resolved interference of HA and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and yeast by a two-step protocol. In the first step, they extracted as much DNA as possible (crude DNA) from a soil sample. I In the second step, the DNA was purified by ion-exchange columns (Qiagen-Tip 500), that removed 97% of the initially co-extracted humic acids. Similarly, major information related to the ionic properties of HSs and/or ion-exchange separation mechanisms, can be found mainly in information resources focused to removal of HSs during natural or waste water treatment, by use of ion-exchange membranes and their fouling by HSs [4,5]. In spite of direct evidence for potential of ion-exchange mechanisms, there is lack of articles on the topics. However, some aspects of the problem were already discussed in Hutta et al. [7] article chapter 6, that reviewed advance of the topic since the review on separation of HS published by Janoš [8] more than 11 years ago. The situation with respect to relatively small progress in the application of ionexchange mechanism for HSs separation still pertains. Exception is the article of Ráczová and Hutta [9], partly covering last two years advances in studies of step gradient ion-exchange liquid chromatography of HSs and profiling of alkali extracts of soils. Based on the preliminary study, we came to the conclusion that very needed is research of behavior and properties of HSs directly in alkaline medium of relatively concentrated solutions of alkaline metal hydroxides. The medium is, by definition of humic substances, also operational medium coming in the very first contact with soil sample and humic substances (aqueous NaOH solution with concentrations from 0.1 to 0.5 mol L-1). NaOH solution is still considered to be the most effective reagent quantitatively isolating HSs from soils [10]. Numerous isolation procedures were also proposed by Schnitzer [11], Orlov [12], Stevenson [13] and Tan [14]. Hayes et al. [15] published isolation of humic substances from soil using aqueous extractants of different pH and XAD resins.The key ingredient in all the HSs isolation methods, regardless the variations, is alkaline solution containing entirely or predominantly NaOH. We decided to develop HPLC methods for separation and characterization of polyelectrolytic humic acids (HA) and fulvic acids (FA) by utilization of alkaline mobile phases having composition as close to the isolation media as is possible. Generally accepted opinion is that FA have higher content of ionisable functional groups (mainly carboxylic groups) than HA, what is supported by studies of Lubal et al. [16] on the acido-basic and complexation properties of humic acids, Gondar et al. [17] and Campitelli [18] who focused to characterization of acid– base properties of FA and HA. Another important source of information for IEC method development is research of HA and FA by electrophoretic methods [19-22] or in a broader sense by electro-separation methods and their combinations with the other separation methods [23,24]. Off-line combination of preparative isotachophoresis and size-exclusion chromatography in analysis of HA was published by Radičová et al. [25]. Very valuable with respect to our work is information provided by Pokorná et al. [26] on the stability of humic acids in alkaline media studied by electrophoresis and mass spectrometry. The article shows that during long lasting storage (up to hundred days) of alkaline HA solutions their degradation was observed. Elaboration of fast isolation and separation methods is therefore needed. Due to high complexity of HSs, the obvious trend is toward combined and comprehensive separation and detection method. Investigation, design and development was published for combinations of liquid chromatography methods (RP and SEC) by Góra et al. [27] and for resolving the chemical heterogeneity of natural organic matter with the aid of comprehensive 2D liquid chromatography

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by Duarte et al [28]. For combined methods, beside older RP-HPLC methods [29]. Recently, hydrophilic interaction liquid chromatography (HILIC) separation mechanism was successfully evaluated also for separation of aquatic HSs by Wang et al. [30]. The intention of our recent work was to evaluate analytical potential of stepwise gradients [26,28] in combination with anion-exchange chromatography in highly alkaline separation medium for analysis and characterization HSs and aqueous NaOH soil extracts. High content of anion ionogenic functional groups in HA and FA retaining chelate forming properties, led our decision to study potential of competitive chelating ligand EDTA as the only buffering component in aqueous mobile phase at pH values higher than 12.0. As far as we know no anionexchange method using step-wise gradient elution of humic substances under alkaline conditions was published by the other authors. We report novel simple anion-exchange HPLC separation method with UVspectrophotometric and fluorimetric detection for the analysis and characterization of commercially available HSs, various types of organic matter extracted by alkali solutions from soil samples and several model aromatic acids representing various negative charge characteristics.

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2. Experimental

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2.1 Chemicals and reagents

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The reagents were of analytical-reagent grade. Water for gradient HPLC was prepared by Labconco Pro-PS unit (Labconco, Kansas City, USA). NaOH (Merck, Darmstadt, Germany) was used for preparation of solutions of humic substances, extraction soils and preparation of standard solutions of aromatic acids. EDTA (Sigma Aldrich, Steinheim, Germany) was used for buffered mobile phases preparation. Commercial humic acids were purchased from Sigma-Aldrich (denoted as SALD) with relative molecular mass 500-1000 (product catalogue Sigma Aldrich, Steinheim, Germany). Humic acid Ecohum (denoted as ECOH) was commercially available fertilizer isolated from peat by ammonia solution at industrial scale. Humic acids from localities Cerová and Suchá Hora, Slovakia (denoted as CERV and SUHO, respectively) were isolated from peat using fractionation procedure recommended by IHSS [31,32]. Soil samples for this study were collected from different localities Slovakia (Gbely – abbr. GBEL, Šajdikové Humence - SAJD, Senec - SENC and Kremnica-Skalka - SKAL) and they were pedologically characterized by Dlapa [33]. Pedologic characteristics of soils are summarized in Table 1. All studied aromatic acids (benzenecarboxylic acid, 2-hydroxy-benzenecarboxylic acid, benzene-1,2-dicarboxylic acid, benzene-1,4-dicarboxylic acid, 2,6-dihydroxy-benzenecarboxylic acid, benzene-1,2,4 tricarboxylic acid, benzene-1,2,5-tricarboxylic acid and benzene-1,2,4,5-tetracarboxylic acid) and hydroxybenzene were obtained from Sigma-Aldrich (Sigma Aldrich, Steinheim, Germany). Table 1

2.2 Instrumentation

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Studies in selected groups of HA retention behavior and retention of other components in soil isolates were carried out by the HPLC system LaChrom (Merck-Hitachi, Darmstadt, Germany). Measured dwell volume of the system (including column) was 1.46 mL and this volume must be considered when gradient mixing profile and chromatogram appearance is compared. Compact Glass Column (30mmx3 mm) filled with Separon HEMA-BIO 1000 DEAE sorbent (60 μm particle diameter, Tessek, Prague, Czech Republic) was utilized. Chromatographic conditions summary is given in Table 2.

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For gradient elution different gradients were tested. They were prepared in HPLC system by programming various gradient shapes by mixing mobile phase A and B.. Mobile phase A composition was: aqueous solution of sodium salt of EDTA (pH 12.0, 5 mmol L-1). Mobile phase B composition was: aqueous solution of sodium salt of EDTA (pH 12.0, 500 mmol L-1). Gradient program (Fig. 3) was set from 0.0 to 1.9 min isocratic 0% B in A; than from 2.0 to 3.9 min isocratic 1% B in A, from 4.0 min to 5.9 min isocratic 2% B in A, from 6.0 min to 7.9 min isocratic 4% B in A, from 8.0 min to 9.9 min isocratic 8% B in A, from 10.0 min to 11.9 min isocratic 16% B in A, from 12.0 min to 13.9 min isocratic 32% B in A, from 14.0 min to 15.9 min isocratic 64% B in A, from 16.0 min to 30.0 min isocratic 100% B in A. Column reequilibration (not shown in Fig. 3) was set from 30.1 to 33.0 min by linear decrease from 100% B in A to 0% B in A and between runs 5 min mobile phase A pumping was maintained at flow rate: 1.0 mL min-1 and column oven temperature 40°C. pH meter InoLab pH 730 (WTW, Weilheim, Germany) with combined glass/argentochloride electrode was used and each pH measurement was corrected with respect to the electrode alkaline error. Table 2

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2.3 Mobile phase buffer preparation Buffer solution A (5.0 mmol L-1 EDTA pH 12.0) was prepared by dilution of buffer solution B with aqueous solution of NaOH pH 12.0 .Buffer solution B (500.0 mmol L-1 EDTA pH 12.0) was prepared by dissolving 146.12 g of EDTA and made up the volume to 1000 mL. Final pH was adjusted to 12.0 by addition NaOH pellets.

2.4 Sample Preparation The stock solutions of HA were prepared daily fresh by dissolution 3.0 mg of HA per 1.00 mL of 0.01 mol L-1 NaOH solution. The isolation procedure was: 1.00 g of sieved soil sample (15 mesh) was added to 10.00 mL of 1.0 mol L-1 NaOH solution and agitated for 30 minutes. After centrifugal separation (3500 rpm, 15 min) liquid phase was decanted and the solid residue was discarded. Prior to analysis the stock solutions and the soil extracts were centrifuged for 15 minutes at 3500 rpm. Solutions of aromatic acids were prepared by dissolution of weighed

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aromatic acids at 1.00 mg mL-1 concentration levels in aqueous solution containing 0.01 mol L-1 NaOH (except benzoic acid prepared at concentration 5.00 mg mL-1).

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3. Results and discussion Acid Features of Humic Substances

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Humic substances are characterized by high polydispersity of almost all properties and features including those useful for their separation, detection and characterization [7]. Authors gathered information on effective charge characteristics of HA and FA from various published sources and found valuable information in articles [17,18]. The modified information is schematically drawn in Figure 1. In reference to Fig. 1 content of carboxylic groups is indirectly proportional to relative molecular mass and their acidity is defined by span of pKa with maximum distribution between pH 3.0 and 5.0. Hydroxyl groups are of three kinds and their total content is given by the sum of their content in phenols, saccharides and reduced hydroquinones. The reactivity of saccharide hydroxyl groups is usually lower than phenolic hydroxyl groups. Weakly acidic phenolic functional groups having pKa between 8 and 10 add characteristic highly negative charge and unique chemical behavior of HSs in medium alkaline solutions. Together with carboxylic groups they determine ion-exchange capacity of HSs in many interactions. Their acido-basic properties, high content and structural positional variability of neighbouring groups give them also metal chelate-forming ability, bonding water molecules by ion-dipol interactions and bridging interactions of metal cations. Rule of thumb is that higher content of aromatic systems in HSs structure results in lower content of carboxylic groups and phenolic functional groups. Hydroxyl groups in saccharides as HSs constituents are very weak acids with pKa within the range 12 to 13 [38-40].

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Figure 1

Apparently, under the common conditions saccharide aliphatic hydroxyl groups have no chance to put in effect of HSs net negative charge increase. However, one must bear in mind that HSs are operationally defined on the basis of their isolation by, for instance, aqueous 0.1 mol L-1 NaOH solution (pH 13.0) from soil. Therefore they should have net negative charge defined by all three types of contributing functional groups mentioned above. All the data and their interpretation can aid in design of isolation and separation procedures, but also in design of ionexchange chromatography and electro-separation methods for separation of HSs. 3.2. Chromatographic separation Bearing in mind the aim of the work, i.e. design and evaluation of anion-exchange chromatography method for separation HSs under very alkaline conditions approaching pH of the most effective extractants of HSs from soil and potentially high effective charge that HSs have under the conditions, we searched for available and effective anionogenic substance as the buffer and elution component of mobile phase. We calculated effective charge vs pH for 10 common anionogenic substances and decided to evaluate EDTA as the only component of

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aqueous mobile phase that exerts both acceptable acido-basic buffering properties and metal chelate forming ability, because under the isolation conditions many metals and non-metals codissolve with HSs. Ethylenediamminetetraacetic acid [EDTA; 2,2',2'',2'''-(ethane-1,2-diyldinitrilo) tetraacetic acid] is widely used to dissolve various inorganic salts including lime, substance accompanying humic substances in certain soil types. Its usefulness in modern separation methods is evident as is discussed in review article of Ráczová [36] on properties, determination and utilization of EDTA. One useful role of EDTA as a hexadentate ligand and chelating agent is in its ability to "sequester" metal ions such as Ca2+ and Fe3+ present frequently in soil samples. After being bound by EDTA, metal ions remain in solution but exhibit diminished reactivity. EDTA in alkaline medium can bear high net charge up to the value of four minus commonly as tetrasodium or tetrapotassium salt, thus creating conditions of relatively high ionic strength at relatively low concentration (no solubility problems) in the aqueous mobile phase. The selection of proper ion-exchanger was influenced partly by previous experience of authors, its commercial availability, beneficial properties and good references. Various bioseparations based on ion-exchange separation mechanisms using macroreticular hydroxyethyl methacrylate (HEMA) ion-exchangers filled in special pressure resistant (up to 20MPa) glass columns are available [34-36]. HEMA is chemically and hydrolytically resistant material for separation bio-macromolecules as well as low molecular mass substances, because it is available with pore diameters having exclusion limits from 40 000 Da to over 1 000 000 Da. According to the authors and manufacturer information [37] the great advantage of HEMA sorbents is its negligible degradation even under strongly basic conditions enabling in-situ column sanitation by 0.1 mol L-1 NaOH solution as is required by FDA protocols. In our preliminary experiments with strong anion exchanger Separon HEMA Bio 1000 Q (30mmx3 mm) and linear gradients of mobile phase A to mobile phase B within 40 minutes we got low chromatographic recovery of each of studied HA falling within range 67% to 84%. Calculation of recovery was based on ratios of overall fluorescence intensity of collected HA in mobile phase after ion-exchange chromatography to the intensity of HA fluorescence when injection volume of HA was diluted to the same volume of mobile phase after HA transport through the HPLC system (FIA mode with detached column, average composition of the mobile phase gradient). The experiments attempted to increase the recovery by column oven temperature increase revealed strange behavior of HA as can be observed in Figure 2. We achieved very moderate increase of recovery of HA from 71% to 85%.

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Figure 2

In the following experiments column oven temperature was fixed to 40°C and we selected Separon HEMA 1000 bio DEAE. We achieved much lower retention of HA in alkaline mobile phase due to the fact that pKa values of (diethyl)aminoethyl groups are around pH 12.3. That facilitates pH manipulation with the aim to decrease HA retention in an anion-exchange mode. After selecting optimized conditions based on humic substances peak separation, the run time of the proposed assay was 38 min with stepwise gradient elution. Figure 3

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Chromatograms of HSs profiling are shown in Figure 3 and they show ability of the devised system to produce characteristic peak-like profiles of HAs of various origin or location as-well-as alkaline extracts of soils collected in various localities in Slovakia. Isolation of HSs was done by extraction utilizing aqueous 1.0 mol L-1 NaOH. Observed peaks are enforced by the step gradient shape. Extracts of arable soils from localities Gbely and Senec gave too low analytical signal (not shown in Fig. 3) in profiles compared to Ecohum (ECOH) and SigmaAldrich (SALD) HA, visible are HA from Sucha hora (SUHO) and Cerova (CERV). The main observed difference is in the proportion of signals attributed to non-retained or weakly retained HSs (of yellow color – beside fluorescence also absorbing light at 420 nm) eluted by 5 mmol L-1 EDTA4- and medium to strongly retained HSs eluted successively by mobile phase containing between 50 mmol L-1 and 500 mmol L-1 EDTA4- at pH 12.0. Chromatographic recovery approached 98%.

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Chromatographic profiles in Figure 4 differentiate among HSs isolated in raw extracts from arable soils (GBEL, SENC) and raw extracts of forest soils (SKAL, SAJD). Due to relatively large particles of the used sorbent the described separation system offers apparently low efficiency, however attempts to increase the transport efficiency by use of 25 µm particles failed, because the apparent efficiency calculated from peak width is the result of nature of humic substances and also shape of step gradient. Separation mechanism from the point-of-view of effective charge of humic substances was studied by the properly selected set of aromatic acids representing net charges 1-, 2-, 3- and 4-. Representatives of the charge 1- at the pH 12.0 are phenol, benzoic acid; charge 2- salicylic acid, phthalic and terephthalic acid; charge 3- are 2,6-dihydroxybenzoic acid, benzene-1,2,4 tricarboxylic acid and benzene-1,2,5-tricarboxylic acid; charge 4- is benzene-1,2,4,5tetracarboxylic acid, that were analyzed in the separation system under almost identical conditions as described for the profiling of the HSs. From the comparison of information coded in Figure 3 and Figure 4 , respectively,by application simple analogy we can conclude that under the actual separation conditions non-retained or weakly retained fraction of humic substances represents substances bearing effective charge from 0 to 1- eluted from the ion exchanger by 5 mmol L-1 EDTA 4-, whereas retained humic substances probably bear effective charge from 3- to more than 4- with the highest signal attributable to the effective charge higher than 4- (eluted by 328 mmol L-1 l EDTA 4-).

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The improved gradient shape resulted also in improvement of such chromatographic figures-of-merit as are retention time stability and peak area reproducibility (see Table 3 and Fig. 5 ) with retention time relative standard deviation lower than ±1% except phenol (±3%) and peak areas RSD better than ±5%. Estimated LODs fall within the limits 31-66 ng per injection and estimated LOQs fall within the limits 100-220 ng per injection. Recovery of aromatic acids evaluated from chromatograms (absorbance at 280 nm) is 99%. Figure 5

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Further testing of the system by probes from the very last period revealed that the anionexchange method is influenced also by some specific interactions in the case of 2,4-dihydroxy benzoate. Anions of aromatic acids containing in the structure that feature are retained at least 10 times longer than already investigated analytes. Separation mechanism of wider group of aromatic acids is now under the investigation and maybe salting-out effects to the HEMA matrix take also place. On the base of the results and after more detailed investigation in near future the authors will try to elucidate the IEC separation mechanism.

4. Conclusion

Acknowledgements

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Novel short column anion exchange chromatography method enables relatively simple profiling HSs, soils alkali extracts and study of retention behavior of selected aromatic acids. Mobile phase composed of EDTA adjusted by NaOH to pH12.0 and more as the part of stepwise gradient (10 steps, 38 min duration) brings benefit for the routine HSs characterization. The advantages of the proposed method are simplicity, reproducibility, repeatability and sufficient precision ensuring that it is suitable for HSs fractionation in combinations with the other HPLC separation mechanisms (e.g. SEC, RP-HPLC, HILIC, IMAC) for comprehensive characterization or fractionation of various types of humic acids, fulvic acids, soil extracts.

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Table 3

The authors are thankful to projects VEGA 1/1349/12, APVV-0583-11 for financial support within the frame of VVCE-0070-07.

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10. K.H. Tan, Ch. 3 Extraction and fractionation of humic substances, in: Humic Matter in Soil and the Environment, Principles and Controversies, Marcel Dekker, Inc., 2003. 11. M. Schnitzer, Organic matter characterization, in: A.L. Page, R.H. Miller, D.R. Keeney (Eds.), Methods of Soil Analysis, Part 2, Agronomy series. No.9. Am. Soc. Agronomy and Soil Sci. Soc. Am., Inc., Publ., Madison, WI. 1982, pp. 581-594. 12. D.S. Orlov, Humus Acids of Soils. Moscow University Press, in: K.H. Tan, (ed.), Amerind Publ. New Delhi, India, 1985. 13. F.J. Stevenson, Humus Chemistry. Genesis, Composition, Reactions. 2nd Ed., John Wiley & Sons, New York, NY 1994. 14. K.H. Tan, Soil Sampling, Preparation, and Analysis. Marcel Dekker, Inc., New York, NY 1996. 15. T.M. Hayes, M.H.B. Hayes, J.O. Skjemstad, R.S. Swift, R.L. Malcolm, Isolation of humic substances from soil using aqueous extractants of different pH and XAD resins, and their characterization by 13C-NMR, in: C.E. Clapp, M.H.B. Hayes, N. Senesi, S.M. Griffith (Eds.), Humic Substances and Organic Matter in Soil and Water Environments: Characteristics, Transformation, and Interactions, Proc. 7th Conf. Intern. Humic Substances Soc., Univ. West Indies, St. Augustine, Trinidad, and Tobago, 3-8 July 1994, International Humic Substances Soc., Inc., Dept. Soil, Water, and Climate, Univ. Minnesota, St. Paul, MN, 1997, pp 13-24. 16. P. Lubal, D. Široký, D. Fetsch, J. Havel, The acidobasic and complexation properties of humic acids. Study of complexation of Czech humic acids with metal ions, Talanta 47, (1998) 401-412. 17. D. Gondar, R. Lopez, S. Fiol, J.M. Antelo, F. Arce, Characterization and acid–base properties of fulvic and humic acids isolated from two horizons of an ombrotrophic peat bog, Geoderma 126 (2005) 367-37418. P.A. Campitelli, M.I. Velasco, S.B. Ceppi, Chemical and physicochemical characteristics of humic acids extracted from compost, soil and amended soil, Talanta 69 (2006) 1234-1239.19. I. Nagyová, D. Kaniansky, Discrete spacers for photometric characterization of humic acids separated by capillary isotachophoresis, J. Chromatogr. A, 916 (2001) 191-200. 20. M.L Pacheco, E.M Peña-Méndez, J Havel, Supramolecular interaction of humic acids with organic and inorganic xenobiotics studied by capillary electrophoresis, Chemosphere 51, (2003) 95-108.

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21. F. d'Orlyé , P.E. Reiller, Contribution of capillary electrophoresis to an integrated vision of humic substance size and charge characterizations, J Colloid Interface Sci 368 (2012) 231-240. 22. M. Tatzber, F. Mutsch, A. Mentler, E. Leitgeb, M. Englisch, M.H. Gerzabek, Capillary electrophoresis characterisation of humic acids: application to diverse forest soil samples, Envir. Chem. 8 (2011) 589-601. 23. O.A. Trubetskoj, C. Richard, G. Guyot, G. Voyard, O.E. Trubetskaya, Analysis of electrophoretic soil humic acids fractions by reversed-phase high performance liquid chromatography with on-line absorbance and fluorescence detection, J. Chromatogr. A 1243 (2012) 62-68. 24. L. Cavani, C. Ciavatta, O.E. Trubetskaya, O.I. Reznikova, G.V. Afanaseva, O.A. Trubetskoj, Capillary zone electrophoresis of soil humic acid fractions obtained by coupling size-exclusion chromatography and polyacrylamide gel electrophoresis, J. Chromatogr. A, 983 (2003) 263-270. 25. Z. Radičová, R. Bodor, R. Góra, M. Hutta, M. Masár, Off-line combination of preparative isotachophoresis and size exclusion chromatography in analysis of humic acids, Chem. Listy 107 (2013) 432-434. 26. L. Pokorná, D. Gajdošova, S. Mikeska, P. Homola, J. Havel, The stability of humic acids in alkaline media, in: E. A. Ghabbour, G. Davies (Eds), Humic Substances: Structures, Models and Functions, Royal Society of Chemistry (Great Britain), 2001, pp 133-149. 27. R. Góra, M. Hutta, P. Rohárik, Characterization and analysis of soil humic acids by off-line combination of wide-pore octadecylsilica column reverse phase high performance liquid chromatography with narrow bore column size-exclusion chromatography and fluorescence detection, J. Chromatogr. A 1220 (2012) 44-49. 28. R.M.B.O. Duarte, A.C. Barros, A.C. Duarte, Resolving the chemical heterogeneity of natural organic matter: New insights from comprehensive two-dimensional liquid chromatography, J. Chromatogr. A 1249 (2012) 138-146. 29. M. Hutta, R. Góra, N ovel stepwise gradient reversed-phase liquid chromatography separations of humic substances, air particulate humic-like substances and lignins, J Chromatogr A 1012 (2003) 67-79. 30. R-Q. Wang, L. Gutierrez, N. S. Choon, J.-P. Croué, Hydrophilic interaction liquid chromatography method for measuring the composition of aquatic humic substances, Anal. Chim. Acta 853 (2015) 608-616. 31. J. Kandráč, M. Hutta, M. Foltin, Preparation and characterization of humic and fulvic acid working standards - practical experience, J Radioanal. Nucl. Chem. 208 (1996) 587592. 32. http://www.humicsubstances.org/soilhafa.html 33. P. Dlapa, Pedologic Report - Characteristics of selected soil profiles for the purpose of studying soil organic mater within the project APVV-0595-07 (Charakteristika vybraných pôdnych profilov pre účely štúdia pôdnej organickej hmoty v rámci projektu APVV-0595-07, 2008, Comenius University in Bratislava, Faculty of Natural Sciences, Department of Soil Science, Bratislava, Slovak republic. 34. http://www.tessek.com/tes3.htm (2014, Dec. 5th). Applications sheets no. 28, 29, 32, 61, 62, 63, 109, 133, 134 35. http://www.tessek.com/cat4.htm (2014, Dec. 5th).

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36. J. Ráczová, M. Hutta, Metódy analýzy reálnych vzoriek na obsah EDTA a prehľad jej využitia v moderných separačných metódach (Methods of Analysis of Real Samples for EDTA and Review of Their Utilization in Modern Separations), Chem. listy 107 (2013) 688-694. (in Slovak) 37. J. Čoupek, I. Vinš, Hydroxyethyl methacrylate-based sorbents for high-performance liquid chromatography of proteins, J Chromatogr. A 658 (1994) 391-398. 38. L. Bhattacharyya, J.S. Rohrer (Eds), Applications of ion chromatography for pharmaceutical and biological products, (1st ed.), Appendix 2 – Dissociation constants (pKa) of common sugars and alcohols, John Wiley & Sons, Inc, 2012.

474 475

39. S. Feng, Ch. Bagia, G. Mpourmpakis, Determination of proton affinities and acidity constants of sugars, J. Phys. Chem. A 117 (2013) 5211−5219.

476 477 478 479

40. K. Inoue, K. Kitahara, Y. Aikawa, S. Arai and T. Masuda-Hanada, HPLC separation of all aldopentoses and aldohexoses on an anion-exchange stationary phase prepared from polystyrene-based copolymer and diamine: the effect of NaOH eluent concentration, Molecules 16 (2011) 5905-5915.

an

us

cr

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465 466 467 468 469 470 471 472 473

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te

d

M

480

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480

Captions

481

Fig. 1. Dependence of net charge of HA and FA on pH isolated from various soil horizons evaluated from data of potentiometric titrations modified according to Fig 4 in [17]. Black areas show regions evaluated from the measured data and dashed area above pH 10 is probable extrapolation with the aid of results of Campitelli [18] Fig. 1.

ip t

482 483 484 485

cr

486 487

us

Fig. 2. Dependence of chromatographic profile of humic acids SUHO and ECOH on column oven temperature in step gradient elution from short column filled by Separon HEMA Bio Q. SUHO represents also typical temperature dependent behavior of SALD HA, whereas SUHO represents also CERV. Mobile phase A = 5 mmol L-1 EDTA, pH 12.0, B = 500 mmol L-1 EDTA, pH 12.0, flow-rate 0.5 ml/ min, fluorimetric detection (ex. 480 nm; em. 530 nm).

an

488 489 490 491 492 493

500 501 502 503 504 505 506 507 508 509 510 511

M

d

te

499

Fig. 3. Chromatographic profiles of selected humic substances (HA Cerova denoted as CERV, HA Aldrich denoted as SALD, HA Ecohum denoted as ECOH and HA Sucha Hora denoted as SUHO). Conditions: flow rate: 1.0 mL min-1, temperature: 40°C, injection volume 25 μl. Duration and shape of used stepwise gradient is illustrated in the figure. For the other conditions, see Section Experimental.

Fig. 4. Chromatographic profiles of humic substances in extracts of soils from locations of Gbely (GBEL), Šajdikové Humence (SAJD), Senec (SENC) and Kremnica-Skalka (SKAL). Shown is the most interesting part of chromatogram. Conditions: flow-rate: 1.0 mL min-1, temperature: 40°C, injection volume 25 μl, stepwise gradient, fluorimetric detection (ex. 480 nm; em. 530 nm). GBEL and SENC denote arable soils, whereas SAJD and SKAL denote forest soils. For the other conditions, see Section Experimental.

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494 495 496 497 498

Fig. 5. Reproducibility of 5 overlayed chromatographic profiles of selected mixed standard of aromatic acids (from 1.00 to 5.00 mg mL-1, each) measured within two days. Gradient elution, pH 12.0, ultraviolet detection (280 nm), temperature: 40°C, injection volume 10 μl, flow rate: 1.0 mL min-1. Abbreviation: Phenolate PHEN, Salicylate SLCL, 2,6-Dihydroxy-benzoate 2,6-DHB, Benzene-1,2,4,5-tetracarboxylate 1,2,4,5 TCBA.

512 513

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513 514 515

ip t

516 517

cr

518 519

us

520

Table 1 Pedological characteristics of selected soil samples according to [33].

524 525 526 527 528 529 530

pH (KCl) 5.77 2.83 7.26 3.72

M

pH (H2O) 6.57 3.96 7.93 3.95

HS Content (% m/m) 5.17 16.9 1.72 26.4

Type of soil smonica umbrisol chernozem andosol

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GBEL SAJD SENC SKAL

Sampling depth (cm) 0-10 0-10 0-10 0-10

te

Locality

d

522 523

an

521

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530

Table 2 Chromatographic conditions for anion-exchange chromatography 30 x3 mm, Separon HEMA-BIO 1000 DEAE 60 μm

Column

ip t

531 532



Fluorescence detection:ex 480 nm and em 530 nm for standard HS and soil extracts 280 nm for polyvalent aromatic carboxylic acids

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cr

Wavelength

an

Photometric detection: 280 nm

Injection volume

25 µl standard HS and soil extracts,

M

10 µl aromatic carboxylic acids solutions

1.0 mL min-1

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Column oven temperature

Run time

533 534 535

te

d

Flow-rate

40°C

38 min (with 2 minute gradient step duration) 47 min (with 3 minute gradient step duration)

536 537 538 539 540

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540 541

Table 3

542

Statistical evaluation of reproducibility of retention times (tR) and reproducibility of peak areas (A) in 5 consecutive runs measured within two days. In the table the concentration of EDTA4means concentration (c(EDTA)) that eluted analyte peak apex

ip t

543 544 545

tR RSD

A

c(EDTA)

(min)

(%)

(mAU.s)

(%)

(mmol L-1)

Phenol

1.067±0.035

±3.28

338.3±9.5

±2.8

5

Benzoic acid

1.998±0.013

±0.65

123.0±4.8

±3.9

5

Salicylic acid

4.340±0.039

±0.90

573.8±20.7 ±3.6

10

Terephthalic acid

4.951±0.026

±0.50

270.6±12.1 ±4.5

10

Phthalic acid

5.101±0.023

±0.45

328.0±14.2 ±4.3

10

2,6-Dihydroxy-benzoic acid

10.27±0.06

±0.58

455.1±22.2 ±4.8

25

±0.53

148.2±5.1

±3.4

25

11.31±0.07

±0.61

165.7±6.1

±3.7

25

16.97±0.07

±0.41

235.0±8.4

3.6

84

Benzene-1,2,4-tricarboxylic acid

Benzene-1,2,4,5tetracarboxylic acid 547 548

11.18±0.06

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Benzene-1,2,5-tricarboxylic acid

an

M

d

Analyte

us

A RSD

te

tR

cr

546

549 550

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Figure

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Figure

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Figure

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Figure

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Figure

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