Environ Sci Pollut Res DOI 10.1007/s11356-015-4188-1
RESEARCH ARTICLE
Evaluation of Suwannee River NOM electrophoretic fractions by RP-HPLC with online absorbance and fluorescence detection Olga E. Trubetskaya & Claire Richard & Oleg A. Trubetskoj
Received: 12 November 2014 / Accepted: 29 January 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract 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 (SECPAGE) 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 455/420 nm and two proteinlike fluorophores with emission maxima at around 300 and 340 nm. The HS-like fluorophore with emission maximum at 435 nm was located in the hydrophilic peak in all the samples.
Responsible editor: Philippe Garrigues O. E. Trubetskaya Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Moscow region, Russia 142290 C. Richard Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448, 63000 Clermont-Ferrand, France C. Richard CNRS, UMR 6296, ICCF, 63171 Aubière, France O. A. Trubetskoj (*) Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow region, Russia 142290 e-mail: [email protected]
Those with maxima at 455–465 nm 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.
Keywords Water NOM . RP-HPLC . SEC-PAGE setup . UV-visible and fluorescence spectroscopy . Protein-like and HS-like fluorophores
Introduction Natural water organic matter (NOM) is a complex mixture of organic biochemicals that can undergo various pathways of diagenetic alteration. The process of diagenesis can create a chain of various hydrophobic/hydrophilic molecules as a result of oxidation/cleavage and condensation reactions. Thus, any effort to understand the ecological fate of these compounds in the environment requires identification of their hydrophobic properties. For this reason, reversed-phase highperformance liquid chromatography (RP-HPLC) is often applied to water NOM to obtain fractions of different hydrophobic properties (Lombardi and Jardim 1999; Egeberg and Alberts, 2002; Parlanti, 2002; Wu et al., 2003; Stenson 2008; Hutta et al., 2011; Li et al., 2014). These data could extend the applicability of RP-HPLC method for research of water NOM chemical structure. On the other hand, they have environmental significance for understanding the nature of chemical interactions at the molecular level between water NOM and other organic constituents, such as hydrophobic organic pollutants capacity, which was mainly based on organic matter polarity.
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Yet, another potential consequence of NOM hydrophobicity distributions may occur in the fluorescence exhibited by these mixtures, and fluorescence properties have often been used to characterize water NOM (Susic and Boto, 1989; Coble, 1996, 2007; Egeberg and Alberts, 2002; Wu 2003; Abtt-Braun, 2004; Park, 2009; Youhei et al., 2008; Huguet et al., 2010; Ma et al., 2010; Li et al., 2014) but it still provides limited information about the localization of NOM fluorophores. Further determination of fluorescent NOM constituents between fractions obtained by RP-HPLC procedure and exhibiting different polarity should be promising for the understanding of the water NOM fluorescence origin. Water NOM is polyelectrolytic, and electrophoretic techniques should be applicable for constituent separation and further characterization. An electrophoretic approach, which has been successfully used in the last 40 years, is the polyacrylamide gel electrophoresis (PAGE). With this method, fractions with distinct electrophoretic mobilities were obtained from water NOM (Duxbury 1989; Baxter and Malysz, 1992; Dunkel et al., 1997; Peurvouri et al. 2001; Parlanti et al., 2002; Trubetskoi and Trubetskaya, 2004; Trubetskoj et al., 2009). The obtained electropherograms are mainly interpreted as fingerprints of the analyzed water NOM. However, we are not aware of any literature data about the characterization of hydrophobic/hydrophilic properties of NOM fractions with different electrophoretic mobilities (EM) and molecular sizes (MS). Such determinations should be useful for the understanding of water NOM structure and properties. Moreover, they can help in clarifying the nature of chemical interactions at the molecular level between water NOM and other organic constituents, such as hydrophobic organic pollutants. We have previously developed an effective method for the fractionation of different humic substances (HS) based on the combined use of conventional preparative size exclusion chromatography (SEC) and analytical PAGE; this procedure was called the SEC-PAGE setup (Trubetskoj et al., 1997). The advantage of this combination rests primarily in the presence of urea, which is added at a level of 7 M in both SEC and PAGE to assist in the rupture of hydrogen bonds and prevents interaction between the fractionated humic material and the solid immobile phase on which the substances are separated. This is accompanied by the disaggregation of HS, which we believe is primarily related to the disruption of hydrogen bonds. The proposed procedure allows separation of primary disaggregated structural humic components, thereby solving some of the key problems occurring in the fractionation of HS, for example, irreversible/reversible chromatographic column adsorption and the influence of PAGE chemical components (e.g., Tris and borate ions) on the composition of HS fractions. On the other hand, 1H-NMR in D2O and solid 13C-NMR of soil HS before and after concentrated urea treatment showed no structural differences (Trubetskoj et al., 2010). SEC-PAGE
setup was first applied to water NOM extracted from natural terrestrial waters of Finland and USA (Peurvuori et al. 2001; Trubetskoj et al., 2009). It was shown that aquatic NOM fraction with highest EM and lowest MS fluoresced intensively and initiated the formation of singlet oxygen. On the contrary, the fluorescence intensity of low EM fractions with the highest and medium MS was insignificant (Trubetskoj et al., 2009). The objectives of the current work was to use RP-HPLC with online absorbance and fluorescence detections for analysis of Suwannee river NOM and its fractions obtained by SEC-PAGE setup for revealing the fluorescent NOM constituents of different polarity between fractions of different EM and MS. The multi-step scheme summarizing the separations and analysis of Suwannee River natural organic matter (SRNOM) is presented on Fig. 1. The NOM isolated from Suwannee River was chosen as one of the standard of the International Humic Substances Society (IHSS). This sample include Suwannee River humic and fulvic acids, thus using of this standard is more important for understanding of the nature of chemical interactions at the molecular level between water NOM and other organic constituents.
Materials and methods Material The NOM sample, isolated by reverse osmosis from Suwannee river water, Georgia, USA (SRNOM), was purchased as a standard material (ref. number 1R101N) from the International Humic Substances Society. Major elemental composition of SRNOM were as follows: C=52.5 %, H= 4.2 %, N=1.1 %, ash content=7.0 %, and water content= 8.2 %. All reagents were of the highest grade available. Water was purified using a Millipore Milli-Q system (Millipore αQ, resistivity 18.2 MΩ cm − 1 , DOC < 0.1 mg L−1). Phosphate buffer (10 mM Na2HPO4/NaH2PO4, pH 6.5) was prepared on reversed osmoses water, obtained with RIOS 5 and Synergy, Millipore. Conventional preparative SEC-PAGE setup Fractionation of SRNOM by SEC-PAGE setup (i.e., using PAGE for subsequent testing of SEC aliquots from different sections of the elution profile) was previously reported (Trubetskoj et al., 1997). Briefly, 20 mg SRNOM were dissolved in 1 mL 7 M urea and loaded onto a Sephadex G-75 (Pharmacia, Sweden) column (1.5×100 cm), equilibrated with the same solution. The void (Vo) and the total (Vt) column volumes were 47 and 160 mL, respectively. The void volume was determined using Dextran Blue 2000. Fractionation ranges for Sephadex G-75 were 80,000 to 3000 for proteins and 50,000 to 1000 for polysaccharides. Flow rate was
Environ Sci Pollut Res Fig. 1 The multi-step scheme summarizing the separations and analysis of SRNOM
SRNOM Conventional preparative SEC-PAGE setup in 7M urea
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Analytical RP-HPLC in 10mM phosphate buffer, pH 6.5, with on-line absorbance detection at 270 nm and fluorescence detection at Ex/Em 270/450nm and 270/330 nm
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Fraction Highest hydrophobicity Weak fluorescence intensity Protein-like and HS-like fluorophores
15 mL h−1. The UV-detector (ISCO, USA) was set at 280 nm. The eluate from Vo (47 mL) to Vt (160 mL) was combined into 10 aliquots, the volume of each aliquot was about 11–13 mL (Fig. 2a); 0.1 mL of each aliquot was added to 0.01 mL of buffer, which contains 0.89 M Tris-borate, 10 % SDS and 10 mM EDTA, pH 8.3, and assayed by PAGE (Fig. 2b). The apparatus was a vertical electrophoresis device (LKB 2001 Vertical Electrophoresis, Sweden) with a gel slab (20 × 20 cm). Acrylamide 9.7 % and bisacrylamide 0.3 % were dissolved in 89 mM Tris-borate buffer with 1 mM EDTA and 7 M urea, pH 8.3. For polymerization, 0.014 mL N,N, N ,N -tetramethylethylendiamine and 0.4 mL 10 % ammonium persulfate were added to 40 mL of acrylamide/ bisacrylamide solution. The electrode buffer had a concentration of 89 mM Tris-borate with 1 mM EDTA, pH 8.3. Electrophoresis was carried out at a room temperature for 1 h at a constant current intensity of 25 mA; during electrophoresis, the voltage gradually increased from 300 to 500 V. On the basis of PAGE analysis (Fig. 2b), three naturally colored fractions (A = aliquot I, B = aliquots II–IV, and C+D = aliquots V–X) originated different EM were detected, concentrated on UF membrane with nominal cut of 5 kDa, dialyzed through 10 kDa cellulose dialysis tubing (Sigma-Aldrich, Moscow, Russia) during 7 days against distilled water, lyophilized, and used for further analyses.
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Fraction Medium hydrophobicity Weak fluorescence intensity Protein-like and HS-like fluorophores
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Fraction Lowest hydrophobicity High fluorescence intensity HS-like fluorophores
Analytical RP-HPLC SRNOM and its fractions were analyzed on a Waters ACQUITYTM Ultra-Performance Liquid Chromatographic system with a cooling autosampler (ACQUITY Sample Manager), a binary pumping module (ACQUITY Solvent Manager), and column oven enabling temperature control of analytical column (Waters Corp., Milford, MA, USA). An analytical column (UPLC© BEH C18, 2.1×100 mm, particle size 1.7 μm - Waters Corp., USA) equipped a precolumn used as stationary phase. The column was maintained at 30 °C. UV-Visible detection was carried out using an ACQUITY photodiode array detector (PDA), designed for wavelengths in the range 210– 400 nm, and RP-HPLC chromatograms with online absorbance at 270 nm were analyzed. The fluorescence of samples was monitored using an ACQUITY Fluorescence detector - FLR (Waters Corp., Milford, MA, USA), connected directly to the waste line of the absorbance PDA detector. The delay between PDA and FLR detection was 0.12 min. FLR detector was designed for the excitation wavelength 270 nm and emission range 290–600 nm. The RP-HPLC chromatograms with online emission at 330 and 450 nm were analyzed. Analyses of blank phosphate buffer solutions (10 mM) were performed before the
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(a)
humic matter was diluted to 3 mL (about 600 times), this allowed the use of PDA detector, which has the maximum absorbance limit of 2 a.u. (1.5 % deviation). Data were collected and processed by chromatographic software Empower2TM (Waters Corp., Milford, MA, USA). The entire cycle of RPHPLC procedures was repeated three times. Deviations between three chromatogram’s profiles of each sample did not exceed 3.0 %.
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Fig. 2 Preparative size exclusion chromatography of 20 mg SRNOM on Sephadex G-75 column (100 cm×1.5 cm) using 7 M urea as eluting medium (a). Letters I–X on the x-axis show the aliquots, analyzed by PAGE (b)
analyses of the SRNOM and its fractions to check the absence of any absorbance and fluorescent peaks in the solvents. Solvent A (methanol, HPLC grade CHROMASOLV®, Sigma-Aldrich) and solvent B (10 mM phosphate buffer, pH 6.5, prepared in Milli-Q water) were used for gradient formation. A stepwise gradient was applied starting at 0.00 min with 0 % of solvent A in solvent B, at 2.22 min=10 % A in B, at 3.33 min= 20 % A in B, at 4.45 min = 30 % A in B, at 5.56 min= 40 % A in B, at 6.67 min = 50 % A in B, at 7.7 min = 60 % A in B, at 11.12 min = 70 % A in B, at 13.89 min=100 % A, at 20 min=0 % A in B, and continuing for another 10 min at flow rate of 0.3 mL min−1. Solutions of SRNOM and fractions A, B, and C+D were prepared by dissolving weighed amounts of dry material in 10 mM phosphate buffer, pH 6.5. To estimate the relative amounts of humic matter eluted and irreversibly absorbed on the column during the RPHPLC analyses, all samples were prepared in the same manner: the absorbance of each sample was adjusted to 6.5 a.u. at 270 nm in a 1-cm quartz cuvette on the basis of dilution 10– 20 times of stock solutions, the volume of injection onto the reversed-phase column was 0.005 mL. All humic matter, which did not irreversibly adsorb on the column, was eluted during the first 10 min. Flow rate was 0.3 mL min−1. It means that injected
UV-visible absorption spectra of SRNOM and its fractions A, B, and C+D were recorded using a Cary 3 spectrophotometer (Varian, Cary, USA) in a 1-cm quartz cuvette from 220 to 700 nm at a concentration of 50 mg L−1 in 10 mM phosphate buffer, pH 6.5. The specific adsorption coefficient at 280 nm (A280) at C=50 mg L−1 was used for fractions comparison. Fluorescence emission spectra of SRNOM and its fractions were recorded using a Cary Eclipse fluorescence spectrophotometer (Varian, Cary, NC, USA) in a 1-cm quartz cuvette. Excitation wavelength was 270 nm, emission spectra were recorded from 310 to 700 nm. In order to minimize the inner filter effect, the solutions were diluted with Milli-Q water to an absorbance of 0.05±0.01 at 270 nm. UV-visible absorption spectra and fluorescence emission spectra of the RP-HPLC peaks were extracted from the data of PDA and FLR detectors. For SRNOM, its fractions and all RP-HPLC peaks the absorbance ratio at 270 and 366 nm (A2/A3) were calculated.
Results and discussion Preparative fractionation of SRNOM by SEC-PAGE setup Preparative SEC-fractionation of SRNOM on the column with Sephadex G-75 in 7 M urea revealed the presence of two broad overlapping chromatographic peaks. First small
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peak (47–60 mL) was eluted at the void column volume, and the second large peak with the shoulder covered almost all fractionation range of the column from 61 to 160 mL (Fig. 2a). The elution profile was divided into 10 aliquots, each of them were analyzed by PAGE (Fig. 2b). The aliquot I (elution volume from 47 to 60 mL) correspond to the excluded peak and formed at the electropherogram start zone A, which do not penetrate
Table 1 Relative contribution of resolved peaks 1–7 on RP-HPLC total chromatograms, ratios A2/ A3 and emission maxima of bulk SRNOM, fractions A, B, and C+ D, and RP-HPLC peaks 1–7, 1a, 3a, and 4a. Specific absorption coefficient at 280 nm (A280) of SRNOM and its fractions A, B, and C+D
sh shoulder at
Sample
into the 10 % PAG. The aliquots II–IV (elution volume from 61 to 93 mL) formed an intensively colored narrow zone in the mid part of PAG and combined into fraction B. The aliquots V–X (elution volume from 94 to 160 mL) forming several colored bands on the mid and bottom of the PAG were combined into fraction C+D. On the basis of the elution orders, we suggest, that the MS of fractions are in the order: A>B>C+D.
Relative contribution (%) of RP-HPLC peaks 1–7
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A2/A3
Emission maximum (nm) at λex =270 nm Protein-like
SRNOM Peak 1 53 Peak 2 19 Peak 3 15 Peak 4 8 Peak 5 4 Peak 6 1 Σ Peaks 2–6 47 High MS fraction A Peak 1 9 Peak 1a – Peak 2 21 Peak 3 33 Peak 3a – Peak 4 22 Peak 4a – Peak 5 10 Peak 6 3 Peak 7 2 Σ Peaks 2–7 91 Medium MS fraction B Peak 1 12 Peak 1a Peak 2 31 Peak 3 32 Peak 3a – Peak 4 17 Peak 4a – Peak 5 5 Peak 6 2 Peak 7 1 Σ Peaks 2–7 88 Low MS fraction C+D Peak 1 52 Peak 2 24 Peak 3 15 Peak 4 6 Peak 5 2 Peak 6 1 Σ Peaks 2–6 48
0.61
3.9 5.3 3.3 3.1 3.4 3.1 3.1
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2.8 3.3 4.4 2.8 2.8 2.7 2.7 2.7 2.8 2.8 2.8
350
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340
0.75
0.89
3.3 4.1 3.1 3.0 3.0 2.8 2.6
HS-like 455 435 450 450 450 450 450 440 435
≈300 455 455 340 460 340 460 460
465 435
≈300 465 465 340 465 340 465 465
465 435 455 sh420 455 sh420 455 sh420 455 sh420 455 sh420
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UV-visible absorption and fluorescent emission spectra of SRNOM and its fractions UV-visible absorption spectra of SRNOM and its fractions A and B appeared to be featureless without major maxima and decreased gradually with increasing wavelength with shoulder between 260–280 nm. On the other hand, fraction C+D has the shoulder between 260–280 nm and a maximum below 220 nm (Fig. 3). The values of specific absorption coefficient at 280 nm (A280) varied in the order C+D>B>SRNOM>A (Fig. 3, Table 1). As currently accepted, the fluorescence of water NOM is due to two main groups of fluorophores (Coble, 1996; Huguet et al., 2010. Boyle et al., 2009; Andrew et al., 2013). One group usually has excitation (Ex) maximum less than 305 nm and emission (Em) maximum less than 380 nm, related to aromatic amino acids, and is often referred to as protein-like fluorophores. The other one with Ex/Em 220– 360/380–470 nm is attributed to HS-like fluorophores of water NOM. However, until now, their relationship with humic components of different MS and EM remains uncertain. Fluorescence emission spectra of SRNOM and its fractions, recorded upon excitation at 270 nm, are given in Fig. 4. For the bulk SRNOM and fraction C+D, the fluorescence emission spectra originated different maxima at 455 and 465 nm, respectively. In contrast, fractions A and B revealed two maxima at 350 and 440 and 340 and 465 nm, respectively. Thus coupling SEC-PAGE allows to separate bulk SRNOM on fractions, which contained different groups of fluorophores. The vast majority of HS-like fluorophores located in low MS fraction C+D, while the high MS fractions A and B contain both protein-like and HS-like fluorophores. The relative concentration of protein-like fluorophores in fraction A was considerably more than in fraction B (Fig. 4, inset). These data indicate that SRNOM is not homogeneous and can be splitted
into several fluorescent sub-fractions with different emission maxima. Thus, overall DOM fluorescence could comes from simple summation of all the fluorescence of the individual fluorophores presented by taking account of the dilution factors. On the other hand, instead of simple summation, the overall fluorescence could arise from charge transfer interactions between fraction moieties within DOM (Boyle et al., 2009). Either of the possible scenarios would invalidate the effectiveness of this fluorophores fractionation method. For more deep investigation of physical–chemical properties of SRNOM and revealing of less complex mixture of NOM compounds, we carried out analytical fractionation of bulk SRNOM and its fraction by RP-HPLC with online absorbance and fluorescence detection. RP-HPLC of SRNOM and its fractions Figure 5 (left column) presents RP-HPLC chromatograms of bulk SRNOM and fractions A, B, and C+D with online absorbance detection at 270 nm. The chromatograms exhibit the resolution of six (SRNOM and fraction C+D) or seven (fractions A and B) peaks. The hydrophobicity of RP-HPLC peaks increased from the first to the last eluted peak due to the increase of methanol concentration in the stepwise separation procedure. For simplification, we refer to the first peak as hydrophilic, because it is eluted in an aqueous phosphate buffer only, whereas the other peaks are assumed as hydrophobic, because they are eluted by methanol at different concentrations (from 10 to 60 %). No material was eluted by methanol
A
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100
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Fig. 4 Emission spectra of SRNOM and its fractions A, B, and C+D setting excitation at 270 nm and using A270=0.05. Inset spectra normalized at the HS-like emission maxima
Fig. 5 RP-HPLC chromatograms of SRNOM and fractions A, B, and C+ D with online absorbance at 270 nm (left column) and fluorescence detection (right column) at Ex/Em wavelengths 270/450 nm (solid line) and 270/330 nm (dashed line). Inset zoom on the 1.5- to 6.5-min zone of the chromatograms
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Relationships between fluorescence properties and polarity of SRNOM and its fractions Figure 5 (right column) presents RP-HPLC chromatograms of SRNOM and its fractions with online fluorescence detection at excitation–emission wavelength pairs (Ex/Em) 270/450 and 270/330 nm for detection of HS-like and protein-like fluorophores, respectively. Data obtained at Ex/Em 270/ 450 nm revealed HS-like fluorescence in peaks 1–6 with highest intensity in hydrophilic peak 1 for all samples, a very weak fluorescence in peak 7 of fractions A and B was not taken into consideration. The fluorescence emission spectra of the RP-HPLC peaks were extracted from the data of FLR detector. The emission maxima at λex =270 nm were presented in Table 1. The maximum of emission in hydrophilic peak 1 of SRNOM and fractions A, B, and C+D was 435 nm. The hydrophobic peaks 2–6 of all the samples had fluorescence maxima, shifted to long wavelength in comparison with hydrophilic peak 1. In fraction A, hydrophobic peaks 2–3 and 4– 6 revealed emission maxima at 455 and 460 nm, respectively. In fraction B, all the hydrophobic peaks revealed emission maximum at 465 nm. In fraction C+D, the hydrophobic peaks showed emission maximum at 455 nm with a shoulder at 420 nm. In SRNOM, all the hydrophobic peaks revealed emission maximum at 450 nm. The fluorescence maxima of fractions A and B (440 and 465 nm, respectively) lay within the range of fluorescence maxima of their RP-HPLC peaks 1– 6 (Table 1). However, fluorescence maxima of bulk SRNOM and fraction C+D exceeded fluorescence maxima values of all the peaks (Table 1). This suggests that a part of fluorophores of SRNOM and fraction C+D irreversibly adsorbed on the C18-column. Setting Ex/Em at 270/330 nm to detect the fluorescence related to aromatic amino acids, often referred to as protein-
Normalized absorbance
at a concentration more than 60 % in all the investigated samples. All RP-HPLC experiments were made in 10 mM phosphate buffer, pH 6.5. The choice of buffer was due to its high buffer capacity and wide application in studies of humic substances by RP-HPLC (Hutta et al., 2011; Wu et al., 2003). Moreover, some preliminary experiments showed that at a more lower pH all NOM matter had been irreversibly adsorbed on the C18-column. Because all samples were injected onto the column under constant conditions with special attention on injection volume and absorbance (see BMaterials and methods^), we had the possibility to estimate relative irreversible adsorption of different samples on the reversed-phase C18-column by comparison of the total chromatogram’s area. This calculus was made using the suggestion that absorption coefficient and fluorescence quantum yield of investigated samples do not change during RP-HPLC. The total area of the chromatographic peaks of SRNOM, fractions B and C+D were rather similar, but the total area of the chromatographic peaks of fraction A was lower by 20 % (Fig. 5, left column). Fraction A seems to be more hydrophobic than fractions B and C+D, but on the basis of these data it was impossible to compare hydrophobicity of fractions B and C+D. On the other hand, in spite of similar relative irreversible adsorption of fractions B and C+ D on C18-column, their eluted material demonstrated significant differences in the ratio of hydrophilic and hydrophobic peaks area (Table 1). Fraction C+D had a relatively higher abundance of hydrophilic constituents, 52 % of total chromatogram’s area was corresponded to peak 1, and areas under hydrophobic peaks 2–6 essentially decreased with the increasing of methanol concentration. Meanwhile, fractions A and B had low content of hydrophilic components—peak 1 contributed to only 9 and 12 % of total chromatogram area, respectively (Fig. 5, left column and Table 1). Analysis of SRNOM and its fractions in part of their relative irreversible adsorption on C18column and relative distribution of eluted from the column matter on hydrophobic and hydrophilic peaks shown, that hydrophobicity of fractions decreased in order: A>B>C+D. It should be noted, that absorption spectra of hydrophilic and hydrophobic RP-HPLC peaks (extracted from the data of PDA detector) in SRNOM and its fractions were featureless and showed a gradual decrease in absorbance with increasing wavelength without major maxima. However, they had differences in the values of absorbance ratio at 270 and 366 nm (A2/ A3) (Table 1). This ratio was considerably higher in all hydrophilic peaks of SRNOM and its fractions in comparison with the same ratio in hydrophobic peaks. This trend was more pronounced in SRNOM and fraction C+D and could be explained by different ratio aromatic/aliphatic compounds in hydrophilic and hydrophobic peaks. The RP-HPLC with using stepwise gradient of methanol allowed us to map the SRNOM and its fractions in terms of distribution of hydrophilic/ hydrophobic components.
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Wavelength, nm Fig. 6 Absorption spectra of fraction A, its RP-HPLC peaks 1a, 3a, and 4a, normalized at A366 nm and blank solution for peak 1a (phosphate buffer at elution volume 1.78 ml). Spectra were extracted from the data of PDA detector. Fraction B and its RP-HPLC peaks 1a, 3a, and 4a demonstrated similar results
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like fluorescence, did not revealed essential signals in the bulk SRNOM chromatogram (Fig. 5, right column). Similar results were reported by other researchers for bulk SRNOM, Suwannee River humic, and fulvic acids (Coble 2007; Wu 2003; Alberts and Takács 2004). However, new peaks occurred in chromatograms of high MS fraction A and medium MS fraction B: a peak 1a at retention time 1.9 min, i.e., in the hydrophilic zone, and peaks 3a and 4a at retention times 4.9 and 5.8 min in the hydrophobic zone of chromatograms. Low MS fraction C+D demonstrated only traces of these peaks (Fig. 5, right column, inset). These peaks showed strong protein-like fluorescence emission maxima at 300–340 nm (Table 1). Interestingly, peaks 3a and 4a had an emission maxima at 340 nm, but peak 1a—about 300 nm (Table 1). It is well known, that protein-like fluorescence emission maxima at around 300–380 nm correspond to aromatic amino acids like as phenylalanine, tyrosine, and tryptophan (Lehninger 2000; Lakowicz 2006). Peak 1a might correspond to tyrosine-like chromophore bounded to rather hydrophilic part of SRNOM, because it demonstrated emission maximum at about 300 nm (Table 1) and absorbance spectrum with two maxima at 220 and 270 nm (Fig. 6)—the two optical characteristics of tyrosine amino acid (Lakowicz 2006). The absorbance spectra of peaks 3a and 4a were featureless without major maxima decreasing gradually with increasing wavelength. We attributed peaks 3a and 4a to a mixture of non polar NOM particles having featureless absorption spectra and protein-like fluorophore(s) with emission maximum(a) around 340 nm.
Conclusion SEC-PAGE setup followed by RP-HPLC allowed us to develop approach of SRNOM separation on less heterogeneous compounds mixture for their further study and structural identification. RP-HPLC on C18-column with online absorbance and fluorescence detection gave possibility to map SRNOM and its different on MS fractions in terms of distribution of hydrophilic/hydrophobic components and fluorophores. Our results show that: (i) Analysis of SRNOM and its fractions in part of their relative irreversible absorption 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: high MS fraction A>medium MS fraction B>low MS fraction C+D; (ii) The SRNOM contained at least three groups of the HSlike fluorophores with different emission maxima: fluorophore(s) with fluorescence emission maximum at 435 nm was located in hydrophilic peak and those with emission maxima at 450–465 nm in the hydrophobic
peaks, the vast majority of HS-like fluorescence located in low MS fraction C+D; (iii) The SRNOM contained at least two groups of the protein-like chromophores, located mainly in high MS fraction A and medium MS fraction B. One group of fluorophore(s) with emission maximum around 300 nm bounded to polar NOM particles and have absorbance spectrum with two maxima at 220 and 270 nm, the second group of fluorophore(s) with the emission maximum around 340 nm bounded to non polar NOM particles and have featureless absorption spectra; The data obtained could be significant for future NOM chemical structural characterization. Acknowledgments The work has been supported by Russian Foundation for Basic Research (projects no 13-05-00241-a and 15-04-00525-a).
References Abbt-Braun G, Lankes U, Frimmel F (2004) Structural characterization of aquatic humic substances—the need for a multiple method approach. Aquat Sci 66:151–170 Alberts JJ, Takács M (2004) Total luminescence spectra of IHSS standard and reference fulvic acids, humic acids and natural organic matter: comparison of aquatic and terrestrial source terms. Org Geochem 35:243–256 Andrew AA, Del Vecchio R, Subramaniam A, Blough NV (2013) Chromophoric dissolved organic matter (CDOM) in the equatorial atlantic ocean: optical properties and their relation to CDOM structure and source. Mar Chem 148:33–43 Baxter RM, Malysz J (1992) Analysis of aquatic humic material and high molecular weight components of bleached kraft mill effluent (BKME) by gradient gel electrophoresis. Chemosphere 24:1745– 1753 Boyle ES, Guerriero N, Thiallet A, Del Vecchio R, Blough NV (2009) Optical properties of humic substances and CDOM: relation to structure. Environ Sci Technol 43:2262–2268 Coble P (1996) Characterization of marine and terrestrial DOM in seawater using excitation-emission matrix spectroscopy. Mar Chem 51: 325–346 Coble PG (2007) Marine optical biogeochemistry: the chemistry of ocean color. Chem Rev 107:402–418 Dunkel R, Rüttinger H-H, Peisker K (1997) Comparative study for the separation of aquatic humic substances by electrophoresis. J Chromatogr A 777:355–362 Duxbury JM (1989) Studies of the molecular size and charge of humic substances by electrophoresis. In: Hayes MHB, MacCarthy P, Malcolm RL, Swift RS (eds) Humic Substances II. Wiley, Chichester, pp 593–620 Egeberg K, Alberts JJ (2002) Determination of hydrophobicity of NOM by RP-HPLC, and the effect of pH and ionic strength. Water Res 36: 4997–5004 Huguet A, Vacher L, Saubusse S, Etcheber H, Abril G, Relexans S, Ibalot F, Parlanti E (2010) New insights into the size distribution of fluorescent dissolved organic matter in estuarine waters. Org Geochem 41:595–510
Environ Sci Pollut Res Hutta M, Góra R, Halko R, Chalanyova M (2011) Some theoretical and practical aspects in the separation of humic substances by combined liquid chromatography methods. J Chromatogr A 1218:8946–8957 Lakowicz JR (2006) Principles of fluorescence spectroscopy. Springer Science, New York Lehninger AL (2000) Principles of biochemistry. Worth Publishers, New York Li W-T, Chen S-Y, Xu Z-X, Li Y, Chuang C-D, Li A-M (2014) Characterization of dissolved organic matter in municipal wastewater using fluorescence PARAFAC analysis and chromatography multi-excitation/emission scan: a comparative study. Environ Sci Technol 48:2603–2609 Lombardi AT, Jardim WF (1999) Fluorescence spectroscopy of high performance liquid chromatography fractionated marine and terrestrial organic materials. Water Res 33:512–520 Ma J, Del-Vecchio R, Golanski KS, Boyle ES, Blough NV (2010) Optical properties of Humic substances and CDOM: effects of borohydride reduction. Environ Sci Technol 44:5395–5402 Park J-H (2009) Spectroscopic characterization of dissolved organic matter and its interactions with metals in surface waters using size exclusion chromatography. Chemosphere 77:485–494 Parlanti E, Morin B, Vacher L (2002) Combined 3D-spectrofluorometry, high performance liquid chromatography and capillary electrophoresis for the characterization of dissolved organic matter in natural waters. Org Geochem 33:221–236 Peurvouri J, Pihlaja K, Trubetskaya O, Reznikova O, Afanas’eva G, Trubetskoj O (2001) SEC-PAGE characterization of lake aquatic humic matter isolated with XAD-resin and tangential membrane ultrafiltration. Int J Environ Anal Chem 80:141–152
Stenson AC (2008) Reversed-phase chromatography fractionation tailored to mass spectral characterization of humic substances. Env Sci Technol 42:2060–2065 Susic M, Boto KG (1989) High-performance liquid chromatographic determination of humic acid in environmental samples at the nanogram level using fluorescence detection. J Chromatography 482: 175–187 Trubetskoi OA, Trubetskaya OE (2004) Comparative electrophoretic analysis of humic substances in soils, river and lake waters. Eur Soil Sci 37:1170–1172 Trubetskoj OA, Trubetskaya OE, Afanas’eva GV, Reznikova OI, SaizJimenez C (1997) Polyacrylamide gel electrophoresis of soil humic acid fractionated by size exclusion chromatography and ultrafiltration. J Chromatogr A 767:285–292 Trubetskoj OA, Trubetskaya OE, Richard C (2009) Photochemical activity and fluorescence of electrophoretic fractions of aquatic humic matter. Water Resour 36:518–524 Trubetskoj OA, Hatcher PG, Trubetskaya OE (2010) 1H-NMR and 13CNMR spectroscopy of chernozem soil humic acid fractionated by combined size-exclusion chromatography and electrophoresis. Chem Ecol 26:315–325 Wu FC, Evans RD, Dillon PJ (2003) Separation and characterization of NOM by high-performance liquid chromatography and on-line three-dimensional excitation emission matrix fluorescence detection. Environ Sci Technol 37:3687–3693 Youhei Y, Jaffé R, Maie N, Tanoue E (2008) Assessing the dynamics of dissolved organic matter (DOM) in coastal environments by excitation emission matrix fluorescence and parallel factor analysis (EEMPARAFAC). Limnol Oceanogr 53:1900–1908
RESEARCH ARTICLE
Evaluation of Suwannee River NOM electrophoretic fractions by RP-HPLC with online absorbance and fluorescence detection Olga E. Trubetskaya & Claire Richard & Oleg A. Trubetskoj
Received: 12 November 2014 / Accepted: 29 January 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract 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 (SECPAGE) 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 455/420 nm and two proteinlike fluorophores with emission maxima at around 300 and 340 nm. The HS-like fluorophore with emission maximum at 435 nm was located in the hydrophilic peak in all the samples.
Responsible editor: Philippe Garrigues O. E. Trubetskaya Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Moscow region, Russia 142290 C. Richard Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448, 63000 Clermont-Ferrand, France C. Richard CNRS, UMR 6296, ICCF, 63171 Aubière, France O. A. Trubetskoj (*) Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow region, Russia 142290 e-mail: [email protected]
Those with maxima at 455–465 nm 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.
Keywords Water NOM . RP-HPLC . SEC-PAGE setup . UV-visible and fluorescence spectroscopy . Protein-like and HS-like fluorophores
Introduction Natural water organic matter (NOM) is a complex mixture of organic biochemicals that can undergo various pathways of diagenetic alteration. The process of diagenesis can create a chain of various hydrophobic/hydrophilic molecules as a result of oxidation/cleavage and condensation reactions. Thus, any effort to understand the ecological fate of these compounds in the environment requires identification of their hydrophobic properties. For this reason, reversed-phase highperformance liquid chromatography (RP-HPLC) is often applied to water NOM to obtain fractions of different hydrophobic properties (Lombardi and Jardim 1999; Egeberg and Alberts, 2002; Parlanti, 2002; Wu et al., 2003; Stenson 2008; Hutta et al., 2011; Li et al., 2014). These data could extend the applicability of RP-HPLC method for research of water NOM chemical structure. On the other hand, they have environmental significance for understanding the nature of chemical interactions at the molecular level between water NOM and other organic constituents, such as hydrophobic organic pollutants capacity, which was mainly based on organic matter polarity.
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Yet, another potential consequence of NOM hydrophobicity distributions may occur in the fluorescence exhibited by these mixtures, and fluorescence properties have often been used to characterize water NOM (Susic and Boto, 1989; Coble, 1996, 2007; Egeberg and Alberts, 2002; Wu 2003; Abtt-Braun, 2004; Park, 2009; Youhei et al., 2008; Huguet et al., 2010; Ma et al., 2010; Li et al., 2014) but it still provides limited information about the localization of NOM fluorophores. Further determination of fluorescent NOM constituents between fractions obtained by RP-HPLC procedure and exhibiting different polarity should be promising for the understanding of the water NOM fluorescence origin. Water NOM is polyelectrolytic, and electrophoretic techniques should be applicable for constituent separation and further characterization. An electrophoretic approach, which has been successfully used in the last 40 years, is the polyacrylamide gel electrophoresis (PAGE). With this method, fractions with distinct electrophoretic mobilities were obtained from water NOM (Duxbury 1989; Baxter and Malysz, 1992; Dunkel et al., 1997; Peurvouri et al. 2001; Parlanti et al., 2002; Trubetskoi and Trubetskaya, 2004; Trubetskoj et al., 2009). The obtained electropherograms are mainly interpreted as fingerprints of the analyzed water NOM. However, we are not aware of any literature data about the characterization of hydrophobic/hydrophilic properties of NOM fractions with different electrophoretic mobilities (EM) and molecular sizes (MS). Such determinations should be useful for the understanding of water NOM structure and properties. Moreover, they can help in clarifying the nature of chemical interactions at the molecular level between water NOM and other organic constituents, such as hydrophobic organic pollutants. We have previously developed an effective method for the fractionation of different humic substances (HS) based on the combined use of conventional preparative size exclusion chromatography (SEC) and analytical PAGE; this procedure was called the SEC-PAGE setup (Trubetskoj et al., 1997). The advantage of this combination rests primarily in the presence of urea, which is added at a level of 7 M in both SEC and PAGE to assist in the rupture of hydrogen bonds and prevents interaction between the fractionated humic material and the solid immobile phase on which the substances are separated. This is accompanied by the disaggregation of HS, which we believe is primarily related to the disruption of hydrogen bonds. The proposed procedure allows separation of primary disaggregated structural humic components, thereby solving some of the key problems occurring in the fractionation of HS, for example, irreversible/reversible chromatographic column adsorption and the influence of PAGE chemical components (e.g., Tris and borate ions) on the composition of HS fractions. On the other hand, 1H-NMR in D2O and solid 13C-NMR of soil HS before and after concentrated urea treatment showed no structural differences (Trubetskoj et al., 2010). SEC-PAGE
setup was first applied to water NOM extracted from natural terrestrial waters of Finland and USA (Peurvuori et al. 2001; Trubetskoj et al., 2009). It was shown that aquatic NOM fraction with highest EM and lowest MS fluoresced intensively and initiated the formation of singlet oxygen. On the contrary, the fluorescence intensity of low EM fractions with the highest and medium MS was insignificant (Trubetskoj et al., 2009). The objectives of the current work was to use RP-HPLC with online absorbance and fluorescence detections for analysis of Suwannee river NOM and its fractions obtained by SEC-PAGE setup for revealing the fluorescent NOM constituents of different polarity between fractions of different EM and MS. The multi-step scheme summarizing the separations and analysis of Suwannee River natural organic matter (SRNOM) is presented on Fig. 1. The NOM isolated from Suwannee River was chosen as one of the standard of the International Humic Substances Society (IHSS). This sample include Suwannee River humic and fulvic acids, thus using of this standard is more important for understanding of the nature of chemical interactions at the molecular level between water NOM and other organic constituents.
Materials and methods Material The NOM sample, isolated by reverse osmosis from Suwannee river water, Georgia, USA (SRNOM), was purchased as a standard material (ref. number 1R101N) from the International Humic Substances Society. Major elemental composition of SRNOM were as follows: C=52.5 %, H= 4.2 %, N=1.1 %, ash content=7.0 %, and water content= 8.2 %. All reagents were of the highest grade available. Water was purified using a Millipore Milli-Q system (Millipore αQ, resistivity 18.2 MΩ cm − 1 , DOC < 0.1 mg L−1). Phosphate buffer (10 mM Na2HPO4/NaH2PO4, pH 6.5) was prepared on reversed osmoses water, obtained with RIOS 5 and Synergy, Millipore. Conventional preparative SEC-PAGE setup Fractionation of SRNOM by SEC-PAGE setup (i.e., using PAGE for subsequent testing of SEC aliquots from different sections of the elution profile) was previously reported (Trubetskoj et al., 1997). Briefly, 20 mg SRNOM were dissolved in 1 mL 7 M urea and loaded onto a Sephadex G-75 (Pharmacia, Sweden) column (1.5×100 cm), equilibrated with the same solution. The void (Vo) and the total (Vt) column volumes were 47 and 160 mL, respectively. The void volume was determined using Dextran Blue 2000. Fractionation ranges for Sephadex G-75 were 80,000 to 3000 for proteins and 50,000 to 1000 for polysaccharides. Flow rate was
Environ Sci Pollut Res Fig. 1 The multi-step scheme summarizing the separations and analysis of SRNOM
SRNOM Conventional preparative SEC-PAGE setup in 7M urea
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Analytical RP-HPLC in 10mM phosphate buffer, pH 6.5, with on-line absorbance detection at 270 nm and fluorescence detection at Ex/Em 270/450nm and 270/330 nm
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15 mL h−1. The UV-detector (ISCO, USA) was set at 280 nm. The eluate from Vo (47 mL) to Vt (160 mL) was combined into 10 aliquots, the volume of each aliquot was about 11–13 mL (Fig. 2a); 0.1 mL of each aliquot was added to 0.01 mL of buffer, which contains 0.89 M Tris-borate, 10 % SDS and 10 mM EDTA, pH 8.3, and assayed by PAGE (Fig. 2b). The apparatus was a vertical electrophoresis device (LKB 2001 Vertical Electrophoresis, Sweden) with a gel slab (20 × 20 cm). Acrylamide 9.7 % and bisacrylamide 0.3 % were dissolved in 89 mM Tris-borate buffer with 1 mM EDTA and 7 M urea, pH 8.3. For polymerization, 0.014 mL N,N, N ,N -tetramethylethylendiamine and 0.4 mL 10 % ammonium persulfate were added to 40 mL of acrylamide/ bisacrylamide solution. The electrode buffer had a concentration of 89 mM Tris-borate with 1 mM EDTA, pH 8.3. Electrophoresis was carried out at a room temperature for 1 h at a constant current intensity of 25 mA; during electrophoresis, the voltage gradually increased from 300 to 500 V. On the basis of PAGE analysis (Fig. 2b), three naturally colored fractions (A = aliquot I, B = aliquots II–IV, and C+D = aliquots V–X) originated different EM were detected, concentrated on UF membrane with nominal cut of 5 kDa, dialyzed through 10 kDa cellulose dialysis tubing (Sigma-Aldrich, Moscow, Russia) during 7 days against distilled water, lyophilized, and used for further analyses.
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Analytical RP-HPLC SRNOM and its fractions were analyzed on a Waters ACQUITYTM Ultra-Performance Liquid Chromatographic system with a cooling autosampler (ACQUITY Sample Manager), a binary pumping module (ACQUITY Solvent Manager), and column oven enabling temperature control of analytical column (Waters Corp., Milford, MA, USA). An analytical column (UPLC© BEH C18, 2.1×100 mm, particle size 1.7 μm - Waters Corp., USA) equipped a precolumn used as stationary phase. The column was maintained at 30 °C. UV-Visible detection was carried out using an ACQUITY photodiode array detector (PDA), designed for wavelengths in the range 210– 400 nm, and RP-HPLC chromatograms with online absorbance at 270 nm were analyzed. The fluorescence of samples was monitored using an ACQUITY Fluorescence detector - FLR (Waters Corp., Milford, MA, USA), connected directly to the waste line of the absorbance PDA detector. The delay between PDA and FLR detection was 0.12 min. FLR detector was designed for the excitation wavelength 270 nm and emission range 290–600 nm. The RP-HPLC chromatograms with online emission at 330 and 450 nm were analyzed. Analyses of blank phosphate buffer solutions (10 mM) were performed before the
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(a)
humic matter was diluted to 3 mL (about 600 times), this allowed the use of PDA detector, which has the maximum absorbance limit of 2 a.u. (1.5 % deviation). Data were collected and processed by chromatographic software Empower2TM (Waters Corp., Milford, MA, USA). The entire cycle of RPHPLC procedures was repeated three times. Deviations between three chromatogram’s profiles of each sample did not exceed 3.0 %.
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Fig. 2 Preparative size exclusion chromatography of 20 mg SRNOM on Sephadex G-75 column (100 cm×1.5 cm) using 7 M urea as eluting medium (a). Letters I–X on the x-axis show the aliquots, analyzed by PAGE (b)
analyses of the SRNOM and its fractions to check the absence of any absorbance and fluorescent peaks in the solvents. Solvent A (methanol, HPLC grade CHROMASOLV®, Sigma-Aldrich) and solvent B (10 mM phosphate buffer, pH 6.5, prepared in Milli-Q water) were used for gradient formation. A stepwise gradient was applied starting at 0.00 min with 0 % of solvent A in solvent B, at 2.22 min=10 % A in B, at 3.33 min= 20 % A in B, at 4.45 min = 30 % A in B, at 5.56 min= 40 % A in B, at 6.67 min = 50 % A in B, at 7.7 min = 60 % A in B, at 11.12 min = 70 % A in B, at 13.89 min=100 % A, at 20 min=0 % A in B, and continuing for another 10 min at flow rate of 0.3 mL min−1. Solutions of SRNOM and fractions A, B, and C+D were prepared by dissolving weighed amounts of dry material in 10 mM phosphate buffer, pH 6.5. To estimate the relative amounts of humic matter eluted and irreversibly absorbed on the column during the RPHPLC analyses, all samples were prepared in the same manner: the absorbance of each sample was adjusted to 6.5 a.u. at 270 nm in a 1-cm quartz cuvette on the basis of dilution 10– 20 times of stock solutions, the volume of injection onto the reversed-phase column was 0.005 mL. All humic matter, which did not irreversibly adsorb on the column, was eluted during the first 10 min. Flow rate was 0.3 mL min−1. It means that injected
UV-visible absorption spectra of SRNOM and its fractions A, B, and C+D were recorded using a Cary 3 spectrophotometer (Varian, Cary, USA) in a 1-cm quartz cuvette from 220 to 700 nm at a concentration of 50 mg L−1 in 10 mM phosphate buffer, pH 6.5. The specific adsorption coefficient at 280 nm (A280) at C=50 mg L−1 was used for fractions comparison. Fluorescence emission spectra of SRNOM and its fractions were recorded using a Cary Eclipse fluorescence spectrophotometer (Varian, Cary, NC, USA) in a 1-cm quartz cuvette. Excitation wavelength was 270 nm, emission spectra were recorded from 310 to 700 nm. In order to minimize the inner filter effect, the solutions were diluted with Milli-Q water to an absorbance of 0.05±0.01 at 270 nm. UV-visible absorption spectra and fluorescence emission spectra of the RP-HPLC peaks were extracted from the data of PDA and FLR detectors. For SRNOM, its fractions and all RP-HPLC peaks the absorbance ratio at 270 and 366 nm (A2/A3) were calculated.
Results and discussion Preparative fractionation of SRNOM by SEC-PAGE setup Preparative SEC-fractionation of SRNOM on the column with Sephadex G-75 in 7 M urea revealed the presence of two broad overlapping chromatographic peaks. First small
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peak (47–60 mL) was eluted at the void column volume, and the second large peak with the shoulder covered almost all fractionation range of the column from 61 to 160 mL (Fig. 2a). The elution profile was divided into 10 aliquots, each of them were analyzed by PAGE (Fig. 2b). The aliquot I (elution volume from 47 to 60 mL) correspond to the excluded peak and formed at the electropherogram start zone A, which do not penetrate
Table 1 Relative contribution of resolved peaks 1–7 on RP-HPLC total chromatograms, ratios A2/ A3 and emission maxima of bulk SRNOM, fractions A, B, and C+ D, and RP-HPLC peaks 1–7, 1a, 3a, and 4a. Specific absorption coefficient at 280 nm (A280) of SRNOM and its fractions A, B, and C+D
sh shoulder at
Sample
into the 10 % PAG. The aliquots II–IV (elution volume from 61 to 93 mL) formed an intensively colored narrow zone in the mid part of PAG and combined into fraction B. The aliquots V–X (elution volume from 94 to 160 mL) forming several colored bands on the mid and bottom of the PAG were combined into fraction C+D. On the basis of the elution orders, we suggest, that the MS of fractions are in the order: A>B>C+D.
Relative contribution (%) of RP-HPLC peaks 1–7
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A2/A3
Emission maximum (nm) at λex =270 nm Protein-like
SRNOM Peak 1 53 Peak 2 19 Peak 3 15 Peak 4 8 Peak 5 4 Peak 6 1 Σ Peaks 2–6 47 High MS fraction A Peak 1 9 Peak 1a – Peak 2 21 Peak 3 33 Peak 3a – Peak 4 22 Peak 4a – Peak 5 10 Peak 6 3 Peak 7 2 Σ Peaks 2–7 91 Medium MS fraction B Peak 1 12 Peak 1a Peak 2 31 Peak 3 32 Peak 3a – Peak 4 17 Peak 4a – Peak 5 5 Peak 6 2 Peak 7 1 Σ Peaks 2–7 88 Low MS fraction C+D Peak 1 52 Peak 2 24 Peak 3 15 Peak 4 6 Peak 5 2 Peak 6 1 Σ Peaks 2–6 48
0.61
3.9 5.3 3.3 3.1 3.4 3.1 3.1
0.26
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350
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340
0.75
0.89
3.3 4.1 3.1 3.0 3.0 2.8 2.6
HS-like 455 435 450 450 450 450 450 440 435
≈300 455 455 340 460 340 460 460
465 435
≈300 465 465 340 465 340 465 465
465 435 455 sh420 455 sh420 455 sh420 455 sh420 455 sh420
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UV-visible absorption and fluorescent emission spectra of SRNOM and its fractions UV-visible absorption spectra of SRNOM and its fractions A and B appeared to be featureless without major maxima and decreased gradually with increasing wavelength with shoulder between 260–280 nm. On the other hand, fraction C+D has the shoulder between 260–280 nm and a maximum below 220 nm (Fig. 3). The values of specific absorption coefficient at 280 nm (A280) varied in the order C+D>B>SRNOM>A (Fig. 3, Table 1). As currently accepted, the fluorescence of water NOM is due to two main groups of fluorophores (Coble, 1996; Huguet et al., 2010. Boyle et al., 2009; Andrew et al., 2013). One group usually has excitation (Ex) maximum less than 305 nm and emission (Em) maximum less than 380 nm, related to aromatic amino acids, and is often referred to as protein-like fluorophores. The other one with Ex/Em 220– 360/380–470 nm is attributed to HS-like fluorophores of water NOM. However, until now, their relationship with humic components of different MS and EM remains uncertain. Fluorescence emission spectra of SRNOM and its fractions, recorded upon excitation at 270 nm, are given in Fig. 4. For the bulk SRNOM and fraction C+D, the fluorescence emission spectra originated different maxima at 455 and 465 nm, respectively. In contrast, fractions A and B revealed two maxima at 350 and 440 and 340 and 465 nm, respectively. Thus coupling SEC-PAGE allows to separate bulk SRNOM on fractions, which contained different groups of fluorophores. The vast majority of HS-like fluorophores located in low MS fraction C+D, while the high MS fractions A and B contain both protein-like and HS-like fluorophores. The relative concentration of protein-like fluorophores in fraction A was considerably more than in fraction B (Fig. 4, inset). These data indicate that SRNOM is not homogeneous and can be splitted
into several fluorescent sub-fractions with different emission maxima. Thus, overall DOM fluorescence could comes from simple summation of all the fluorescence of the individual fluorophores presented by taking account of the dilution factors. On the other hand, instead of simple summation, the overall fluorescence could arise from charge transfer interactions between fraction moieties within DOM (Boyle et al., 2009). Either of the possible scenarios would invalidate the effectiveness of this fluorophores fractionation method. For more deep investigation of physical–chemical properties of SRNOM and revealing of less complex mixture of NOM compounds, we carried out analytical fractionation of bulk SRNOM and its fraction by RP-HPLC with online absorbance and fluorescence detection. RP-HPLC of SRNOM and its fractions Figure 5 (left column) presents RP-HPLC chromatograms of bulk SRNOM and fractions A, B, and C+D with online absorbance detection at 270 nm. The chromatograms exhibit the resolution of six (SRNOM and fraction C+D) or seven (fractions A and B) peaks. The hydrophobicity of RP-HPLC peaks increased from the first to the last eluted peak due to the increase of methanol concentration in the stepwise separation procedure. For simplification, we refer to the first peak as hydrophilic, because it is eluted in an aqueous phosphate buffer only, whereas the other peaks are assumed as hydrophobic, because they are eluted by methanol at different concentrations (from 10 to 60 %). No material was eluted by methanol
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Fig. 4 Emission spectra of SRNOM and its fractions A, B, and C+D setting excitation at 270 nm and using A270=0.05. Inset spectra normalized at the HS-like emission maxima
Fig. 5 RP-HPLC chromatograms of SRNOM and fractions A, B, and C+ D with online absorbance at 270 nm (left column) and fluorescence detection (right column) at Ex/Em wavelengths 270/450 nm (solid line) and 270/330 nm (dashed line). Inset zoom on the 1.5- to 6.5-min zone of the chromatograms
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1a
60
0 2
3
40
4
5
6
Retention time, min
1
3a 4a
20
2
1a
1
2
3
4
5
6
7
8
9
10
0
1
2
Retention time, min
3
4
3
5
7
6
4
5
6
Fluorescence intensity
Fluorescence intensity
80 0,2
3
4
0,1
1 5 6
7
1
2
3
4
5
6
9
7
8
9
60
1a
40
2
3
4
1
20
2 3
1a
0
3a 4a 4 5
1
2
3
4
5
6
6
7
7
8
9
3 4
Fluorescence intensity
Fluorescence intensity
2
80 60
3
4
10
0
40
2
2
20
4
5
6
7
Retention time, min
8
9
10
5
0
1
2
3
4
4
4 6
0 3
3
5
Retention time, min
3
6
0,0 2
10
2
20
1
100
1
6
Retention tim, min
0,2
0
5
Retention time, min
10
1
5
4a
4
Fraction C+D
0,1
3 3a
2
0
Retention time, min
0,3
10
10
0
0,0 0
8
20
100
0,3
2
7
Retention time, min
Fraction B
Absorbance at 270 nm
3
2
0
0,0
Absorbance at 270 nm
4a
10
5
6
7
Retention time, min
8
9
10
6
Environ Sci Pollut Res
Relationships between fluorescence properties and polarity of SRNOM and its fractions Figure 5 (right column) presents RP-HPLC chromatograms of SRNOM and its fractions with online fluorescence detection at excitation–emission wavelength pairs (Ex/Em) 270/450 and 270/330 nm for detection of HS-like and protein-like fluorophores, respectively. Data obtained at Ex/Em 270/ 450 nm revealed HS-like fluorescence in peaks 1–6 with highest intensity in hydrophilic peak 1 for all samples, a very weak fluorescence in peak 7 of fractions A and B was not taken into consideration. The fluorescence emission spectra of the RP-HPLC peaks were extracted from the data of FLR detector. The emission maxima at λex =270 nm were presented in Table 1. The maximum of emission in hydrophilic peak 1 of SRNOM and fractions A, B, and C+D was 435 nm. The hydrophobic peaks 2–6 of all the samples had fluorescence maxima, shifted to long wavelength in comparison with hydrophilic peak 1. In fraction A, hydrophobic peaks 2–3 and 4– 6 revealed emission maxima at 455 and 460 nm, respectively. In fraction B, all the hydrophobic peaks revealed emission maximum at 465 nm. In fraction C+D, the hydrophobic peaks showed emission maximum at 455 nm with a shoulder at 420 nm. In SRNOM, all the hydrophobic peaks revealed emission maximum at 450 nm. The fluorescence maxima of fractions A and B (440 and 465 nm, respectively) lay within the range of fluorescence maxima of their RP-HPLC peaks 1– 6 (Table 1). However, fluorescence maxima of bulk SRNOM and fraction C+D exceeded fluorescence maxima values of all the peaks (Table 1). This suggests that a part of fluorophores of SRNOM and fraction C+D irreversibly adsorbed on the C18-column. Setting Ex/Em at 270/330 nm to detect the fluorescence related to aromatic amino acids, often referred to as protein-
Normalized absorbance
at a concentration more than 60 % in all the investigated samples. All RP-HPLC experiments were made in 10 mM phosphate buffer, pH 6.5. The choice of buffer was due to its high buffer capacity and wide application in studies of humic substances by RP-HPLC (Hutta et al., 2011; Wu et al., 2003). Moreover, some preliminary experiments showed that at a more lower pH all NOM matter had been irreversibly adsorbed on the C18-column. Because all samples were injected onto the column under constant conditions with special attention on injection volume and absorbance (see BMaterials and methods^), we had the possibility to estimate relative irreversible adsorption of different samples on the reversed-phase C18-column by comparison of the total chromatogram’s area. This calculus was made using the suggestion that absorption coefficient and fluorescence quantum yield of investigated samples do not change during RP-HPLC. The total area of the chromatographic peaks of SRNOM, fractions B and C+D were rather similar, but the total area of the chromatographic peaks of fraction A was lower by 20 % (Fig. 5, left column). Fraction A seems to be more hydrophobic than fractions B and C+D, but on the basis of these data it was impossible to compare hydrophobicity of fractions B and C+D. On the other hand, in spite of similar relative irreversible adsorption of fractions B and C+ D on C18-column, their eluted material demonstrated significant differences in the ratio of hydrophilic and hydrophobic peaks area (Table 1). Fraction C+D had a relatively higher abundance of hydrophilic constituents, 52 % of total chromatogram’s area was corresponded to peak 1, and areas under hydrophobic peaks 2–6 essentially decreased with the increasing of methanol concentration. Meanwhile, fractions A and B had low content of hydrophilic components—peak 1 contributed to only 9 and 12 % of total chromatogram area, respectively (Fig. 5, left column and Table 1). Analysis of SRNOM and its fractions in part of their relative irreversible adsorption on C18column and relative distribution of eluted from the column matter on hydrophobic and hydrophilic peaks shown, that hydrophobicity of fractions decreased in order: A>B>C+D. It should be noted, that absorption spectra of hydrophilic and hydrophobic RP-HPLC peaks (extracted from the data of PDA detector) in SRNOM and its fractions were featureless and showed a gradual decrease in absorbance with increasing wavelength without major maxima. However, they had differences in the values of absorbance ratio at 270 and 366 nm (A2/ A3) (Table 1). This ratio was considerably higher in all hydrophilic peaks of SRNOM and its fractions in comparison with the same ratio in hydrophobic peaks. This trend was more pronounced in SRNOM and fraction C+D and could be explained by different ratio aromatic/aliphatic compounds in hydrophilic and hydrophobic peaks. The RP-HPLC with using stepwise gradient of methanol allowed us to map the SRNOM and its fractions in terms of distribution of hydrophilic/ hydrophobic components.
Peak 1a
0,004
Fr.A, peaks 3a, 4a
0,002
0,000 Phosphate buffer
220
240
260
280
300
320
340
360
Wavelength, nm Fig. 6 Absorption spectra of fraction A, its RP-HPLC peaks 1a, 3a, and 4a, normalized at A366 nm and blank solution for peak 1a (phosphate buffer at elution volume 1.78 ml). Spectra were extracted from the data of PDA detector. Fraction B and its RP-HPLC peaks 1a, 3a, and 4a demonstrated similar results
Environ Sci Pollut Res
like fluorescence, did not revealed essential signals in the bulk SRNOM chromatogram (Fig. 5, right column). Similar results were reported by other researchers for bulk SRNOM, Suwannee River humic, and fulvic acids (Coble 2007; Wu 2003; Alberts and Takács 2004). However, new peaks occurred in chromatograms of high MS fraction A and medium MS fraction B: a peak 1a at retention time 1.9 min, i.e., in the hydrophilic zone, and peaks 3a and 4a at retention times 4.9 and 5.8 min in the hydrophobic zone of chromatograms. Low MS fraction C+D demonstrated only traces of these peaks (Fig. 5, right column, inset). These peaks showed strong protein-like fluorescence emission maxima at 300–340 nm (Table 1). Interestingly, peaks 3a and 4a had an emission maxima at 340 nm, but peak 1a—about 300 nm (Table 1). It is well known, that protein-like fluorescence emission maxima at around 300–380 nm correspond to aromatic amino acids like as phenylalanine, tyrosine, and tryptophan (Lehninger 2000; Lakowicz 2006). Peak 1a might correspond to tyrosine-like chromophore bounded to rather hydrophilic part of SRNOM, because it demonstrated emission maximum at about 300 nm (Table 1) and absorbance spectrum with two maxima at 220 and 270 nm (Fig. 6)—the two optical characteristics of tyrosine amino acid (Lakowicz 2006). The absorbance spectra of peaks 3a and 4a were featureless without major maxima decreasing gradually with increasing wavelength. We attributed peaks 3a and 4a to a mixture of non polar NOM particles having featureless absorption spectra and protein-like fluorophore(s) with emission maximum(a) around 340 nm.
Conclusion SEC-PAGE setup followed by RP-HPLC allowed us to develop approach of SRNOM separation on less heterogeneous compounds mixture for their further study and structural identification. RP-HPLC on C18-column with online absorbance and fluorescence detection gave possibility to map SRNOM and its different on MS fractions in terms of distribution of hydrophilic/hydrophobic components and fluorophores. Our results show that: (i) Analysis of SRNOM and its fractions in part of their relative irreversible absorption 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: high MS fraction A>medium MS fraction B>low MS fraction C+D; (ii) The SRNOM contained at least three groups of the HSlike fluorophores with different emission maxima: fluorophore(s) with fluorescence emission maximum at 435 nm was located in hydrophilic peak and those with emission maxima at 450–465 nm in the hydrophobic
peaks, the vast majority of HS-like fluorescence located in low MS fraction C+D; (iii) The SRNOM contained at least two groups of the protein-like chromophores, located mainly in high MS fraction A and medium MS fraction B. One group of fluorophore(s) with emission maximum around 300 nm bounded to polar NOM particles and have absorbance spectrum with two maxima at 220 and 270 nm, the second group of fluorophore(s) with the emission maximum around 340 nm bounded to non polar NOM particles and have featureless absorption spectra; The data obtained could be significant for future NOM chemical structural characterization. Acknowledgments The work has been supported by Russian Foundation for Basic Research (projects no 13-05-00241-a and 15-04-00525-a).
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