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A new analytical approach for humin determination in sediments and soils
Talanta 71 (2007) 1444–1448
Short communication
A new analytical approach for humin determination in sediments and soils N. Calace ∗ , B.M. Petronio, S. Persia, M. Pietroletti, D. Pacioni Department of Chemistry, University “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy Received 17 March 2006; received in revised form 15 May 2006; accepted 30 June 2006 Available online 14 August 2006
Abstract In this work a new analytical approach is proposed for the recovery of humin present in soil and sediments. The procedure is based on microwave oven treatment for humin deashing. In this way both the treatment time and the concentration of the HCl/HF mixture are significantly reduced (minutes rather than hours, 10% rather than concentrated). By means of the proposed scheme organic matter present in sediment and soil samples can be subdivided into the different fractions (hydrophobic and hydrophilic compounds, fulvic and humic acids, humin) making up the balance of organic carbon. Results obtained for samples characterised by different organic carbon content showed a loss of carbon ranging between 20% and 30%, consistent with previous reports about humin deashing. © 2006 Elsevier B.V. All rights reserved. Keywords: Humin; Separation; Microwave oven
1. Introduction Natural organic carbon can be divided up into labile forms, subjected to bioassimilation and biotransformation, and nonlabile forms. Since labile forms are subject to microbial attack, it is more likely that refractory materials, present in uncharacterised organic matter pool, are buried. The study of factors that control organic matter burial and preservation in sediments and soils has led to the development of several techniques to characterise bulk organic matter [1–3]. Nevertheless knowledge in this field is not exhaustive, and so quantifying and structurally elucidating the uncharacterised organic matter pool remains one of the greatest challenges in marine and terrestrial organic geochemistry today. Humic substances represent a fraction of this uncharacterised organic matter pool and consist of a complex mixture of molecules whose molecular weight varies from 0.5 to 300 kDa. They can be subdivided into three categories that are operationally defined and based upon their solubility in water: (a) fulvic acids, tend to be smaller in molecular weight, soluble in water at all pHs; (b) humic acids are larger and often colloidal, insoluble at pHs lower than two; (c) humin, the highest in molecular weight and carbon content, is strongly associated
∗
Corresponding author. Tel.: +39 0649913723; fax: +39 0649913723. E-mail address: [email protected] (N. Calace).
0039-9140/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2006.06.040
with the mineral matrix and insoluble at any pH. Humic and fulvic acids are subjected to chemical transformations whereas humin is highly refractory. As a consequence of humin insolubility, the physical and chemical nature of humin is the least understood of the three fractions. Moreover, it is known that humin includes unaltered and less-altered biopolymers such as lignin and polysaccharides [4], mineral-bound lipids and humic acid-like materials [5], kerogen and black carbon [6]. It also contains acid-hydrolysable structure segments, carbohydrates, proteins and esters that can be removed by acid treatment [7]. By definition, humin consists of the solid residue that remains after centrifugation of the alkali extract of the sample. To separate humin from the inorganic matrix (mainly clays and oxides) the residue is generally subjected to deashing. The ideal treatment should be capable of dissolving a substantial proportion of the mineral matrix without significantly altering the chemical structure of organic compounds. Durand and Nicaise [8] carried out the demineralisation process with a concentrated HCl/HF mixture, but in this case a loss of organic matter was observed. Consequently, the HF concentration was reduced (10%), increasing the number of treatments, depending on organic matter concentration [9,10]. Generally six treatments were found to be enough. Using 15% HF the loss of organic matter increased [11]. The number of extractions can be reduced by increasing the extraction time. Two treatments with HF 10% at room temperature for 12 h are
N. Calace et al. / Talanta 71 (2007) 1444–1448
generally sufficient, while three extractions are necessary for samples with low organic carbon content and high concentration of hard oxide minerals [11,12]. Silicates were also eliminated by dissolution in hot HCl/HF mixture: at 70 ◦ C for 24 h [13] and at 60 ◦ C for 20 h [6]. For the above treatments the loss of organic matter ranges from 5% to 20.5%. Often subsequent treatment with HCl 6 M is carried out to hydrolyse labile biopolymers not removed by HF/HCl [14] and to remove CaF2 formed during the demineralisation procedure [6]. Mineral residues are also separated from organic matter by HCl (10%) and HF (70%) mixture shacking, centrifuging, ultrasonic vibration and ZnBr2 floatation [15]. Densimetric separation after exhaustive treatments with concentrated HF/HCl was also employed by Augris et al. [16]. It should be noted that all the above reported procedures are characterised by a significant number of extractions and long extraction times. In recent years there has been growing interest in the use of microwave heating in analytical chemistry. Microwave-assisted acid solubilisation is a highly suitable method for the digestion of complex matrices such as soil and sediments. Operationally, the procedure allows shorter digestion times and needs smaller quantities of acids, and so a number of potential applications of microwave processing have been investigated. These include microwave-assisted ore grinding, microwave assisted reduction of metal oxides, microwave assisted drying and anhydration; microwave assisted mineral leaching, etc. [17–23]. In this paper we propose a new procedure in which a microwave energy is employed for the digestion of the inorganic fraction present in the matrix alone. In particular the HF/HCl treatment for humin deashing [15] was carried out in a microwave oven. In this way treatment time lasts minutes rather than hours. Finally, sediment and soil samples were analysed according to the fractionation scheme proposed by us, making up the balance of organic carbon. The scheme allows subdivision into different fractions of organic compounds present both in sediments and in soils. In particular hydrophobic and hydrophilic compounds, fulvic and humic acids, and humin are separated [24–30]. 2. Experimental 2.1. Materials Marine sediments were taken from cores sampled in the Ross Sea (Antarctica). Antarctic sediments are characterised by a high silica content due to the presence of siliceous diatoms and silicoflagellates, and a low organic carbon content, prevalently associated with minerals. In particular, the superficial layer of a core characterised by non-transformed biogenic silica and soluble in basic medium (sediment 1) was used, and two lower layers of other cores in which siliceous minerals prevail (sediments 2 and 3). Sediment 1 (74◦ 00 S, 174◦ 48 E), located in the centre of the northern part of the Joides Basin at a depth of 582 m, represents a setting characterised by high biosiliceous sediment accumulation. Sediment 2 (75◦ 54 S, 177◦ 34 E), was collected
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at a depth of 628 m in an area near the continental shelf break in a central Ross Sea basin and sediment 3 (75◦ 04 S, 164◦ 138 E), located in the western Ross Sea at a depth of 950 m, was collected in an area representative of an environment with high energy and reworked sediments. A soil sample collected in an agricultural area located near Rome was also analysed. 2.2. Methodology for sediment and soil characterisation A Labconco 77400 lyophiliser (Labconco, Kansas City, MO) was used for sample lyophilisation. Grain size composition was obtained using common sieving methods (Table 1). Total organic carbon (TOC) content was determined with a Carlo Erba EA11110 CHNS-O Element Analyser (Carlo Erba, Milan, Italy). A 100 mg aliquot of dried sediment was acidified with 500 l of concentrated HCl (Merck, Germany) in order to eliminate the carbonate fraction, then freeze-dried and re-weighed. The freeze-dried residue was then analysed for TOC. The measurements were repeated six times and the relative standard deviations were less than 5%. Carbonate content was determined as difference between the total carbon content of sample, determined with elemental analyser, and TOC. 2.3. Methodology for humin deashing Humin deashing optimisation was carried out using 8.0 g aliquots of lyophilised sediment 3 (Table 1) after extraction of soluble organic fractions (hydrophobic, hydrophilic, humic and fulvic fractions). The total organic carbon remaining in the insoluble residue was 2.0 ± 0.12 mg g−1 . The decision to optimise the humin deashing procedure with sediment 3 was due to the low organic carbon and high silicates content. The insoluble residue was treated with 50 ml of a 1 N HCl and 10% HF (v/v) solution in a microwave oven (10 min at 30 W power, 12 min at 80 W). The maximum temperature reached in the vessels was 70 ◦ C. In order to test this procedure, the humin deashing methodology based on HF/HCl solution maintained at room temperature for 24 h [15], was also performed. In order to eliminate the minerals associated with the humin fraction as far as possible, a further step based on densimetric separation was performed [31]. The residue was thus treated: (1) with ZnCl2 solution in order to separate humin (lighter fraction) from residual minerals. The ZnCl2 treatment (10 ml Table 1 Grain size composition of sediments and soil samples % of
Sediment 1
Sediment 2
Sediment 3
Soil
Coarse sand Fine sand Silt Clay Carbonate Total organic carbon Total nitrogen
0 32.6 0 67.4 0 0.89 0.13
3.5 50.5 0 46.5 0 0.54 <0.1
10.1 36.7 0 52.3 0 0.36 <0.1
18.0 34.4 25.0 22.6 n.d. 4.9 0.48
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N. Calace et al. / Talanta 71 (2007) 1444–1448
solution) was repeated as long as the light fraction was present. The organic fraction was precipitated by diluting the ZnCl2 solution with distilled water; (2) with ZnCl2 solution. The organic fraction precipitated by diluting the ZnCl2 solution with distilled water was then separated and treated three times with HCl 6 M (each time 10 ml); (3) three times with HCl 6 M (each time 10 ml), then the residue was treated with ZnCl2 (d = 2 g cm−3 ) solution. The humin (Hm) obtained as precipitate from (1) to (3) was dried under vacuum at 100 ◦ C and then analysed for organic carbon content. The water used for rinsing and dilution was organic-free UVoxidised water dispensed by a Milli-Q Gradient A10 instrument from (Millipore, Bedford, MA). Glassware was cleaned by soaking in a 10% HNO3 bath for 24 h, rinsing thoroughly with water and muffling at 350 ◦ C for 3 h. All reagents were analytical grade. 2.4. Fractionation of organic carbon Fractionation of organic carbon was carried out according to the following procedure:
were separated, suspended in deionised water and purified by dialysis against deionised water using tubular membranes (Spectra/Por® , Spectrum Laboratories Inc., Rancho Dominguez, CA) with a molecular weight size of 1000 Da. They were then freeze-dried and analysed for organic carbon content by elemental analysis. Fulvic acids remaining in solution after acidification at pH 1 were purified using an Amicon stirred ultrafiltration cell, model 8400 (Millipore, Milan, Italy) fitted at the bottom with a 500 Da membrane disc. (4) To obtain the humin (Hm) fraction the residual sediment was treated twice with 50 ml of a 1 N HCl and 10% HF (v/v) solution in a microwave oven (10 min at 30 W power, 12 min at 80 W). The residue was then treated with ZnCl2 solution and humin (light fraction d < 2 g cm−3 ) was separated from mineral residue (heavy fraction d > 2 g cm−3 ). The Hm fraction was then treated three times with 6 M HCl (each time 10 ml at room temperature), rinsed with distilled water and dried under vacuum at 100 ◦ C. The organic carbon content of the humin fraction was determined by elemental analysis. 3. Results and discussion 3.1. Humin deashing
(1) To obtain the hydrophobic (Hb) fraction, lyophilised sediment (ca. 10 g) was extracted for 24 h at room temperature with a CH2 Cl2 /CH3 OH (2:1, v/v) mixture in a 1:5 (w/v) solid/liquid ratio. Two consecutive extractions of 24 h were carried out in a continuous rotating agitator. After centrifugation for 30 min at 4000 rpm, the supernatants were dried at 60 ◦ C in a crucible under vacuum in a desiccator, previously brought to constant weight in a desiccator at 100 ◦ C, and then weighed. The organic carbon content of the Hb fraction was determined by elemental analysis. (2) To obtain the hydrophilic (Hp) fraction, the residual sediment was treated with 90 ml of a 0.5 N HCl solution for 24 h in a continuous rotating agitator and, after centrifugation for 30 min at 2000 rpm, the acid solution was made up to a volume of 100 ml with distilled water. Aliquots of 20 ml were dried under vacuum in a desiccator at 60 ◦ C in a pre-weighed crucible. (3) To obtain humic materials the residual sediment was treated with 90 ml of a 0.5 N NaOH solution for 24 h in a continuous rotating agitator. After centrifugation for 30 min at 2000 rpm, the alkaline supernatant, containing humic and fulvic acids, was acidified at pH 1 by dropwise addition of concentrated HCl in order to separate acid-soluble fulvic acids (FA) from precipitating humic acids (HA). Both acid (step 2) and alkaline extractions (step 3) were alternatively repeated on residual sediment until the alkaline solution was colourless. The organic carbon content of the Hp fraction was determined by elemental analysis and only the first 20 ml aliquot yielded a detectable organic carbon content. In the case of humic substance extraction all the alkaline solutions were mixed. Humic acids, precipitated after acidification to pH 1 of the alkaline solution,
The results for the solid/liquid ratio needed for humin deashing are presented in Fig. 1. Results highlighted that the liquid amount used for humin deashing did not influence the yield of the treatment. For this reason we chose to use a solid/liquid ratio of 1:5 (w/v). The time and power values for microwave oven operation were selected so as to reach the maximum temperature of 70 ◦ C in the vessels. This temperature was chosen to take into account that the addition of the HF/HCl solution to sediment would increase the temperature in the mixture up to 70 ◦ C due to the exothermic reaction of silicate dissolution and so that the temperature value would not exceed 70 ◦ C in order to prevent the possible oxidation of organic compounds [32]. Fig. 2 shows the deashing results obtained from the microwave oven treatment. After two treatments with 50 ml of a 1 N HCl and 10% HF (v/v) solution in a microwave oven,
Fig. 1. Residue percentage vs. the liquid amount used for humin deashing.
N. Calace et al. / Talanta 71 (2007) 1444–1448
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Fig. 4. The residue percentage and the carbon percentage after densimetric separation according to treatment 1 (ZnCl2 ), treatment 2 (HCl before ZnCl2 ) and treatment 3 (HCl after ZnCl2 ). Fig. 2. The residue percentage and the carbon percentage as function of the number of treatments with HF/HCl solution in microwave oven.
Fig. 3. The residue percentage and the carbon percentage as function of the number of treatments with HF/HCl solution at room temperature.
the residue and the carbon percentage (9% and 2.2%, respectively) of the insoluble residue did not change. The calculated amount of organic carbon of the humin fraction was about 1.5 ± 0.064 mg g−1 , which accounts for 75% of the total organic carbon content of the sample (2.00 ± 0.12). On the other hand, the residue and carbon percentage of the residue obtained by treatments with HF/HCl solution at room temperature reached a plateaux after six treatments (Fig. 3). The residue and carbon percentage of the residue were 5% and 2.8%, respectively, which correspond to a humin carbon amount of 1.1 ± 0.042 mg g−1 . Results obtained from two procedures do not show any significant differences (95% at the confidence level) in humin deashing yield but comparing the results of the two procedures it is evident that the employment
of the microwave oven merely speeds up the dissolution of the mineral matrix. However, even if the amount of recovered humin carbon is higher, the carbon percentage of the residue is also low (∼2.2%), indicating a strong presence of the mineral fraction. Consequently, to increase the purification level a further step was taken, based on a densimetric separation of organic and inorganic fractions and on treatment with HCl 6 M to remove the CaF2 formed during the demineralisation process. With densimetric separation alone no increase in carbon percentage is observed, while carbon percentage significantly increases when HCl is employed (Fig. 4). The best results are obtained with the procedure (2), performing HCl treatment after densimetric separation. The amount of calculated organic carbon was practically the same (1.4 ± 0.18 mg g−1 ) for the procedures (1)–(3) and was the same as that calculated after microwave deashing treatments. This finding showed that the different treatments take effect only on the mineral fraction and that the recovery percentage of humin organic carbon depends mainly on microwave assisted acid treatment. 3.2. Organic carbon fractionation procedure Results obtained by the organic carbon fractionation (Table 2) on matrices characterised by different organic carbon content showed a loss of carbon ranging between 20% and 30%, this is consistent with previous reports by several researchers for humin deashing [6,8,11,13]. A large part of the carbon loss is due to the humin deashing step. As a result, elemental carbon analysis of all the samples studied were carried out soon after the alkaline extraction and before the humin deashing treatment (Table 3) and results show that the loss of carbon after humic substance extraction is negligible.
Table 2 Total organic carbon and carbon amount in the different fractions TOC (mg g−1 ) Sediment 1 Sediment 2 Sediment 3 Soil
8.9 5.4 3.6 48.9
± ± ± ±
0.3 0.4 0.4 0.2
Hb (mg g−1 ) 0.170 0.080 0.070 0.26
± ± ± ±
0.008 0.005 0.004 0.01
Hp (mg g−1 ) 0.16 0.11 0.11 0.42
± ± ± ±
0.01 0.01 0.01 0.01
HA (mg g−1 ) 1.5 0.75 0.45 8.6
± ± ± ±
0.1 0.06 0.03 0.1
FA (mg g−1 ) 4.0 2.1 0.9 16.7
± ± ± ±
0.4 0.4 0.3 0.5
Hm (mg g−1 ) 0.64 0.71 1.4 12.3
± ± ± ±
0.09 0.09 0.2 0.3
C loss (%) 28 31 19 22
± ± ± ±
10 18 25 2
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N. Calace et al. / Talanta 71 (2007) 1444–1448
Table 3 Total organic carbon, organic carbon before humin deashing and after alkaline treatments, and organic carbon as the sum of the fractions before humin deashing TOC Sediment 1 Sediment 2 Sediment 3 Soil
8.9 5.4 3.6 48.9
TOC − OCafter
OCafter ± ± ± ±
0.3 0.4 0.4 0.2
2.9 2.8 2.0 26.1
± ± ± ±
0.1 0.2 0.1 0.5
6.0 2.6 1.6 22.8
± ± ± ±
0.4 0.6 0.5 0.7
OC Hb + Hp + FA + HA 5.8 3.0 1.5 26
± ± ± ±
0.5 0.5 0.4 1
It should be considered that during the humin deashing procedure, some hydrolysable fragments of organic compounds could be solubilised. Moreover, organic molecules strictly bound to mineral matrix can be solubilised following dissolution of the inorganic matrix, which eliminates their protective support and so may release organic compounds into solution. Some researchers [33,34] have pointed out that the potential for alteration and losses is particularly significant for ‘immature’ organic matter in aquatic environments such as that found in surface sediments, which are usually younger than deep sediments. Indeed the trend in the organic carbon losses was found to be related to the age of sediments (samples 1 and 2 are much younger than sediment 3) although tests were performed on only a few samples. 4. Conclusion The results obtained with the approach proposed for the solubilisation of the inorganic matrix of sediments and soils show that microwave-assisted deashing is a viable alternative to traditional systems. The deashing procedure, based on the use of a microwave energy, makes the dissolution process faster and the treatment time is significantly reduced from hours to minutes. The fractionation scheme proposed could be applied also to samples in which organic carbon content is low. The balance of organic carbon shows that a large part of the carbon loss is due to the humin deashing step. Carbon loss, does not differ from the values reported by certain authors, cannot be ascribed to the use of the microwave oven and can probably be ascribed to hydrolysis processes related to the HF/HCl mixture. Acknowledgement This work was funded by the Italian National Research Programme for Antarctica (PNRA), Sector 9: Chimica degli ambienti polari. Research Project 2004/9.1. References [1] R.G. Keil, F.S. Hu, E.C. Tsamakis, J.I. Hedges, Nature 369 (1994) 639. [2] S.G. Wakeham, C. Lee, J.I. Hedges, P.J. Hernes, M.L. Peterson, Geochim. Cosmochim. Acta 61 (1997) 5363.
[3] J.I. Hedges, F.S. Hu, A.H. Devol, H.E. Hartnett, R.G. Keil, Am. J. Sci. 299 (1999) 529. [4] P.G. Hatcher, I.A. Breger, G.E. Maciel, N.M. Szeverenyi, in: G.R. Aiken, D.M. McKenight, R.L. Wershaw, P. MacCarty (Eds.), Humic Substances in Soil, Sediment and Water. Geochemistry, Isolation and Characterisation, John Wiley & Sons, New York, 1985, pp. 275–302. [5] J. Rice, P. MacCarthy, Environ. Sci. Technol. 24 (1990) 1875. [6] J. Song, P. Peng, W. Huang, Environ. Sci. Technol. 36 (2002) 3960. [7] M.H. Engel, S.A. Macko, Organic Geochemistry: Principles and Applications, Plenum Press, New York, 1993. [8] B. Durand, G. Nicaise, in: B. Durand (Ed.), Procedures for Kerogen Isolation, Kerogen. Techniq, Paris, 1980, pp. 35–53 (Chapter 2). [9] C.M. Preston, R.H. Newman, Geoderma 68 (1995) 229. [10] J.O. Skjemstad, P. Clarke, J.A. Tailor, J.M. Oades, R.H. Newman, Aust. J. Soil Res. 32 (1994) 1215. [11] Y. Gelinas, J.A. Baldock, J.I. Hedges, Org. Geochem. 32 (2001) 677. [12] M.W.I. Schmidt, H. Knicker, P.G. Hatcher, I. K¨ogel-Knabner, Eur. J. Soil Sci. 48 (1997) 319. [13] M. Khaddor, M. Ziyad, J. Joffre, A. Ambles, Chem. Geol. 186 (2002) 17. [14] H. Knicker, J.C. del Rio, P.G. Hatcher, R.D. Minard, Org. Geochem. 32 (2001) 397. [15] H.K. Karapanagioti, J. Childs, D.A. Sabatini, Environ. Sci. Technol. 35 (2001) 4684. [16] N. Augris, J. Balesdent, A. Mariotti, S. Derenne, C. Largeau, Org. Geochem. 28 (1998) 119. [17] A. Abu-Sambra, J.S. Morris, S.R. Koirtyohann, Anal. Chem. 47 (1975) 1475. [18] H.M. Kingston, L.B. Jassei, Introduction to Microwave Sample Preparation: Theory and Practice, 1st ed., American Chemical Society, Washington, DC, 1988. [19] H. Matusiewicz, R.E. Sturgeon, Prog. Anal. Spectrosc. 12 (1989) 21. [20] M.H. Feinberg, Analysis 19 (1991) 47. [21] H.M. Kingston, S.J. Haswell, Microwave-enhanced Chemistry, Fundamentals, Sample Preparation and Applications, American Chemical Society, Washington, DC, 1997. [22] S.M. Bradshaw, E.J. Vanwyk, J.B. Deswaedt, J. Microw. Power Electromag. Energy 32 (1997) 131. [23] M.F. Diprose, Plant Soil 229 (2001) 271. [24] M.Y. Sun, S.G. Wakeham, C. Lee, Geochim. Cosmochim. Acta 61 (1997) 341. [25] E.M. Thurman, R.L. Malcolm, Environ. Sci. Technol. 15 (1981) 463. [26] R.V. Tyson, Org. Geochem. 32 (2001) 333–339. [27] M. Vandenbroucke, R. Pelet, Y. Debyser, in: G.R. Aiken, D.M. McKnight, R.L. Wershaw, P. McCarthy (Eds.), Humic Substances in Soil, Sediment and Water, John Wiley & Sons, New York, 1995, pp. 249–273. [28] Y. Zegouagh, S. Derenne, C. Largeau, P. Bertrand, M.A. Sicre, A. Saliot, B. Rousseau, Org. Geochem. 30 (1999) 83. [29] N. Calace, F. De Paolis, F. Minniti, B.M. Petronio, Talanta 47 (1998) 803. [30] N. Calace, M. Capolei, M. Lucchese, B.M. Petronio, Talanta 49 (1999) 277. [31] N. Poirier, S. Derenne, J.N. Rouzaud, C. Largeau, A. Mariotti, J. Balesdent, J. Maquet, Org. Geochem. 31 (2000) 813. [32] A. Ambles, P. Jambu, J.C. Jacquesy, E. Parlanti, B. Secouet, Soil Sci. 156 (1993) 49. [33] L.M. Mayer, Geochim. Cosmochim. Acta 58 (1994) 1271. [34] R.G. Keil, E. Tsamakis, C.B. Fuh, J.C. Giddings, J.I. Hedges, Geochim. Cosmochim. Acta 58 (1994) 879.
Short communication
A new analytical approach for humin determination in sediments and soils N. Calace ∗ , B.M. Petronio, S. Persia, M. Pietroletti, D. Pacioni Department of Chemistry, University “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy Received 17 March 2006; received in revised form 15 May 2006; accepted 30 June 2006 Available online 14 August 2006
Abstract In this work a new analytical approach is proposed for the recovery of humin present in soil and sediments. The procedure is based on microwave oven treatment for humin deashing. In this way both the treatment time and the concentration of the HCl/HF mixture are significantly reduced (minutes rather than hours, 10% rather than concentrated). By means of the proposed scheme organic matter present in sediment and soil samples can be subdivided into the different fractions (hydrophobic and hydrophilic compounds, fulvic and humic acids, humin) making up the balance of organic carbon. Results obtained for samples characterised by different organic carbon content showed a loss of carbon ranging between 20% and 30%, consistent with previous reports about humin deashing. © 2006 Elsevier B.V. All rights reserved. Keywords: Humin; Separation; Microwave oven
1. Introduction Natural organic carbon can be divided up into labile forms, subjected to bioassimilation and biotransformation, and nonlabile forms. Since labile forms are subject to microbial attack, it is more likely that refractory materials, present in uncharacterised organic matter pool, are buried. The study of factors that control organic matter burial and preservation in sediments and soils has led to the development of several techniques to characterise bulk organic matter [1–3]. Nevertheless knowledge in this field is not exhaustive, and so quantifying and structurally elucidating the uncharacterised organic matter pool remains one of the greatest challenges in marine and terrestrial organic geochemistry today. Humic substances represent a fraction of this uncharacterised organic matter pool and consist of a complex mixture of molecules whose molecular weight varies from 0.5 to 300 kDa. They can be subdivided into three categories that are operationally defined and based upon their solubility in water: (a) fulvic acids, tend to be smaller in molecular weight, soluble in water at all pHs; (b) humic acids are larger and often colloidal, insoluble at pHs lower than two; (c) humin, the highest in molecular weight and carbon content, is strongly associated
∗
Corresponding author. Tel.: +39 0649913723; fax: +39 0649913723. E-mail address: [email protected] (N. Calace).
0039-9140/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2006.06.040
with the mineral matrix and insoluble at any pH. Humic and fulvic acids are subjected to chemical transformations whereas humin is highly refractory. As a consequence of humin insolubility, the physical and chemical nature of humin is the least understood of the three fractions. Moreover, it is known that humin includes unaltered and less-altered biopolymers such as lignin and polysaccharides [4], mineral-bound lipids and humic acid-like materials [5], kerogen and black carbon [6]. It also contains acid-hydrolysable structure segments, carbohydrates, proteins and esters that can be removed by acid treatment [7]. By definition, humin consists of the solid residue that remains after centrifugation of the alkali extract of the sample. To separate humin from the inorganic matrix (mainly clays and oxides) the residue is generally subjected to deashing. The ideal treatment should be capable of dissolving a substantial proportion of the mineral matrix without significantly altering the chemical structure of organic compounds. Durand and Nicaise [8] carried out the demineralisation process with a concentrated HCl/HF mixture, but in this case a loss of organic matter was observed. Consequently, the HF concentration was reduced (10%), increasing the number of treatments, depending on organic matter concentration [9,10]. Generally six treatments were found to be enough. Using 15% HF the loss of organic matter increased [11]. The number of extractions can be reduced by increasing the extraction time. Two treatments with HF 10% at room temperature for 12 h are
N. Calace et al. / Talanta 71 (2007) 1444–1448
generally sufficient, while three extractions are necessary for samples with low organic carbon content and high concentration of hard oxide minerals [11,12]. Silicates were also eliminated by dissolution in hot HCl/HF mixture: at 70 ◦ C for 24 h [13] and at 60 ◦ C for 20 h [6]. For the above treatments the loss of organic matter ranges from 5% to 20.5%. Often subsequent treatment with HCl 6 M is carried out to hydrolyse labile biopolymers not removed by HF/HCl [14] and to remove CaF2 formed during the demineralisation procedure [6]. Mineral residues are also separated from organic matter by HCl (10%) and HF (70%) mixture shacking, centrifuging, ultrasonic vibration and ZnBr2 floatation [15]. Densimetric separation after exhaustive treatments with concentrated HF/HCl was also employed by Augris et al. [16]. It should be noted that all the above reported procedures are characterised by a significant number of extractions and long extraction times. In recent years there has been growing interest in the use of microwave heating in analytical chemistry. Microwave-assisted acid solubilisation is a highly suitable method for the digestion of complex matrices such as soil and sediments. Operationally, the procedure allows shorter digestion times and needs smaller quantities of acids, and so a number of potential applications of microwave processing have been investigated. These include microwave-assisted ore grinding, microwave assisted reduction of metal oxides, microwave assisted drying and anhydration; microwave assisted mineral leaching, etc. [17–23]. In this paper we propose a new procedure in which a microwave energy is employed for the digestion of the inorganic fraction present in the matrix alone. In particular the HF/HCl treatment for humin deashing [15] was carried out in a microwave oven. In this way treatment time lasts minutes rather than hours. Finally, sediment and soil samples were analysed according to the fractionation scheme proposed by us, making up the balance of organic carbon. The scheme allows subdivision into different fractions of organic compounds present both in sediments and in soils. In particular hydrophobic and hydrophilic compounds, fulvic and humic acids, and humin are separated [24–30]. 2. Experimental 2.1. Materials Marine sediments were taken from cores sampled in the Ross Sea (Antarctica). Antarctic sediments are characterised by a high silica content due to the presence of siliceous diatoms and silicoflagellates, and a low organic carbon content, prevalently associated with minerals. In particular, the superficial layer of a core characterised by non-transformed biogenic silica and soluble in basic medium (sediment 1) was used, and two lower layers of other cores in which siliceous minerals prevail (sediments 2 and 3). Sediment 1 (74◦ 00 S, 174◦ 48 E), located in the centre of the northern part of the Joides Basin at a depth of 582 m, represents a setting characterised by high biosiliceous sediment accumulation. Sediment 2 (75◦ 54 S, 177◦ 34 E), was collected
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at a depth of 628 m in an area near the continental shelf break in a central Ross Sea basin and sediment 3 (75◦ 04 S, 164◦ 138 E), located in the western Ross Sea at a depth of 950 m, was collected in an area representative of an environment with high energy and reworked sediments. A soil sample collected in an agricultural area located near Rome was also analysed. 2.2. Methodology for sediment and soil characterisation A Labconco 77400 lyophiliser (Labconco, Kansas City, MO) was used for sample lyophilisation. Grain size composition was obtained using common sieving methods (Table 1). Total organic carbon (TOC) content was determined with a Carlo Erba EA11110 CHNS-O Element Analyser (Carlo Erba, Milan, Italy). A 100 mg aliquot of dried sediment was acidified with 500 l of concentrated HCl (Merck, Germany) in order to eliminate the carbonate fraction, then freeze-dried and re-weighed. The freeze-dried residue was then analysed for TOC. The measurements were repeated six times and the relative standard deviations were less than 5%. Carbonate content was determined as difference between the total carbon content of sample, determined with elemental analyser, and TOC. 2.3. Methodology for humin deashing Humin deashing optimisation was carried out using 8.0 g aliquots of lyophilised sediment 3 (Table 1) after extraction of soluble organic fractions (hydrophobic, hydrophilic, humic and fulvic fractions). The total organic carbon remaining in the insoluble residue was 2.0 ± 0.12 mg g−1 . The decision to optimise the humin deashing procedure with sediment 3 was due to the low organic carbon and high silicates content. The insoluble residue was treated with 50 ml of a 1 N HCl and 10% HF (v/v) solution in a microwave oven (10 min at 30 W power, 12 min at 80 W). The maximum temperature reached in the vessels was 70 ◦ C. In order to test this procedure, the humin deashing methodology based on HF/HCl solution maintained at room temperature for 24 h [15], was also performed. In order to eliminate the minerals associated with the humin fraction as far as possible, a further step based on densimetric separation was performed [31]. The residue was thus treated: (1) with ZnCl2 solution in order to separate humin (lighter fraction) from residual minerals. The ZnCl2 treatment (10 ml Table 1 Grain size composition of sediments and soil samples % of
Sediment 1
Sediment 2
Sediment 3
Soil
Coarse sand Fine sand Silt Clay Carbonate Total organic carbon Total nitrogen
0 32.6 0 67.4 0 0.89 0.13
3.5 50.5 0 46.5 0 0.54 <0.1
10.1 36.7 0 52.3 0 0.36 <0.1
18.0 34.4 25.0 22.6 n.d. 4.9 0.48
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solution) was repeated as long as the light fraction was present. The organic fraction was precipitated by diluting the ZnCl2 solution with distilled water; (2) with ZnCl2 solution. The organic fraction precipitated by diluting the ZnCl2 solution with distilled water was then separated and treated three times with HCl 6 M (each time 10 ml); (3) three times with HCl 6 M (each time 10 ml), then the residue was treated with ZnCl2 (d = 2 g cm−3 ) solution. The humin (Hm) obtained as precipitate from (1) to (3) was dried under vacuum at 100 ◦ C and then analysed for organic carbon content. The water used for rinsing and dilution was organic-free UVoxidised water dispensed by a Milli-Q Gradient A10 instrument from (Millipore, Bedford, MA). Glassware was cleaned by soaking in a 10% HNO3 bath for 24 h, rinsing thoroughly with water and muffling at 350 ◦ C for 3 h. All reagents were analytical grade. 2.4. Fractionation of organic carbon Fractionation of organic carbon was carried out according to the following procedure:
were separated, suspended in deionised water and purified by dialysis against deionised water using tubular membranes (Spectra/Por® , Spectrum Laboratories Inc., Rancho Dominguez, CA) with a molecular weight size of 1000 Da. They were then freeze-dried and analysed for organic carbon content by elemental analysis. Fulvic acids remaining in solution after acidification at pH 1 were purified using an Amicon stirred ultrafiltration cell, model 8400 (Millipore, Milan, Italy) fitted at the bottom with a 500 Da membrane disc. (4) To obtain the humin (Hm) fraction the residual sediment was treated twice with 50 ml of a 1 N HCl and 10% HF (v/v) solution in a microwave oven (10 min at 30 W power, 12 min at 80 W). The residue was then treated with ZnCl2 solution and humin (light fraction d < 2 g cm−3 ) was separated from mineral residue (heavy fraction d > 2 g cm−3 ). The Hm fraction was then treated three times with 6 M HCl (each time 10 ml at room temperature), rinsed with distilled water and dried under vacuum at 100 ◦ C. The organic carbon content of the humin fraction was determined by elemental analysis. 3. Results and discussion 3.1. Humin deashing
(1) To obtain the hydrophobic (Hb) fraction, lyophilised sediment (ca. 10 g) was extracted for 24 h at room temperature with a CH2 Cl2 /CH3 OH (2:1, v/v) mixture in a 1:5 (w/v) solid/liquid ratio. Two consecutive extractions of 24 h were carried out in a continuous rotating agitator. After centrifugation for 30 min at 4000 rpm, the supernatants were dried at 60 ◦ C in a crucible under vacuum in a desiccator, previously brought to constant weight in a desiccator at 100 ◦ C, and then weighed. The organic carbon content of the Hb fraction was determined by elemental analysis. (2) To obtain the hydrophilic (Hp) fraction, the residual sediment was treated with 90 ml of a 0.5 N HCl solution for 24 h in a continuous rotating agitator and, after centrifugation for 30 min at 2000 rpm, the acid solution was made up to a volume of 100 ml with distilled water. Aliquots of 20 ml were dried under vacuum in a desiccator at 60 ◦ C in a pre-weighed crucible. (3) To obtain humic materials the residual sediment was treated with 90 ml of a 0.5 N NaOH solution for 24 h in a continuous rotating agitator. After centrifugation for 30 min at 2000 rpm, the alkaline supernatant, containing humic and fulvic acids, was acidified at pH 1 by dropwise addition of concentrated HCl in order to separate acid-soluble fulvic acids (FA) from precipitating humic acids (HA). Both acid (step 2) and alkaline extractions (step 3) were alternatively repeated on residual sediment until the alkaline solution was colourless. The organic carbon content of the Hp fraction was determined by elemental analysis and only the first 20 ml aliquot yielded a detectable organic carbon content. In the case of humic substance extraction all the alkaline solutions were mixed. Humic acids, precipitated after acidification to pH 1 of the alkaline solution,
The results for the solid/liquid ratio needed for humin deashing are presented in Fig. 1. Results highlighted that the liquid amount used for humin deashing did not influence the yield of the treatment. For this reason we chose to use a solid/liquid ratio of 1:5 (w/v). The time and power values for microwave oven operation were selected so as to reach the maximum temperature of 70 ◦ C in the vessels. This temperature was chosen to take into account that the addition of the HF/HCl solution to sediment would increase the temperature in the mixture up to 70 ◦ C due to the exothermic reaction of silicate dissolution and so that the temperature value would not exceed 70 ◦ C in order to prevent the possible oxidation of organic compounds [32]. Fig. 2 shows the deashing results obtained from the microwave oven treatment. After two treatments with 50 ml of a 1 N HCl and 10% HF (v/v) solution in a microwave oven,
Fig. 1. Residue percentage vs. the liquid amount used for humin deashing.
N. Calace et al. / Talanta 71 (2007) 1444–1448
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Fig. 4. The residue percentage and the carbon percentage after densimetric separation according to treatment 1 (ZnCl2 ), treatment 2 (HCl before ZnCl2 ) and treatment 3 (HCl after ZnCl2 ). Fig. 2. The residue percentage and the carbon percentage as function of the number of treatments with HF/HCl solution in microwave oven.
Fig. 3. The residue percentage and the carbon percentage as function of the number of treatments with HF/HCl solution at room temperature.
the residue and the carbon percentage (9% and 2.2%, respectively) of the insoluble residue did not change. The calculated amount of organic carbon of the humin fraction was about 1.5 ± 0.064 mg g−1 , which accounts for 75% of the total organic carbon content of the sample (2.00 ± 0.12). On the other hand, the residue and carbon percentage of the residue obtained by treatments with HF/HCl solution at room temperature reached a plateaux after six treatments (Fig. 3). The residue and carbon percentage of the residue were 5% and 2.8%, respectively, which correspond to a humin carbon amount of 1.1 ± 0.042 mg g−1 . Results obtained from two procedures do not show any significant differences (95% at the confidence level) in humin deashing yield but comparing the results of the two procedures it is evident that the employment
of the microwave oven merely speeds up the dissolution of the mineral matrix. However, even if the amount of recovered humin carbon is higher, the carbon percentage of the residue is also low (∼2.2%), indicating a strong presence of the mineral fraction. Consequently, to increase the purification level a further step was taken, based on a densimetric separation of organic and inorganic fractions and on treatment with HCl 6 M to remove the CaF2 formed during the demineralisation process. With densimetric separation alone no increase in carbon percentage is observed, while carbon percentage significantly increases when HCl is employed (Fig. 4). The best results are obtained with the procedure (2), performing HCl treatment after densimetric separation. The amount of calculated organic carbon was practically the same (1.4 ± 0.18 mg g−1 ) for the procedures (1)–(3) and was the same as that calculated after microwave deashing treatments. This finding showed that the different treatments take effect only on the mineral fraction and that the recovery percentage of humin organic carbon depends mainly on microwave assisted acid treatment. 3.2. Organic carbon fractionation procedure Results obtained by the organic carbon fractionation (Table 2) on matrices characterised by different organic carbon content showed a loss of carbon ranging between 20% and 30%, this is consistent with previous reports by several researchers for humin deashing [6,8,11,13]. A large part of the carbon loss is due to the humin deashing step. As a result, elemental carbon analysis of all the samples studied were carried out soon after the alkaline extraction and before the humin deashing treatment (Table 3) and results show that the loss of carbon after humic substance extraction is negligible.
Table 2 Total organic carbon and carbon amount in the different fractions TOC (mg g−1 ) Sediment 1 Sediment 2 Sediment 3 Soil
8.9 5.4 3.6 48.9
± ± ± ±
0.3 0.4 0.4 0.2
Hb (mg g−1 ) 0.170 0.080 0.070 0.26
± ± ± ±
0.008 0.005 0.004 0.01
Hp (mg g−1 ) 0.16 0.11 0.11 0.42
± ± ± ±
0.01 0.01 0.01 0.01
HA (mg g−1 ) 1.5 0.75 0.45 8.6
± ± ± ±
0.1 0.06 0.03 0.1
FA (mg g−1 ) 4.0 2.1 0.9 16.7
± ± ± ±
0.4 0.4 0.3 0.5
Hm (mg g−1 ) 0.64 0.71 1.4 12.3
± ± ± ±
0.09 0.09 0.2 0.3
C loss (%) 28 31 19 22
± ± ± ±
10 18 25 2
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Table 3 Total organic carbon, organic carbon before humin deashing and after alkaline treatments, and organic carbon as the sum of the fractions before humin deashing TOC Sediment 1 Sediment 2 Sediment 3 Soil
8.9 5.4 3.6 48.9
TOC − OCafter
OCafter ± ± ± ±
0.3 0.4 0.4 0.2
2.9 2.8 2.0 26.1
± ± ± ±
0.1 0.2 0.1 0.5
6.0 2.6 1.6 22.8
± ± ± ±
0.4 0.6 0.5 0.7
OC Hb + Hp + FA + HA 5.8 3.0 1.5 26
± ± ± ±
0.5 0.5 0.4 1
It should be considered that during the humin deashing procedure, some hydrolysable fragments of organic compounds could be solubilised. Moreover, organic molecules strictly bound to mineral matrix can be solubilised following dissolution of the inorganic matrix, which eliminates their protective support and so may release organic compounds into solution. Some researchers [33,34] have pointed out that the potential for alteration and losses is particularly significant for ‘immature’ organic matter in aquatic environments such as that found in surface sediments, which are usually younger than deep sediments. Indeed the trend in the organic carbon losses was found to be related to the age of sediments (samples 1 and 2 are much younger than sediment 3) although tests were performed on only a few samples. 4. Conclusion The results obtained with the approach proposed for the solubilisation of the inorganic matrix of sediments and soils show that microwave-assisted deashing is a viable alternative to traditional systems. The deashing procedure, based on the use of a microwave energy, makes the dissolution process faster and the treatment time is significantly reduced from hours to minutes. The fractionation scheme proposed could be applied also to samples in which organic carbon content is low. The balance of organic carbon shows that a large part of the carbon loss is due to the humin deashing step. Carbon loss, does not differ from the values reported by certain authors, cannot be ascribed to the use of the microwave oven and can probably be ascribed to hydrolysis processes related to the HF/HCl mixture. Acknowledgement This work was funded by the Italian National Research Programme for Antarctica (PNRA), Sector 9: Chimica degli ambienti polari. Research Project 2004/9.1. References [1] R.G. Keil, F.S. Hu, E.C. Tsamakis, J.I. Hedges, Nature 369 (1994) 639. [2] S.G. Wakeham, C. Lee, J.I. Hedges, P.J. Hernes, M.L. Peterson, Geochim. Cosmochim. Acta 61 (1997) 5363.
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