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The separation of suspended particulate material into organic, mineral and organo-mineral fractions
PII: S0043-1354(97)00347-3
Wat. Res. Vol. 32, No. 5, pp. 1725±1731, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/98 $19.00 + 0.00
TECHNICAL NOTE THE SEPARATION OF SUSPENDED PARTICULATE MATERIAL INTO ORGANIC, MINERAL AND ORGANOMINERAL FRACTIONS J. J. C. DAWSON* and M. F. BILLETT Department of Plant and Soil Science, Cruickshank Building, University of Aberdeen, Aberdeen, AB24 3UU, U.K. (First received August 1996; accepted in revised form August 1997) AbstractÐA method for the separation of particulates in river waters into three chemically distinct fractions has been optimised and evaluated. The method involves centrifugation using heavy liquid density gradients with exclusion densities of 1.61 and 1.80 g/cm3. An arti®cial mixture of organic and mineral materials and a sample of river water particulates, have been fractionated using this technique. Compared to other fractionation techniques, the method involves little sample contamination, achieves separation of mineral free organic matter and is relatively cheap. Suggestions for improvements to the technique, further applications and possible uses are discussed. # 1998 Elsevier Science Ltd. All rights reserved Key wordsÐparticulates, fractionation, density-centrifugation, organic-mineral interactions
INTRODUCTION
Although the transport of elements in a particulate form down a river system is an important transfer mechanism for many elements, the chemistry of the particulates involved is poorly understood because of sampling and analytical problems. Studies have shown that elements such as C, P, Fe and Al, transported in particulate form, can constitute >25% of their total riverine ¯ux (Likens et al., 1977). Particulate transport also depends upon stream conditions, catchment type and land-use (Hope et al., 1994). Organic particulate material in rivers is derived from a number of sources which include plant litter, microbial biomass and its by-products and precipitated dissolved organic material (Sollins et al., 1985). Inorganic particulates originate from weathering and the dissolution of minerals. The fate of the individual fractions diers; organic matter is more likely to be recycled by biota, whereas inorganic material will be stored in sediments. Most studies of element transfer on particulates are based on total loads, concentrating on particulate organic matter and other total elemental ¯uxes such as C, N, P and trace elements. Relatively little is known about whether elements are transported either on organic or inorganic particulates. This in*Author to whom all correspondence should be addressed. E-mail [email protected].
formation is important, because it not only provides information on the provenance of the various chemically distinct forms of particulates, but also on their relative importance in the transport of speci®c elements. In addition, the presence of a speci®c element in the organic or inorganic fraction will determine the fate of that element in the aquatic environment. Various methods have been used to separate organic material in soil which could potentially be applied to the aquatic environment (Table 1). However, they have a number of problems: namely they are (i) laborious; (ii) use hazardous chemicals and (iii) cause chemical contamination of particulate fractions. The method described in this paper, which is based on density-centrifugation, is potentially the least damaging to particulates, least time consuming, cost-eective and causes minimal contamination. This enables subsequent total element analysis of the particulate fractions. The main problem with organic matter fractionation involves the incomplete separation of organic from mineral material. This is particularly dicult because of thin layer coatings of strongly adsorbed organic matter on mineral particles (Sollins et al., 1985; Arshad and Lowe, 1966). To dissociate this particular type of organic matter without eecting the chemical associations involved is not feasible. Furthermore, as these complexes are strongly integrated, separation in a natural system is unlikely.
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Technical Note Table 1. Soil fractionation methods used for the separation of organic matter Soil fractionation method
Sequential extraction Loss-on-ignition Wet acid oxidation using K2Cr2O4 Extraction at high pressures and temperatures Chemical fractionation with Na4P2O7 and NaOH (also HF-HCl). Low temperature ashing Density-centrifugation
A method has therefore been developed and optimised for the separation of particulates by densitycentrifugation into three fractions; organic, organomineral and mineral. METHODS
The density-centrifugation procedure uses sodium iodide (NaI) to separate standard mixtures and a river particulate sample into distinct fractions. The following section includes a description of (i) particulate materials used, (ii) the density-centrifugation method, (iii) optimisation of the method and (iv) evaluation of the method. Materials Materials naturally found in particulates were used as standards in a series of experiments to optimise and evaluate the technique. Plant litter was chosen as a source of organic matter and K feldspar, biotite and muscovite as inorganic materials. All standard materials were ovendried at 508C, ground to <100 mm and stored in a desiccator. Particles of size <100 mm are small enough to prevent sizeable aggregates forming and large enough to prevent adherence to the sides of tubes (Cotter-Howells, 1993; Henley, 1977). A near-bank, silt-rich water sample was collected after arti®cial resuspension, from the River Dee in NE Scotland. It was centrifuged (4000 rpm., 15 min) and the supernatant decanted through a 0.8 mm Millipore isopore polycarbonate membrane ®lter to retain any particulates that had not collected in the residue. The residue and any coarse material (>2 mm) were oven-dried at 508C, ground to pass a 100 mm sieve and stored in a desiccator.
Fig. 1. Diagram of a Hutton Tube used in the density-centrifugation method. A density gradient of 1.00±1.61 g/cm3 exists between the top of the Hutton Tube and the central constriction.
Reference Schnitzer and Schuppli, 1989; Tessier et al., 1979 Woodroe, 1985; Keeling, 1962; Ball, 1964 Shaw, 1959 Schnitzer et al., 1991 Arshad and Lowe, 1966 De Kimpe and Schnitzer, 1990; Gleit and Holland, 1962 Cambardella and Elliott, 1993; Stepanov, 1981; Turchenek and Oades, 1979; Spycher and Young, 1977
Between 5±10 mg of the standard materials, the particulate sample and each separated fraction were analysed for C and N by a Carlo Erba Element NCS analyser (Fisons). Loss-on-ignition was determined for the plant litter and the particulate sample to give an estimate of % organic matter (Ball, 1964). Density-centrifugation method Separation of low-density organic (O) fraction. Ten ml of NaI solution (density = 1.61 g/cm3) were added to a Hutton Tube (Cotter-Howells, 1993; Henley, 1977) containing 0.30 g of sample material (Fig. 1). The upper section of the tube contains a density gradient up to the exclusion density and the lower section contains the required exclusion density. Separation is enhanced by the gradient enabling particulate samples to disperse over the density range above the exclusion density. This prevents a compact layer or mat of low density material forming on top of the heavy liquid which can trap and prevent the heavier mineral material from sinking, hence causing mineral contamination of the organic matter fraction (CotterHowells, 1993). Stirring and ultrasound (u/s) were then applied to the tube (30 min) to aid de-¯occulation (Spycher et al., 1983; Stepanov, 1981). Two ml of 0.1 M NaOH was added to prevent ¯occulation re-occurring and also to help produce the density gradient. The sample was subsequently centrifuged at 2500 rpm for 30 min. (Cotter-Howells, 1993). After centrifugation, the supernatant in the upper section of the tube containing the separated low density material was decanted and ®ltered through a 0.7 mm, Whatman glass micro-®bre ®lter (GF/F). The plunger prevents any high density material from the bottom of the tube contaminating the low density material in the upper compartment. The low density material (O fraction) was washed with 250 ml of 1.0 M NaCl and de-ionized water to remove NaI (Sollins et al., 1985). After the ®rst separation stage, the majority of each sample was washed immediately from the ®lter. The O fraction was freeze-dried and stored in a desiccator prior to determination of C and N. The density-centrifugation fractionation procedure was repeated a further 2 times to maximise recovery of the O fraction. Separation of organo-mineral (OM) fraction from the mineral (M) fraction. Once the O fraction had been decanted, the remaining NaI in the lower compartment of the Hutton tube was passed through a 0.8 mm Millipore isopore polycarbonate membrane ®lter. This enabled the density of the heavy liquid to be changed and avoided sample loss. The material remaining on the ®lter was washed back into the Hutton tube with a higher density (1.80 g/cm3) NaI solution. A glass-®bre ®lter was not used as the micro-®bres permanently retain some of the sample which is therefore lost from subsequent fractionation. The O fraction separation procedure was repeated in its entirety with the higher density NaI. The lower density material in the upper compartment of the tube was termed the organo-mineral (OM) fraction and the residue at the bottom of the tube was called the mineral (M) fraction. The OM and M fractions were washed separately with 250 ml
Technical Note
1727
of NaCl and de-ionized water, freeze-dried and stored in a desiccator prior to determination of C and N. Optimisation of the method Density-centrifugation fractionation under various conditions was carried out using standard plant litter and feldspar mixtures in a 1:1 (by weight) ratio to determine the optimum fractionation conditions. The %C and N content of the separated low density O fraction was measured and compared with that of untreated plant litter and plant litter following density-centrifugation, to determine the best conditions for fractionation (least contamination). The ideal number of separation stages was determined using the optimized conditions for O-fraction separation. Four replicates with ®ve separation stages were carried out for three dierent, 1:1 ratio organic/mineral mixtures (plant litter:feldspar; plant litter:biotite and plant litter:muscovite). The weight of the low-density O fractions was measured after each separation stage to give the ®nal weight of the O fraction separated and hence, the percentage recovery of the plant litter standard. Evaluation of the method Once the method had been optimized, it was used to separate a 1:1 mixture of plant litter:feldspar and the River Dee particulate sample. Each separated fraction was weighed and its %C and N content determined. These values were used to calculate (i) the percentage weight of each fraction as a total of the original mixture/sample weight; (ii) the % of total C in each fraction and (iii) the % of total N in each fraction.
RESULTS AND DISCUSSION
Analysis of materials The C and N content of the materials used is listed in Table 2. The plant litter standard contained 93.64% organic material and the standard minerals very low amounts of C and N. The River Dee particulate sample contained 3.30 %C and 0.18 %N. Optimisation of fractionation conditions and number of separation stages The results of the eight optimisation experiments which are shown in Fig. 2, indicate that the purest fractionated organic matter was produced by the following conditions: 1.61 g/cm3 NaI density, 100 mm sieved sample, 30 min u/s, a 0.1 M NaOH gradient and 30 min centrifugation at 2500 rpm. The conditions produced C and N values in the O fraction, obtained from a 1:1 plant litter:feldspar mixture, of 49.00 2 1.70 %C and 1.20 2 0.03 %N; these were similar to values measured in the O frac-
Fig. 2. A comparison of mean %C (a) and %N (b) in the separated low density O fractions following dierent fractionation conditions. These are: 1. untreated plant litter standard (n = 16); 2. plant litter standard only following density-centrifugation technique (n = 8). Experiments using a 1:1 mixture of plant litter and feldspar are: 3. 200 mm sample; 10 min. u/s; 0.1 M NaOH gradient and d = 1.61 g/cm3, (n = 8). 4. 100 mm sample; 30 min. u/s; 0.1 M NaOH gradient and d = 1.63 g/cm3, (n = 8). 5. 100 mm sample; 30 min. u/s; 0.1 M NaOH gradient and d = < 1.60 g/cm3. 6. 100 mm sample; no u/s; 0.2 M NaOH injected with sample beneath NaI surface and d = 1.63 g/cm3, (n = 4). 7. 100 mm sample; no u/s; 0.2 M NaOH injected with sample beneath NaI surface and d = 1.61 g/cm3, (n = 4). 8. 100 mm sample; 30 min. u/s; 0.1 M NaOH gradient and d = 1.61 g/cm3, (n = 4). All mean values 295% c.i. P < 0.05.
tion of fractionated pure plant litter standards (49.50 20.3%C and 1.10 2 0.05 %N). Similar C and N concentrations (50.49 20.99 %C and 1.26 2 0.07 %N) were also observed with these conditions in O fractions separated from 1:1 mixtures
Table 2. Mean %C, N and organic matter content of untreated standard materials and the River Dee particulate sample
Plant litter Feldspar Biotite Muscovite R. Dee
Mean % carbon
Mean % nitrogen
Mean % organic matter
48.09 20.48 (n = 16) 0.05 2 0.01 (n = 16) 1.06 2 0.02 (n = 16) 0.34 2 0.01 (n = 16) 3.30 20.13 (n = 8)
1.202 0.04 (n = 16) 0.002 0.00 (n = 16) 0.012 0.00 (n = 16) 0.012 0.00 (n = 16) 0.182 0.01 (n = 8)
93.64 2 0.14 (n = 4) n.d. n.d. n.d. 7.382 0.06 (n = 4)
Mean values 295% c.i. (con®dence intervals) P < 0.05. n.d. = not determined.
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Technical Note
have as strong an interaction or as high an organic content as in vivo samples. This suggests that the recovery of free organic matter (material <1.61 g/ cm3) will be higher using standards compared to ``natural'' samples. Evaluation of the method The fractionation of a standard mixture of plant litter with feldspar (Fig. 4) produced a dierent result from that of the particulate sample (Fig. 5), both in terms of the weight of each fraction recovered and the %C and N content of each fraction. Plant litter:feldspar mixture. The weight of the O (<1.61 g/cm3) and OM (1.61±1.80 g/cm3) fractions recovered were 35.14% and 10.70% of the total sample weight, respectively. The O fraction contained more than 70% of the total C and N content of the sample; 80±90% of the total C and N is accounted for in both the O and OM fractions of the mixtures. The high density M fraction (>1.80 g/cm3) accounted for 41.73% of the total sample weight but was mainly mineral material, as can be deduced from the low amounts of C (3.33%) and N (4.48%) present.
Fig. 3. The % of plant litter recovered at each density-centrifugation separation stage for 1:1 mixtures of plant litter:feldspar; plant litter:biotite and plant litter:muscovite. (n = 4), mean values 295% c.i. P < 0.05.
of plant litter with biotite or muscovite (data not shown). Figure 3 shows the mean % recovery of the lowdensity O fraction calculated as a percentage of the original plant litter and added as part of three dierent standard 1:1 mixtures. The optimum number of separation stages required to separate the majority of free organic matter was three. Most of the free organic matter was recovered during the ®rst separation stage, which yielded a mean recovery between the three mixtures of 56.90 22.73% of the organic matter. The remaining four separations only recovered a further 11.70% organic matter. Reducing the number of separation stages from ®ve decreased the time for fractionation by 40% whilst not signi®cantly aecting the % recovery. Some plant litter in the standard mixtures will combine with the minerals to form organo-mineral complexes. Thus, a 100% recovery of the plant litter would not be expected at a density of 1.61 g/cm3. The similarity in the results between the three plant litter and mineral mixtures suggests a similar intensity of interaction between plant litter and each mineral. However, 1:1 standard mixtures will not
Fig. 4. The % dry weight and C and N content of the O, OM and M fractions separated from a 1:1 mixture of plant litter and feldspar. All values are expressed as a % of the total dry weight and total C and N content of the untreated sample.
Technical Note
Particulate sample. These results were distinctly dierent from the standard mixture. The O and OM fractions contained 41.98% and 33.88% of the total C and N of the sample respectively, although these two fractions constituted only ca. 5% of the total sample weight. The heaviest fraction was the M fraction containing 91.89% of the total sample and 44.49% of the total C. However, this result is somewhat misleading because the M fraction was composed of largely mineral material, with C constituting only 1.56% of the M fraction.
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Comparison of results. The O and OM fractions, calculated as a % of the total weight, were larger in the standard mixture, than the particulate sample. This was expected as the original untreated particulate sample contained only 3.30 %C; the standard mixture contained 24.00 %C. Bulking may therefore, be necessary for samples containing low concentrations of organic matter in order to produce enough sample in the O and OM fractions for subsequent analysis. The small concentration of C in relation to the high concentration of mineral ma-
Fig. 5. The % dry weight and C and N content of the O, OM and M fractions separated from an untreated River Dee particulate-rich sample. All values are expressed as a % of the total dry weight and total C and N content of the untreated sample.
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Technical Note
terial measured in the particulate sample, implies that a large proportion of the organic matter is adsorbed onto mineral particles. In a 1:1 mixture of standard materials, this will not be the case as much of the organic material will be free and easily separated at 1.61 g/cm3. In either sample, the organic matter still has to be relatively free of mineral material for it to enter the O or OM fractions (<1.80 g/cm3). The % of total C present in the particulate M fraction (44.5%) showed that, where strong interactions occur between organic and mineral particles, a density >1.80 g/cm3 will be required to produce a pure mineral fraction. This result also shows that organo-mineral material is a larger proportion of the total sample than the data ®rst suggests. The M fraction of the standard mixture consisted of relatively pure mineral material with 3.33% of the total C present. Again, this was due to the initial large concentrations of organic matter present in a free organic form (O fraction) or associated with mineral particles (OM fraction). Improvements to the method Problems may occur if NaI were to be used to separate fractions which were then analysed for + 2+ and exchangeable ions, e.g. PO3ÿ 4 , K , Ca 2+ Mg , as the ionic strength and pH of the NaI solution could cause desorption of ions. A decrease in %N of the O fraction from fractionated plant litter compared to untreated plant litter (Fig. 2b), suggests that some N loss occurred during fractionation; this was not observed for C. A reason for the N loss could be desorption as it solubilizes more easily than C and is lost more readily into solution. Although the loss is ca. 10% of the total N, greater losses would occur with more exchangeable ions. The desorption of N could also be the reason for a greater proportion of N loss, compared to C, in the unrecovered fraction. A possible problem may also occur with the use of NaOH. This can change the structure of the humic and fulvic components of the O fraction by hydrolysis of esters. However, our results suggest that the solubility of C has not beeen eected by possible structural changes. Furthermore, not all of the organic matter was removed at a density of <1.80 g/cm3. This was particularly noticeable for the particulate sample (Fig. 5), where adherence of organic material to minerals was far stronger. Cambardella and Elliott (1993) have shown that 18% of soil C and 25% of soil N is associated with ®ne silt particles with a density between 2.07 and 2.21 g/cm3. A greater density is required to separate a particulate fraction with little or no organic matter present. It would also be possible to separate two OM fractions using this technique; (i) a 1.61±1.80 g/cm3 fraction which is mainly organic matter and (ii) a 1.80±2.25 g/cm3 fraction which is mainly mineral material. These two OM fractions are probably as chemically dis-
tinct from each other as they are from the O and M fractions. One of the diculties in using NaI is that densities >1.80 g/cm3 cannot easily be achieved. Alternatives to NaI include heavy organic liquids, e.g. tetrabromoethane or sodium metatungstate. The advantages of sodium metatungstate are that it is non-toxic; extremely soluble in water; does not exhibit equilibria with other ionic species in solution; produces a neutral solution with water although it is stable in the pH range 2±10; has low viscosity over a wide density range; is comparatively cheap and reusable. Sodium metatungstate can also achieve densities up to 3.12 g/cm3 at 258C and is clearly an ideal heavy liquid for future work in this area (Plewinsky and Kamps, 1984). Applications of the method The technique described here, possibly further improved using sodium metatungstate, could be applied to both water particulate and soil analysis. It could also be used in the analysis of sediments in order to identify the fate of particular elements and compounds such as P, pesticides and heavy metals in the aquatic environment. The high speci®c surface area of colloids and particulates make them ecient adsorbents for metals, trace elements and pollutants (Stumm, 1992). The likely fate of these ions and molecules in the environment is largely determined by their association with organic, organo-mineral or mineral particles. CONCLUSIONS
The separation of free organic material has been achieved with negligible contamination by organomineral and mineral matter, using an optimized density-centrifugation method. Three separation stages are required to ensure an optimum recovery of each fraction. An alternative to the use of NaI will be required to (i) separate and analyse speci®c exchangeable cations and anions due to possible desorption from particulate surfaces caused by ionic NaI and (ii) increase the fractionation density (possibly up to 3.0 g/cm3) to remove the last remnants of organic material still present in the ``mineral'' fraction.The technique is recommended for the fractionation of water particulates, soils and sediment samples and their subsequent element analysis. REFERENCES
Arshad M. A. and Lowe L. E. (1966) Fractionation and characterisation of naturally occurring organo-clay complexes. Soil Sci. Soc. Am. Proc. 30, 731±735. Ball D. F. (1964) Loss-on-ignition as an estimate of organic matter and organic carbon in non-calcareous soils. J. Soil Sci. 15, 84±92. Cambardella C. A. and Elliott E. T. (1993) Methods for physical separation and characterisation of soil organic matter fractions. Geoderma 56, 449±457.
Technical Note Cotter-Howells J. (1993) Separation of high density minerals from soil. Sci. Total Environ. 132, 93±98. De Kimpe C. R. and Schnitzer M. (1990) Low-temperature ashing of humic and fulvic acid. Soil Sci. Soc. Am. J. 54, 399±403. Gleit C. E. and Holland W. D. (1962) Use of electrically excited oxygen for the low temperature decomposition of organic substances. Anal. Chem. 34, 1454±1457. Henley K. J. (1977) Improved heavy-liquid separation at ®ne particle sizes. Am. Mineral. 62, 377±381. Hope D., Billett M. F. and Cresser M. S. (1994) A review of the export of carbon in river water: ¯uxes and processes. Environ. Pollut. 84, 301±324. Keeling P. S. (1962) Some experiments on the low temperature removal of carbonaceous material from clays. Clay Min. Bull. 28, 155±158. Likens G. E., Bormann F. H., Pierce R. S., Eaton J. S. and Johnson N. M. (1977) Biogeochemistry of a Forested Ecosystem. Springer-Verlag, New York, U.S.A. Plewinsky B. and Kamps R. (1984) Sodium metatungstate, a new medium for binary and ternary density gradient centrifugation. Makromol. Chem. 185, 1429±1439. Schnitzer M., Schulten H. R., Schuppli P. and Angers D. A. (1991) Organic matter extraction from soils with high pressure and temperatures. Soil Sci. Soc. Am. J. 55, 102±108. Schnitzer M. and Schuppli P. (1989) Method for the sequential extraction of organic matter from soils and soil fractions. Soil Sci. Soc. Am. J. 53, 1418±1424.
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Shaw K. (1959) Determination of organic carbon in soil and plant material. J. Soil Sci. 10, 316±326. Sollins P., Glassman C. A. and Dahm C. N. (1985) Composition and possible origin of detrital material in streams. Ecology 66, 297±299. Spycher G., Sollins P. and Rose S. (1983) Carbon and nitrogen in the light fraction of a forest soil: vertical distribution and seasonal patterns. Soil Sci. 135, 79±87. Spycher G. and Young J. L. (1977) Density fractionation of water-dispersible soil organic-mineral particles. Comm. Soil Plant Anal. 8, 37±48. Stepanov I. S. (1981) Physical methods for extracting fractions of organo-mineral substances from soils. Sov. Soil Sci. 13, 106±119. Stumm W. (1992). Chemistry of the Solid-Water Interface. Processes at the Mineral-Water and Particle-Water Interface in Natural Systems. John Wiley, New York, U.S.A. Tessier A., Campbell P. G. C. and Bisson M. (1979) Sequential extraction procedure for the speciation of particulate trace metals. Anal. Chem. 51, 844±851. Turchenek L. W. and Oades J. M. (1979) Fractionation of organo-mineral complexes by sedimentation and density techniques. Geoderma 21, 311±343. Woodroe C. D. (1985) Studies of a mangrove basin, Tu Crater, New Zeland: III. The ¯ux of organic and inorganic particulate matter. Estuar., Coast. Shelf Sci. 20, 447±461.
Wat. Res. Vol. 32, No. 5, pp. 1725±1731, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/98 $19.00 + 0.00
TECHNICAL NOTE THE SEPARATION OF SUSPENDED PARTICULATE MATERIAL INTO ORGANIC, MINERAL AND ORGANOMINERAL FRACTIONS J. J. C. DAWSON* and M. F. BILLETT Department of Plant and Soil Science, Cruickshank Building, University of Aberdeen, Aberdeen, AB24 3UU, U.K. (First received August 1996; accepted in revised form August 1997) AbstractÐA method for the separation of particulates in river waters into three chemically distinct fractions has been optimised and evaluated. The method involves centrifugation using heavy liquid density gradients with exclusion densities of 1.61 and 1.80 g/cm3. An arti®cial mixture of organic and mineral materials and a sample of river water particulates, have been fractionated using this technique. Compared to other fractionation techniques, the method involves little sample contamination, achieves separation of mineral free organic matter and is relatively cheap. Suggestions for improvements to the technique, further applications and possible uses are discussed. # 1998 Elsevier Science Ltd. All rights reserved Key wordsÐparticulates, fractionation, density-centrifugation, organic-mineral interactions
INTRODUCTION
Although the transport of elements in a particulate form down a river system is an important transfer mechanism for many elements, the chemistry of the particulates involved is poorly understood because of sampling and analytical problems. Studies have shown that elements such as C, P, Fe and Al, transported in particulate form, can constitute >25% of their total riverine ¯ux (Likens et al., 1977). Particulate transport also depends upon stream conditions, catchment type and land-use (Hope et al., 1994). Organic particulate material in rivers is derived from a number of sources which include plant litter, microbial biomass and its by-products and precipitated dissolved organic material (Sollins et al., 1985). Inorganic particulates originate from weathering and the dissolution of minerals. The fate of the individual fractions diers; organic matter is more likely to be recycled by biota, whereas inorganic material will be stored in sediments. Most studies of element transfer on particulates are based on total loads, concentrating on particulate organic matter and other total elemental ¯uxes such as C, N, P and trace elements. Relatively little is known about whether elements are transported either on organic or inorganic particulates. This in*Author to whom all correspondence should be addressed. E-mail [email protected].
formation is important, because it not only provides information on the provenance of the various chemically distinct forms of particulates, but also on their relative importance in the transport of speci®c elements. In addition, the presence of a speci®c element in the organic or inorganic fraction will determine the fate of that element in the aquatic environment. Various methods have been used to separate organic material in soil which could potentially be applied to the aquatic environment (Table 1). However, they have a number of problems: namely they are (i) laborious; (ii) use hazardous chemicals and (iii) cause chemical contamination of particulate fractions. The method described in this paper, which is based on density-centrifugation, is potentially the least damaging to particulates, least time consuming, cost-eective and causes minimal contamination. This enables subsequent total element analysis of the particulate fractions. The main problem with organic matter fractionation involves the incomplete separation of organic from mineral material. This is particularly dicult because of thin layer coatings of strongly adsorbed organic matter on mineral particles (Sollins et al., 1985; Arshad and Lowe, 1966). To dissociate this particular type of organic matter without eecting the chemical associations involved is not feasible. Furthermore, as these complexes are strongly integrated, separation in a natural system is unlikely.
1725
1726
Technical Note Table 1. Soil fractionation methods used for the separation of organic matter Soil fractionation method
Sequential extraction Loss-on-ignition Wet acid oxidation using K2Cr2O4 Extraction at high pressures and temperatures Chemical fractionation with Na4P2O7 and NaOH (also HF-HCl). Low temperature ashing Density-centrifugation
A method has therefore been developed and optimised for the separation of particulates by densitycentrifugation into three fractions; organic, organomineral and mineral. METHODS
The density-centrifugation procedure uses sodium iodide (NaI) to separate standard mixtures and a river particulate sample into distinct fractions. The following section includes a description of (i) particulate materials used, (ii) the density-centrifugation method, (iii) optimisation of the method and (iv) evaluation of the method. Materials Materials naturally found in particulates were used as standards in a series of experiments to optimise and evaluate the technique. Plant litter was chosen as a source of organic matter and K feldspar, biotite and muscovite as inorganic materials. All standard materials were ovendried at 508C, ground to <100 mm and stored in a desiccator. Particles of size <100 mm are small enough to prevent sizeable aggregates forming and large enough to prevent adherence to the sides of tubes (Cotter-Howells, 1993; Henley, 1977). A near-bank, silt-rich water sample was collected after arti®cial resuspension, from the River Dee in NE Scotland. It was centrifuged (4000 rpm., 15 min) and the supernatant decanted through a 0.8 mm Millipore isopore polycarbonate membrane ®lter to retain any particulates that had not collected in the residue. The residue and any coarse material (>2 mm) were oven-dried at 508C, ground to pass a 100 mm sieve and stored in a desiccator.
Fig. 1. Diagram of a Hutton Tube used in the density-centrifugation method. A density gradient of 1.00±1.61 g/cm3 exists between the top of the Hutton Tube and the central constriction.
Reference Schnitzer and Schuppli, 1989; Tessier et al., 1979 Woodroe, 1985; Keeling, 1962; Ball, 1964 Shaw, 1959 Schnitzer et al., 1991 Arshad and Lowe, 1966 De Kimpe and Schnitzer, 1990; Gleit and Holland, 1962 Cambardella and Elliott, 1993; Stepanov, 1981; Turchenek and Oades, 1979; Spycher and Young, 1977
Between 5±10 mg of the standard materials, the particulate sample and each separated fraction were analysed for C and N by a Carlo Erba Element NCS analyser (Fisons). Loss-on-ignition was determined for the plant litter and the particulate sample to give an estimate of % organic matter (Ball, 1964). Density-centrifugation method Separation of low-density organic (O) fraction. Ten ml of NaI solution (density = 1.61 g/cm3) were added to a Hutton Tube (Cotter-Howells, 1993; Henley, 1977) containing 0.30 g of sample material (Fig. 1). The upper section of the tube contains a density gradient up to the exclusion density and the lower section contains the required exclusion density. Separation is enhanced by the gradient enabling particulate samples to disperse over the density range above the exclusion density. This prevents a compact layer or mat of low density material forming on top of the heavy liquid which can trap and prevent the heavier mineral material from sinking, hence causing mineral contamination of the organic matter fraction (CotterHowells, 1993). Stirring and ultrasound (u/s) were then applied to the tube (30 min) to aid de-¯occulation (Spycher et al., 1983; Stepanov, 1981). Two ml of 0.1 M NaOH was added to prevent ¯occulation re-occurring and also to help produce the density gradient. The sample was subsequently centrifuged at 2500 rpm for 30 min. (Cotter-Howells, 1993). After centrifugation, the supernatant in the upper section of the tube containing the separated low density material was decanted and ®ltered through a 0.7 mm, Whatman glass micro-®bre ®lter (GF/F). The plunger prevents any high density material from the bottom of the tube contaminating the low density material in the upper compartment. The low density material (O fraction) was washed with 250 ml of 1.0 M NaCl and de-ionized water to remove NaI (Sollins et al., 1985). After the ®rst separation stage, the majority of each sample was washed immediately from the ®lter. The O fraction was freeze-dried and stored in a desiccator prior to determination of C and N. The density-centrifugation fractionation procedure was repeated a further 2 times to maximise recovery of the O fraction. Separation of organo-mineral (OM) fraction from the mineral (M) fraction. Once the O fraction had been decanted, the remaining NaI in the lower compartment of the Hutton tube was passed through a 0.8 mm Millipore isopore polycarbonate membrane ®lter. This enabled the density of the heavy liquid to be changed and avoided sample loss. The material remaining on the ®lter was washed back into the Hutton tube with a higher density (1.80 g/cm3) NaI solution. A glass-®bre ®lter was not used as the micro-®bres permanently retain some of the sample which is therefore lost from subsequent fractionation. The O fraction separation procedure was repeated in its entirety with the higher density NaI. The lower density material in the upper compartment of the tube was termed the organo-mineral (OM) fraction and the residue at the bottom of the tube was called the mineral (M) fraction. The OM and M fractions were washed separately with 250 ml
Technical Note
1727
of NaCl and de-ionized water, freeze-dried and stored in a desiccator prior to determination of C and N. Optimisation of the method Density-centrifugation fractionation under various conditions was carried out using standard plant litter and feldspar mixtures in a 1:1 (by weight) ratio to determine the optimum fractionation conditions. The %C and N content of the separated low density O fraction was measured and compared with that of untreated plant litter and plant litter following density-centrifugation, to determine the best conditions for fractionation (least contamination). The ideal number of separation stages was determined using the optimized conditions for O-fraction separation. Four replicates with ®ve separation stages were carried out for three dierent, 1:1 ratio organic/mineral mixtures (plant litter:feldspar; plant litter:biotite and plant litter:muscovite). The weight of the low-density O fractions was measured after each separation stage to give the ®nal weight of the O fraction separated and hence, the percentage recovery of the plant litter standard. Evaluation of the method Once the method had been optimized, it was used to separate a 1:1 mixture of plant litter:feldspar and the River Dee particulate sample. Each separated fraction was weighed and its %C and N content determined. These values were used to calculate (i) the percentage weight of each fraction as a total of the original mixture/sample weight; (ii) the % of total C in each fraction and (iii) the % of total N in each fraction.
RESULTS AND DISCUSSION
Analysis of materials The C and N content of the materials used is listed in Table 2. The plant litter standard contained 93.64% organic material and the standard minerals very low amounts of C and N. The River Dee particulate sample contained 3.30 %C and 0.18 %N. Optimisation of fractionation conditions and number of separation stages The results of the eight optimisation experiments which are shown in Fig. 2, indicate that the purest fractionated organic matter was produced by the following conditions: 1.61 g/cm3 NaI density, 100 mm sieved sample, 30 min u/s, a 0.1 M NaOH gradient and 30 min centrifugation at 2500 rpm. The conditions produced C and N values in the O fraction, obtained from a 1:1 plant litter:feldspar mixture, of 49.00 2 1.70 %C and 1.20 2 0.03 %N; these were similar to values measured in the O frac-
Fig. 2. A comparison of mean %C (a) and %N (b) in the separated low density O fractions following dierent fractionation conditions. These are: 1. untreated plant litter standard (n = 16); 2. plant litter standard only following density-centrifugation technique (n = 8). Experiments using a 1:1 mixture of plant litter and feldspar are: 3. 200 mm sample; 10 min. u/s; 0.1 M NaOH gradient and d = 1.61 g/cm3, (n = 8). 4. 100 mm sample; 30 min. u/s; 0.1 M NaOH gradient and d = 1.63 g/cm3, (n = 8). 5. 100 mm sample; 30 min. u/s; 0.1 M NaOH gradient and d = < 1.60 g/cm3. 6. 100 mm sample; no u/s; 0.2 M NaOH injected with sample beneath NaI surface and d = 1.63 g/cm3, (n = 4). 7. 100 mm sample; no u/s; 0.2 M NaOH injected with sample beneath NaI surface and d = 1.61 g/cm3, (n = 4). 8. 100 mm sample; 30 min. u/s; 0.1 M NaOH gradient and d = 1.61 g/cm3, (n = 4). All mean values 295% c.i. P < 0.05.
tion of fractionated pure plant litter standards (49.50 20.3%C and 1.10 2 0.05 %N). Similar C and N concentrations (50.49 20.99 %C and 1.26 2 0.07 %N) were also observed with these conditions in O fractions separated from 1:1 mixtures
Table 2. Mean %C, N and organic matter content of untreated standard materials and the River Dee particulate sample
Plant litter Feldspar Biotite Muscovite R. Dee
Mean % carbon
Mean % nitrogen
Mean % organic matter
48.09 20.48 (n = 16) 0.05 2 0.01 (n = 16) 1.06 2 0.02 (n = 16) 0.34 2 0.01 (n = 16) 3.30 20.13 (n = 8)
1.202 0.04 (n = 16) 0.002 0.00 (n = 16) 0.012 0.00 (n = 16) 0.012 0.00 (n = 16) 0.182 0.01 (n = 8)
93.64 2 0.14 (n = 4) n.d. n.d. n.d. 7.382 0.06 (n = 4)
Mean values 295% c.i. (con®dence intervals) P < 0.05. n.d. = not determined.
1728
Technical Note
have as strong an interaction or as high an organic content as in vivo samples. This suggests that the recovery of free organic matter (material <1.61 g/ cm3) will be higher using standards compared to ``natural'' samples. Evaluation of the method The fractionation of a standard mixture of plant litter with feldspar (Fig. 4) produced a dierent result from that of the particulate sample (Fig. 5), both in terms of the weight of each fraction recovered and the %C and N content of each fraction. Plant litter:feldspar mixture. The weight of the O (<1.61 g/cm3) and OM (1.61±1.80 g/cm3) fractions recovered were 35.14% and 10.70% of the total sample weight, respectively. The O fraction contained more than 70% of the total C and N content of the sample; 80±90% of the total C and N is accounted for in both the O and OM fractions of the mixtures. The high density M fraction (>1.80 g/cm3) accounted for 41.73% of the total sample weight but was mainly mineral material, as can be deduced from the low amounts of C (3.33%) and N (4.48%) present.
Fig. 3. The % of plant litter recovered at each density-centrifugation separation stage for 1:1 mixtures of plant litter:feldspar; plant litter:biotite and plant litter:muscovite. (n = 4), mean values 295% c.i. P < 0.05.
of plant litter with biotite or muscovite (data not shown). Figure 3 shows the mean % recovery of the lowdensity O fraction calculated as a percentage of the original plant litter and added as part of three dierent standard 1:1 mixtures. The optimum number of separation stages required to separate the majority of free organic matter was three. Most of the free organic matter was recovered during the ®rst separation stage, which yielded a mean recovery between the three mixtures of 56.90 22.73% of the organic matter. The remaining four separations only recovered a further 11.70% organic matter. Reducing the number of separation stages from ®ve decreased the time for fractionation by 40% whilst not signi®cantly aecting the % recovery. Some plant litter in the standard mixtures will combine with the minerals to form organo-mineral complexes. Thus, a 100% recovery of the plant litter would not be expected at a density of 1.61 g/cm3. The similarity in the results between the three plant litter and mineral mixtures suggests a similar intensity of interaction between plant litter and each mineral. However, 1:1 standard mixtures will not
Fig. 4. The % dry weight and C and N content of the O, OM and M fractions separated from a 1:1 mixture of plant litter and feldspar. All values are expressed as a % of the total dry weight and total C and N content of the untreated sample.
Technical Note
Particulate sample. These results were distinctly dierent from the standard mixture. The O and OM fractions contained 41.98% and 33.88% of the total C and N of the sample respectively, although these two fractions constituted only ca. 5% of the total sample weight. The heaviest fraction was the M fraction containing 91.89% of the total sample and 44.49% of the total C. However, this result is somewhat misleading because the M fraction was composed of largely mineral material, with C constituting only 1.56% of the M fraction.
1729
Comparison of results. The O and OM fractions, calculated as a % of the total weight, were larger in the standard mixture, than the particulate sample. This was expected as the original untreated particulate sample contained only 3.30 %C; the standard mixture contained 24.00 %C. Bulking may therefore, be necessary for samples containing low concentrations of organic matter in order to produce enough sample in the O and OM fractions for subsequent analysis. The small concentration of C in relation to the high concentration of mineral ma-
Fig. 5. The % dry weight and C and N content of the O, OM and M fractions separated from an untreated River Dee particulate-rich sample. All values are expressed as a % of the total dry weight and total C and N content of the untreated sample.
1730
Technical Note
terial measured in the particulate sample, implies that a large proportion of the organic matter is adsorbed onto mineral particles. In a 1:1 mixture of standard materials, this will not be the case as much of the organic material will be free and easily separated at 1.61 g/cm3. In either sample, the organic matter still has to be relatively free of mineral material for it to enter the O or OM fractions (<1.80 g/cm3). The % of total C present in the particulate M fraction (44.5%) showed that, where strong interactions occur between organic and mineral particles, a density >1.80 g/cm3 will be required to produce a pure mineral fraction. This result also shows that organo-mineral material is a larger proportion of the total sample than the data ®rst suggests. The M fraction of the standard mixture consisted of relatively pure mineral material with 3.33% of the total C present. Again, this was due to the initial large concentrations of organic matter present in a free organic form (O fraction) or associated with mineral particles (OM fraction). Improvements to the method Problems may occur if NaI were to be used to separate fractions which were then analysed for + 2+ and exchangeable ions, e.g. PO3ÿ 4 , K , Ca 2+ Mg , as the ionic strength and pH of the NaI solution could cause desorption of ions. A decrease in %N of the O fraction from fractionated plant litter compared to untreated plant litter (Fig. 2b), suggests that some N loss occurred during fractionation; this was not observed for C. A reason for the N loss could be desorption as it solubilizes more easily than C and is lost more readily into solution. Although the loss is ca. 10% of the total N, greater losses would occur with more exchangeable ions. The desorption of N could also be the reason for a greater proportion of N loss, compared to C, in the unrecovered fraction. A possible problem may also occur with the use of NaOH. This can change the structure of the humic and fulvic components of the O fraction by hydrolysis of esters. However, our results suggest that the solubility of C has not beeen eected by possible structural changes. Furthermore, not all of the organic matter was removed at a density of <1.80 g/cm3. This was particularly noticeable for the particulate sample (Fig. 5), where adherence of organic material to minerals was far stronger. Cambardella and Elliott (1993) have shown that 18% of soil C and 25% of soil N is associated with ®ne silt particles with a density between 2.07 and 2.21 g/cm3. A greater density is required to separate a particulate fraction with little or no organic matter present. It would also be possible to separate two OM fractions using this technique; (i) a 1.61±1.80 g/cm3 fraction which is mainly organic matter and (ii) a 1.80±2.25 g/cm3 fraction which is mainly mineral material. These two OM fractions are probably as chemically dis-
tinct from each other as they are from the O and M fractions. One of the diculties in using NaI is that densities >1.80 g/cm3 cannot easily be achieved. Alternatives to NaI include heavy organic liquids, e.g. tetrabromoethane or sodium metatungstate. The advantages of sodium metatungstate are that it is non-toxic; extremely soluble in water; does not exhibit equilibria with other ionic species in solution; produces a neutral solution with water although it is stable in the pH range 2±10; has low viscosity over a wide density range; is comparatively cheap and reusable. Sodium metatungstate can also achieve densities up to 3.12 g/cm3 at 258C and is clearly an ideal heavy liquid for future work in this area (Plewinsky and Kamps, 1984). Applications of the method The technique described here, possibly further improved using sodium metatungstate, could be applied to both water particulate and soil analysis. It could also be used in the analysis of sediments in order to identify the fate of particular elements and compounds such as P, pesticides and heavy metals in the aquatic environment. The high speci®c surface area of colloids and particulates make them ecient adsorbents for metals, trace elements and pollutants (Stumm, 1992). The likely fate of these ions and molecules in the environment is largely determined by their association with organic, organo-mineral or mineral particles. CONCLUSIONS
The separation of free organic material has been achieved with negligible contamination by organomineral and mineral matter, using an optimized density-centrifugation method. Three separation stages are required to ensure an optimum recovery of each fraction. An alternative to the use of NaI will be required to (i) separate and analyse speci®c exchangeable cations and anions due to possible desorption from particulate surfaces caused by ionic NaI and (ii) increase the fractionation density (possibly up to 3.0 g/cm3) to remove the last remnants of organic material still present in the ``mineral'' fraction.The technique is recommended for the fractionation of water particulates, soils and sediment samples and their subsequent element analysis. REFERENCES
Arshad M. A. and Lowe L. E. (1966) Fractionation and characterisation of naturally occurring organo-clay complexes. Soil Sci. Soc. Am. Proc. 30, 731±735. Ball D. F. (1964) Loss-on-ignition as an estimate of organic matter and organic carbon in non-calcareous soils. J. Soil Sci. 15, 84±92. Cambardella C. A. and Elliott E. T. (1993) Methods for physical separation and characterisation of soil organic matter fractions. Geoderma 56, 449±457.
Technical Note Cotter-Howells J. (1993) Separation of high density minerals from soil. Sci. Total Environ. 132, 93±98. De Kimpe C. R. and Schnitzer M. (1990) Low-temperature ashing of humic and fulvic acid. Soil Sci. Soc. Am. J. 54, 399±403. Gleit C. E. and Holland W. D. (1962) Use of electrically excited oxygen for the low temperature decomposition of organic substances. Anal. Chem. 34, 1454±1457. Henley K. J. (1977) Improved heavy-liquid separation at ®ne particle sizes. Am. Mineral. 62, 377±381. Hope D., Billett M. F. and Cresser M. S. (1994) A review of the export of carbon in river water: ¯uxes and processes. Environ. Pollut. 84, 301±324. Keeling P. S. (1962) Some experiments on the low temperature removal of carbonaceous material from clays. Clay Min. Bull. 28, 155±158. Likens G. E., Bormann F. H., Pierce R. S., Eaton J. S. and Johnson N. M. (1977) Biogeochemistry of a Forested Ecosystem. Springer-Verlag, New York, U.S.A. Plewinsky B. and Kamps R. (1984) Sodium metatungstate, a new medium for binary and ternary density gradient centrifugation. Makromol. Chem. 185, 1429±1439. Schnitzer M., Schulten H. R., Schuppli P. and Angers D. A. (1991) Organic matter extraction from soils with high pressure and temperatures. Soil Sci. Soc. Am. J. 55, 102±108. Schnitzer M. and Schuppli P. (1989) Method for the sequential extraction of organic matter from soils and soil fractions. Soil Sci. Soc. Am. J. 53, 1418±1424.
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Shaw K. (1959) Determination of organic carbon in soil and plant material. J. Soil Sci. 10, 316±326. Sollins P., Glassman C. A. and Dahm C. N. (1985) Composition and possible origin of detrital material in streams. Ecology 66, 297±299. Spycher G., Sollins P. and Rose S. (1983) Carbon and nitrogen in the light fraction of a forest soil: vertical distribution and seasonal patterns. Soil Sci. 135, 79±87. Spycher G. and Young J. L. (1977) Density fractionation of water-dispersible soil organic-mineral particles. Comm. Soil Plant Anal. 8, 37±48. Stepanov I. S. (1981) Physical methods for extracting fractions of organo-mineral substances from soils. Sov. Soil Sci. 13, 106±119. Stumm W. (1992). Chemistry of the Solid-Water Interface. Processes at the Mineral-Water and Particle-Water Interface in Natural Systems. John Wiley, New York, U.S.A. Tessier A., Campbell P. G. C. and Bisson M. (1979) Sequential extraction procedure for the speciation of particulate trace metals. Anal. Chem. 51, 844±851. Turchenek L. W. and Oades J. M. (1979) Fractionation of organo-mineral complexes by sedimentation and density techniques. Geoderma 21, 311±343. Woodroe C. D. (1985) Studies of a mangrove basin, Tu Crater, New Zeland: III. The ¯ux of organic and inorganic particulate matter. Estuar., Coast. Shelf Sci. 20, 447±461.