Isolation of IHSS Soil Fulvic and Humic Acids

Isolation of IHSS Soil Fulvic and Humic Acidsexerpt from -- Swift, R.S. 1996 Organic matter characterization (chap 35). pp. 10181020. In D.L. Sparks et al. (eds) Methods of soil analysis. Part 3. Chemical methods. Soil Sci. Soc. Am. Book Series: 5. Soil Sci. Soc. Am. Madison, WI © Soil Science Society of America, used with permission. Reprints can be obtained from: Roger.S. Swift, CSIRO, PMB 2, Glen Osmond 5064, Australia. International Humic Substance Society Method. A number of methods for the extraction of humic substances from soil using sodium hydroxide solution have been published. These methods are generally successful and yield comparable results. The following is a method which has been developed by the International Humic Substance Society (IHSS) as an acceptable method for the extraction of humic substances from soils. It has been clearly stated by IHSS that this is not meant to be a recommended or approved method, but a method that has been found to be satisfactory for most soil types and one which can be performed in most laboratories. It produces relatively high yields and can be used as a standard method for comparisons between and within laboratories. An important component of this method is the use of an adsorbent resin in the purification process. This can be replaced by dialysis if the resin is unavailable (See note about dialysis at end of text). Materials 1. Hydrochloric acid (HCl), 1 M, 6 M 2. Sodium hydroxide, 1 M, 0.1 M 3. Potassium hydroxide (KOH), 0.1 M 4. Potassium chloride (KCl) 5. Hydrofluoric acid (HF) concentrated, 0.3 M 6. XAD-8 resin (Rohm & Haas Co., Philadelphia, PA) 7. Visking dialysis tubing (Visking Co., Chicago, IL) [MWCO (molecular weight cut-off)] 10,000 dalton Method Remove roots and sieve the dried soil sample to pass a 2.0-mm sieve. Equilibrate the sample to a pH value between 1 to 2 with 1 M HCl at room temperature. Adjust the solution volume with 0.1 MHCl to provide a final concentration that has a ratio of 10 mL liquid/1 g dry sample. Shake the suspension for 1 h and then separate the supernatant from the residue by decantation after allowing the solution to settle or by low speed centrifugation. Save the supernatant (FA Extract 1) for the isolation of fulvic acid using XAD-8. Neutralize the soil residue with 1 M NaOH to pH = 7.0 then add 0.1 M NaOH under an atmosphere of N2 to give a final extractant to soil ratio of 10:1. Extract the suspension under N2 with intermittent shaking for a minimum of 4 h. Allow the alkaline suspension to settle overnight and collect the supernatant by means of decantation or centrifugation. Acidify the supernatant with 6 M HCl with constant stirring to pH = 1.0 and then allow the suspension to stand for 12 to 16 h. Centrifuge to separate the humic acid (precipitate) and fulvic acid (supernatant - FA Extract 2) fractions. Redissolve the humic acid fraction by adding a minimum volume of 0.1 M KOH under N2. Add solid KCl to attain a concentration of 0.3 M [K+] and then centrifuge at high speed to remove the suspended solids. Reprecipitate the humic acid by adding 6 M HCl with constant stirring to pH = 1.0 and allow the suspension to stand again for 12 to 16 h. Centrifuge and discard the supernatant. Suspend the humic acid precipitate in 0.1 M HCl/0.3 M HF solution in a plastic container and shake overnight at room Sci.1 M NaOH followed by two column volumes of distilled H2O.0. Note on HF treatment: More recently a filtration step has been added for removal of clays from the NaOH soil extract. Because it is sometimes difficult to obtain. Kuwatsuka. If it is not possible to purify the fulvic acid using resin treatments.). 150:763-770. If there is a significant concentration of polyvalent cations such as A13+ present.test with silver nitrate AgNO3. methyl methacrylate ester resin (see "Fractionation of Humic Substances Adsorption"). E. 1990. These methods and methods used to store the resin are detailed by Thurman & Malcolm (1981). followed by 2 to 3 column volumes of distilled H2O. Pass the supernatant designated "FA Extract 1" through a column of XAD-8 (0.65 column volumes of distilled H2O. as it is likely to contain significant amounts of unbound soil polysaccharide.1 M NaOH. a fraction obtained in this way should be referred to as a fulvic fraction. Technol. Back elute the XAD-8 column with 1 column volume of 0. Extensive purification procedures of the resins are required before use.temperature. Soil Sci. 15:463-466. Repeat the back elution and acidification as for "FA Extract 1" above. rather than fulvic acid. Freeze dry the humic acid. CA) using three times the mole of Na ions in solution]. Characterization of electrophoretic fractions of humic substances with different electrofocusing behavior. Plant Nutr. Immediately acidify the solution with 6 M HCl to pH = 1. Pass the eluate through H+-saturated cation exchange resin [Bio-Rad AG-MP-5 (Bio-Rad. A. J. Watanabe. 37:611-617. G. Technically. these may form insoluble metal-humate complexes as the solution is neutralized. Fractionation of soil fulvic acids using polyvinyl-pyrrolidone and their ionization difference spectra. Comments XAD-8 is a nonionic. Soil Sci. exhaustive dialysis against distilled H2O is an alternative but less satisfactory method of purification. Preparative isolation of aquatic humic substances. Discard the effluent. The solution volume should be sufficient to maintain the fulvic acid in solution.. macroporous (pore size 25 µm). Bragato. rinse the XAD-8 column containing sorbed fulvic acid with 0.65 column volumes of distilled H2O. Combine the final eluates from each of the fulvic acid extracts and pass this solution through XAD-8 resin in a glass column (column volume should be one-fifth of sample volume). if necessary. Therefore. and R. before finally dialyzing against distilled H2O. Comellas. until the ash content is below 1%. Transfer the precipitate to a Visking dialysis tube by slurrying with water and dialyze against distilled water until the dialysis water gives a negative Cl.2 micrometer polyethersulfone membrane filter (Gelman Supor) under pressure (nitrogen gas). the dialysis should be carried out against dilute HC1 initially until the concentration of any polyvalent cations has been significantly reduced. This removes removes ash without HF. A. 1981. Centrifuge and repeat the HCl/HF treatment. and L. it may be necessary to use an alternative resin such as Polyclar. 1990.15 mL of resin per gram of initial sample dry weight at a flow rate of 15 bed volumes per h). 1991. Add concentrated HF to a final concentration of 0. REFERENCES De Nobili. which is a crosslinked poly(vinylpyrrolidone) (PVP) (De Nobili et al...0 mL of resin per gram of initial sample dry weight).M. Back elute with 1 column volume of 0. Puigbo. Alcaniz. (See note about dialysis at end of text. Environ. Richmond.M.. Rinse with 0. and S. Watanabe & Kuwatsuka.3 M HF. The extract should be filtered twice through the same membrane. Thurman. Pass the supernatant designated "FA Extract 2" through a column of XAD-8 (1. Malcolm. Freeze dry the eluate to recover the H+-saturated fulvic acid.L. The partial plugging of the filter with the first filtration helps remove . M. 1991) or other equivalent resin. The NaOH extract is filtered through a 0. fine clay and eliminates the need for the KOH-KCl and HF-HCl treatments to reduce the ash content. Research on dialysis of fulvic acid shows much of fulvic acid will pass through commercially available dialysis membranes. Note on dialysis of fulvic acids. Dialysis is probably not a good substitute for the resin treatment. . Sampai tahap ini. Ekstraksi asam basa didasarkan atas kelarutan asam humat yang larut dalam alkali/basa dan mengendap dalam asam. sedangkan untuk memisahkan bahan humat dan nonhumat yang mengendap dilakukan dengan sentrifuse kembali. dimana semakin jernih berarti kandungan asam fulvatnya semakin rendah dan sebaliknya. Endapan asam humat kemudian di suspensi dengan larutan HCl 5% + HF 5% ( 50 ml ) untuk membersihkan kandungan abu yang terdapat dalam larutan dan kemudian di sentrifugal ( purifikasi ). Endapan asam humat dipisahkan dari supernatan yang mengandung asam fulvat dengan sentrifugal. 10-20 gram pupuk Kompos. Timbang 10-20 gram tanah ( Top Soil dan Sub Soil ) < 2 mm ( 0-10 cm dan 10-20 cm ). Setelah selesai pengocokan dibiarkan selama minimal 1 x 24 jam. 4. kemudian disentrifuse dan hasil sentrifuse ditera 500 ml. Supernatan di asamkan dengan HCl 6 N hingga pH 1. sedangkan asam humat dapat diperkirakan berdasarkan jumlah endapannya.0 sambil dilakukan pengadukan ( fraksionasi ). Bahan humat yang dihasilkan diturunkan pH-nya dengan HCl 0. 10-20 cm gram pupuk kandang ( Sapi dan Ayam ). Kemudian di kocok selama 2 jam atau lebih dengan mesin pengocok ( shaker ). 5. sehingga cairan terbagi menjadi dua bagian yaitu yang mengendap sebagai asam humat dan yang larut sebagai asam fulvat. Untuk memisahkan bahan humat dan nonhumat yang terapung diatasnya dilakukan penyaringan dengan buncher filter.1 N sampai pH 2. Penghitungan persentase ( berat ) asam humat yang terdapat pada bahan Pemisahan asam humat dan asam fulvat dari senyawa humat (bahan humat) dilakukan dengan cara ekstraksi asam basa. 6. Endapan asam humat di cuci dengan air aquades ( 3x ) ( furifikasi ).1 N. 2. 7.Cara Kerja 1. dan kemudian suspense yang mengendap dibuang dan supernatant yang berwarna gelap dipindahkan ke erlenmeyer yang lain. Tambahkan pada masing-masing sampel 50-100 ml NaOH 0. 9.5M ( ekstraksi ). 3. Masing sampel dimasukkan ke dalam Erlenmeyer. sedangkan asam fulvat dapat larut dalam alkali maupun asam. 8. Asam fulvat dapat diperkirakan berdasarkan tingkat kekeruhan/kejernihan cairan. Contoh tanah kering 60ºC ditimbang 5 gram lalu dikocok dengan 2 x 250 ml NaOH 0. 10. Asam humat (bagian yang mengendap) . 10-20 gram tanah Gambut. kandungan asam humat dan asam fulvat sudah dapat diperkirakan secara kualitatif. Asam humat yang diperoleh di keringkan dalam oven dengan suhu 0 40 C. dapat diketahui secara kuantitatif dengan cara disentrifuse 3500 rpm selama 15 menit dari bahan humat pada pH 2 . . kemudian dikeringkan dalam oven 60ºC sampai bobotnya konstan lalu ditimbang untuk mengetahui jumlah asam humat yang dihasilkan. sehingga diperoleh endapan asam humat dan cairan di atas endapan (asam fulvat) yang kemudian dipisahkan. Asam humat hasil sentrifuse dimasukan secara kuantitatif ke dalam cawan porselin yang sudah diketahui bobot kosongnya.