Modification of Butters-Chenery method for determination of total sulfur in plants and soil.

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1 Modification of Butters-Chenery method for determination of total sulfur in plants and soil. Krzysztof Bielecki, Grzegorz Kulczycki Department of Plant Nutrition, Wrocław University of Environmental and Life Sciences, Poland Reliable determinations of the sulfur content in soil and plants is important for preserving the environment where ever it would be considered a toxic element and alternatively it is necessary for obtaining optimal yields of appropriate quality as a needed element for plant nutrition. Emissions of sulfur oxides into the atmosphere mainly from burning solid fuels and natural gas for energy and technological processes, have a negative impact on the environment. Despite a significant reduction in the emissions of sulfur oxides in Poland 1, constant monitoring of the content of this element in soils and plants is still required. The reduction of sulfur oxide emissions in recent years led to an increase in arable area, where sulfur deficiency in soil and plants was found 2. Research that has been done on this subject has led to modifications in the Butters-Chenery method for measuring the content of sulfur in plants and soils. There are fast and relatively easy methods for the turbidimetric determination of sulfur 3. Many alternative methods have been described in the literature that take into account both the mineralization of the sample (the oxidation of sulfur compounds) and a very important step obtaining a barium sulfate suspension 4-7,11. One of commonly used method for determining the total content of sulfur in plants and soils is the Butters-Chenery method 8,9. This method consists of oxidizing the sulfur contained in organic compounds and then turbidimetric measurement of the sulfates produced, precipitated as barium sulfate. However, the procedure to obtain suspension of barium sulfate is quite complicated. It requires solid BaCl 2 with a precise degree of crushing and mixing the solution several times. This study modified the original method by using a barium chloride solution and Tween 80 (suspension stabilizer) in place of solid barium chloride and gum arabic 10. Experimental Preliminary research Chemicals (analytical grade): magnesium sulfate, nitric acid, hydrochloric acid, phosphoric acid, acetic acid, barium chloride in the form of BaCl 2 2H 2 O. Preliminary experiments were performed using standard solutions of sulfur concentrations of 0-30 g/cm 3 S-SO 4 made with MgSO 4. Each of the solutions also contained Mg(NO 3 ) 2 at a concentration of 0.09 mol/dm3 and a mixture of acids, ether 2.5% HNO 3 or 2.5% HCl. The concentrations of acid mixtures were 2.5% HNO 3, 1.7% H 3 PO 4 and 5% CH 3 COOH. The barium reagent (BR) was made in two variants; 1 dm 3 reagent contained 40 g of BaCl 2 2H 2 O and 200 cm 3 of Tween 80 (Merck) or 0.05% gum arabic. The BaSO 4 suspension was obtained by mixing 3 cm 3 of the standard solution and 1 cm 3 of the barium reagent. The absorbance of the resulting BaSO 4 suspension was measured for 400 nm with a frequency of one minute using a UV-VIS spectrophotometer Evolution 600 with a cuvette changer. Vegetation experiments The plant material and soil used to determine the total sulfur content were taken from a pot experiment with white mustard. Soil was fertilized with sulfate and elemental sulfur at a dose of 300 mg per pot (5 kg of soil). Sulfate sulfur was used in the form of ammonium sulfate, potassium sulfate, and hydrated calcium sulfate. The elemental sulfur had a particle diameter of less than 0.1, , and more than 0.5 mm and was used in granular form as Vigor-S fertilizer (Siarkopol Tarnobrzeg). The experiment was performed in four replicates and the plants were grown to full maturity. The content of

2 total sulfur was measured in the plant material (seeds and straw) and the soil was sampled at the end of vegetation with the modified version of the Butters and Chenery method. The modified method of determining the content of sulfur in plant material began with digesting the sample using HNO 3 and Mg(NO 3 ) 2 8. Then, the residue was dissolved in 5 cm 3 of 25% HNO 3 (modified method - HNO 3 ) or in 5 cm 3 of 25% HCl (modified method - HCl) and diluted to a volume of 50 cm 3. 3 cm 3 of the resulting solution was mixed with 1 cm 3 of the barium reagent containing Tween 80. After 30 min, the absorbance of the resulting suspension BaSO 4 was measured at a wavelength of 400 nm. The sulfur content was read from a calibration curve for concentrations of 0-30 g/cm 3 S-SO 4. The modified method for measuring the sulfur content in the soil began with digestion of the sample using Mg(NO 3 ) 2 8, and then dissolving the residue in 5 cm 3 of 31% HNO 3 and diluting it to a volume of 50 cm 3. 3 cm 3 of the resulting solution was mixed with 1 cm 3 of the barium reagent containing Tween 80. After 30 min, the absorbance of the resulting BaSO 4 suspension was measured at a wavelength of 400 nm. The sulfur content was read from the calibration curve. The results were subjected to statistical analysis (ANOVA) using Statistica 10. Results The results of the impact of acid mixtures on the formation of the BaSO4 suspension is shown in Fig. 1. In the absence of acids, barium sulfate precipitated rapidly. After 5 minutes at the highest concentration of sulfates, the value of the absorbance of the suspension reached a maximum and then very quickly decreased. At a concentration of 6 g/cm3 S-SO 4 the precipitation process was much slower and the absorption maximum was reached after 20 min and remained constant for a considerable amount of time. Acidification significantly decreased the rate of the formation of BaSO 4. At the lowest concentration of sulfate ions, the greatest value of absorbance of the suspension was reached after approx. 100 minutes. With increasing concentrations of sulfate ions, the barium sulfate precipitated faster, and the absorbance value stabilized after approx. 30 min. The influence of acid on the formation of the barium sulfate suspension is shown in Fig. 2. Nitric acid at a concentration of 2.5% significantly slowed down the formation of barium sulfate at the lowest concentrations of sulfate ions. This effect was not observed when BaSO 4 precipitation occurred in the presence of 2.5% HCl. At higher concentrations of sulfate ions, the rate of suspension formation was only somewhat related to the type of acid. However, the maximum value of absorbance of the suspension was greater when a solution of nitric acid was used. The presence of hydrochloric acid stabilized the degree of turbidity of the suspension. The influence of the stabilizer on the formation of the barium sulfate suspension is shown in Fig. 3. The stability of the resulting barium sulfate suspension at the lowest concentration of sulfate ions was independent of the type of stabilizer. At the highest concentration of sulfate ions, detergent Tween 80 noticeably stabilized the absorbance of the suspension in comparison with gum arabic. The total sulfur content in the mustard plants depended on the type of sulfur compound used and the particle size of the elemental sulfur. Different sulfur fertilization resulted in plant material with a substantial difference in sulfur content (1.24% S seeds, straw 0.37% S). This enabled the comparison of tested methods for high and low content of this element in plants. The average inter-sulfur content in the mustard seeds was approx. 1.17% (Fig. 4). Statistical analysis showed no significant difference in the mean values obtained with the method used. The average intersulfur content in the mustard straws was approx. 0.28% (Fig. 5). Statistical analysis showed no significant difference in the mean values obtained with the method used. The average inter-sulfur content determined by the Butters-Chenery method was 14.5 mgs/100 g of soil (Fig. 6). The average value obtained with a modified version of the method was much higher and reached 25.4 mgs/100 g of soil.

3 A comparison of the two methods for measuring sulfur content in the soil was made using a LECO analyzer. The results are shown in Table 1. The results obtained using the modified method were consistent with those obtained using a sulfur analyzer. The values obtained by the Butters-Chenery method were approx. 50% lower. Summary The results of this study showed that phosphoric acid and acetic acid were not necessary for the precipitation of the BaSO 4 barium reagent to determine the content of sulfur in plant material. The Butters-Chenery method uses these acids due to their positive impact on the solubility of solid BaCl 2 8. Hydrochloric acid may successfully be used instead of a nitric acid solution to dissolve the residue after digestion of the plant material. While the absorbance values of the resulting suspension in the presence of nitric acid are larger than in the presence of hydrochloric acid, this acid has a better stabilizing effect on the suspension of barium sulfate. A significant difference is the fact that there is easier dissolution in the hydrochloric acid solution of the precipitate obtained after the digestion of the plant material. Statistical analysis confirms that replacement nitric acid for hydrochloric acid has no influence on the results obtained by Butters-Chenery and modified method. Use of the barium reagent containing detergent Tween 80 considerably simplified the step of obtaining a stable suspension of barium sulfate and significantly reduced the time of analysis. The Butters-Chenery method for determining the content of sulfur in soil requires a 31% nitric acid solution in the final step of mineralization in order to oxidize the sulfur compounds. It is not possible to replace this solution with hydrochloric acid, as in the case for plant material. Since a higher concentration of nitric acid is needed, the precipitation of the barium sulfate is more difficult after the barium reagent is added. When the sulfur concentration in the solution was less than 6 mg/cm 3 S-SO 4 BaSO 4 there was practically no precipitation. A solution to this problem would be to add a certain amount of potassium sulfate to the test samples 10. References 1. A. Kaczor, M.S. Brodowska, Proceedings of ECOpole , W. Grzebisz, K. Przygocka-Cyna, Nawozy i Nawożenie 2003,17, S. Kalembasa, Nawozy i Nawożenie 2004, 18, P. Lundquist, J. Mårtensonn, B. Sörbo, S. Öhman, Clin. Chem. 1980, 26, M.L. Garrido, Analyst 1964, 89, P.N. Matur, D.D. Dharma, G.L. Jain, Z. Pflanzenernahrung. Bodenk. 1988, 151, B. Sörbo, Methods Enzymol. 1987, 143, 3 8. B. Butters, E.M. Chenery, Analyst. 1959, 84, A. Ostrowska, S. Gawliński, Z. Szczubiałka, Metody analizy i oceny właściwości gleb i roślin, IOŚ, Warszawa, B.F. Quin, P.H. Woods, Comm. in Soil Sci. and Plant Anal. 1976, 7, K. Boratyński, A. Grom, M. Ziętecka, Rocz. Glebozn. 1975, XXVI, 121.

4 Fig. 1. Effect of sulfate ions concentrations and acids on turbidity of BaSO 4 suspension (acids 2.5% HNO 3, 1,7% H 3 PO 4, 5% CH 3 COOH) Fig. 2. Effect of sulfate ions concentrations and acids on turbidity of BaSO 4 suspension

5 Fig. 3. Effect of sulfate ions concentrations and stabilizer on turbidity of BaSO 4 Fig. 4. Average amount of sulfur in mustard seeds determined by various methods

6 Fig. 5. Average amount of sulfur in mustard plants determined by various methods Fig. 6. Average amount of sulfur in soil determined by various methods

7 Table 1. Average amount of sulfur in soil determined by various methods [mg/100 g] Obiekty Metoda Buttersa i Chenery`ego Metoda zmodyfikowana Analizator siarki LECO Bez siarki 11,0 27,7 25,5 Siarka elementarna <0,1 mm 14,0 25,4 26,2 Wigor-S 14,0 26,1 27,8 CaSO 4 2H 2 O 15,0 27,7 29,2

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