DTPA extraction from micronutrients.doc

DTPA Extraction for Fe, Zn, Cu, Mn and DTPA-Sorbital Extraction for Fe, Zn, Cu, Mn and BTony Provin and Hailin ZhangApplication and PrincipleFirst developed and published by Lindsay and Norvell (1978) for extracting micronutrients in neutral and calcareous soils, the DTPA extraction method has gained significant adoption by agricultural laboratories. While originally conceived to address the over-estimation of micronutrients by acid extracts in calcareous soils, the equilibrium/chelation mechanism coupled by the use ICP analysis has proven useful in nearly all pH ranges. Plant available boron in the western United States has historically been analyzed via refluxing boiling water, a procedure requiring significant technician involvement and safety hazards. Vaughan and Howe (1994) reported on the potential for using boron chelates instead of refluxing water. As a result, Miller et al. (2000) documented the inclusion of the sugar sorbital in the DTPA extractant for plant available Fe, Zn, Cu, Mn and B simultaneous extraction and determination.Equipment and Apparatus1. Digital balance (0.0000 gram resolution) 2. Digital balance (0.00 gram resolution)3. 100 ml graduated cylinder4. 500 ml graduated cylinder5. 20 L poly carboy6. 1.5-2” Teflon stir bar7. Inline (180 rpm-2-2.5” throw or orbital shaker (180 rpm with 1” throw) either shaker must be fitted with appropriate sample vessel holders8. 250 ml Erlenmeyer flask or similar extracting vessel9. Filter rack10. Dispenser capable of rapid dispensing of 10-20 ml of extractant to 0.1ml resolution11. Calibrated pH meterReagents1. Deionized water2. Diethylenetriaminepentaacetic acid (DTPA)3. Triethanolamine (TEA)4. Calcium chloride (CaCl2)5. HCl (12M)6. ICP or ICP/MS grade 1000 ppm Zn, Fe, Cu, Mn and B standards dissolved in HCl or nitric acid7. In addition to above, for DTPA-Sorbital method include: Sorbitol (D-Sorbital, 98%+)DTPA extractant composition1. 0.005M DTPA2. 0.01M calcium chloride (CaCl2)3. 0.1M TCA (triethanolamine, (HOCH2CH2)3N)To prepare 20L of extract:1. Carboy calibration: On a digital balance, weigh 15,000 and 20,000 g (0.0 g) deionized water (diH2O) into a 20 L carboy. Draw a mark on the carboy at the water level and label 15L and 20 L, respectively.2. Place a 1.5” stir bar into the calibrated carboy. Add diH2O into the carboy to the 15L mark.3. Using a digital balance, weigh 39.89 g (0.00 g) diethylenetriaminepentaacetic acid (DTPA) and add to the carboy. Weigh 29.44 g (0.00 g) calcium chloride (CaCl2) and add to the carboy. Measure 265 mL triethanolamine (TEA) with a 500 mL graduated cylinder and add it to the carboy. Measure 85 mL HCl (12M) with a 100 mL graduated cylinder and add it to the carboy.4. Add diH2O to bring the volume to the 20 L mark.5. Place carboy on a magnetic stirrer and stir overnight (minimum of 8 hr) at a speed of 600 rpm.6. Measure pH of the extractant. Adjust the pH to 7.3 (repeat pH measurement 30 minutes of mixing). Add 9 mL HCl (12M) per each pH unit over 7.3. Add 9 mL TEA per each pH unit under 7.3 DTPA-Sorbital extractant composition1. 0.005M DTPA2. 0.01M calcium chloride (CaCl2)3. 0.1M TCA (triethanolamine, (HOCH2CH2)3N)4. 0.20M D-Sorbitol, 98% minimumTo prepare 20L of extract:1. Carboy calibration. On a digital balance, weigh 15,000 and 20,000 g (0.0 g) deionized water (diH2O) into a 20 L carboy. Draw a mark on the carboy at the water level and label 15L and 20 L, respectively.2. Place a 1.5” stir bar into the calibrated carboy. Add diH2O into the carboy to the 15L mark. 3. Weigh 39.89 g (0.00 g) diethylenetriaminepentaacetic acid (DTPA) and add to the carboy. Weigh 29.44 g (0.00 g) calcium chloride (CaCl2) and add to the carboy. Measure 265 mL triethanolamine (TEA) with a 500 mL graduated cylinder and add to the carboy. Measure 85 mL HCl with a 100 mL graduated cylinder and add to the carboy. Add 728.8 g Sorbital to the carboy. 4. Add diH2O to the 20 L mark.5. Place carboy on a magnetic stirrer and stir overnight (minimum of 8 hr) at a speed of 600 rpm.6. Measure pH of the extractant. Adjust the pH to 7.3 (repeat pH measurement after 30 minutes of mixing). Add 9 mL HCl (12M) per pH unit over 7.3. Add 9 mL TEA per pH unit under 7.3.Note: Small quantities of white precipitates will likely remain in the bottom of the carboy. This material is insoluble contaminates for the DTPA reagent and will not pose an analytical issue provided the precipitates are not brought into suspension during the dispensing operation. The extract can be filtered if precipitates become a problem during dispensing, however the carboy should be rinsed and acid washed between batches.Standard PreparationAll standards should be made from NIST traceable ICP/ICP-MS grade primary standards. The following four or five multi-element standards and associated blank-matrix solution will provide a wide adequate elemental concentration ranges for most soils including heavily manured and garden soils.1. Fill a 1000 mL Class A volumetric flask with about 250 mL of the DTPA or DTPA-Sorbital Extractant2. Using Class A pipettes, transfer the appropriate volume of each primary standard to the volumetric flask.3. Bring to volume with the extraction Solution. Cap flask and invert and shake 20 times. Transfer standard to labeled polypropylene or similar sealable containers.StandardFeZnMnCuB*mls 1000 ppm stdFinalConc.ppmmls 1000 ppm stdFinalConc.ppmmls 1000 ppm stdFinal Conc.ppmmls 1000 ppm stdFinal Conc.ppmmls 1000 ppm std Final Conc.ppm15.05.01.0*0.11.0*0.11.01.01.0*0.1210.010.05.0*0.55.0*0.55.05.01.01.0320.020.01.01.01.01.010.010.05.05.045.0*50.02.02.02.02.020.020.010.010.0*10,000 ppm Fe standard reduces dilution of using extraction solution.*100 ppm working standards*Boron is added to standard for DTPA-Sorbitol method only.ProcedureExtraction1. Either gravimetric or volumetric sampling maybe used as long as a 1:2 ratio of soil to extractant is maintained. a. Ensure clean and dry extracting cups or flasks are available.b. Label extraction containers with appropriate laboratory number or other sample information.c. Weigh or transfer 20 g of soil to appropriate extraction cup or flask. Laboratories that primarily process light textured soils may elect to use 10 gram sample, however these laboratories must ensure adequate filtrate for ICP determination.2. Dispense 40 mL of the DTPA or DTPA-Sorbital extracting solution into each sample cup using the appropriate calibrated dispenser.3. Shake the samples for 2 hr using the 200 rpm shaker table.FiltrationFollowing the two hour shaking period, decant samples into appropriate filters/filter assemblies using medium-fine (Whatman No. 2 equiv.) porosity filter paper.Transfer filtrate to number/referenced test tubes for ICP analyses. AnalysisPlace standards and samples in appropriate order on to ICP sample racks. Calibrate and standardize ICP and method as appropriate per laboratory QA/QC protocols and manufacturers recommendations.Notes on analysis:Low level detection for copper can sometimes be challenging due to the matrix and its tendency to solublize organic carbon compounds. While well tuned radial ICPs are capable of achieving reporting limits of sub 0.05 ppm Cu, many laboratories prefer to utilized their axial ICPs in order to ensure low detection levels of Cu. The soluble carbon species have a strong tendency to coat the aspiration tube of axial torches and many laboratories find they must switch out or clean torches every 150-350 samples, or prior to analyzing other matrixes.CalculationsThe dilution factor for this method is 2. Normal reporting for micronutrient concentrations is in parts per million (ppm or mg/kg) on the soil basis. Therefore, the final ICP analysis result of the extract is multiplied by 2 to provide mg/kg of soil.Analytical PerformanceAccuracy and precision of the method is dependent on several factors including: fineness of the soil pulverization, sample weight taken for extraction and ICP configuration. The table below illustrates the long-term statistics of 1000 analyses of a 2 mm sieved check sample with modest to high levels of Fe, Zn, Mn and Cu. All analyses were performed using an axial ICP with cross-flow nebulizer.FeZnMnCuB*mg/kgmg/kgmg/kgmg/kgmg/kgMean43.071.6051.900.680.50Std Dev.4.7530.2787.8930.0840.04*B was extracted by DTPA-sorbital with 30 analyses in one month.The use of modern CCD ICPs has largely eliminated interference issues with DTPA analysis. While historic issues between Zn interferences on Cu lines in older photomultiplier tube ICPs were potentially troubling, modern CCD instruments afford multiple interference free lines. The limiting factor in DTPA analysis on an ICP is the presence of significant concentrations of organic carbon. During introduction into the plasma, significant CO2 is released and could result in line interferences if the laboratory is using lower wavelength lines. The organic carbon should not pose an issue for metal analysis in the visible spectrum. InterpretationRegional or sub-regional correlation and calibration are required to utilize any soil test method. The table below provides a starting point for laboratories conducting the method.FeZnMnCuBRatingmg/kgmg/kgmg/kgmg/kgmg/kgEx. Low0.1V. Low0.330.05Low2.19-3.190.18-0.220.33-0.660.05-71.000.160.5Ex. Low (corn and sorghum).80Effects of StorageThe DTPA solution and standards are relatively stable provided the laboratory takes steps to prevent evaporation and limits exposure of the solutions to sunlight. If properly stored, the standards and extraction are ok to use even after 5 weeks. The introduction of sorbitol into the DTPA can limit the shelf life of the standards and extractant du