Elemental Analysis of Plant Material


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Elemental Analysis of Plant Material

  1. 1. Plant Mineral Analysis The Hebrew University of Jerusalem Faculty of Agricultural, Food and Environmental Quality Sciences Rehovot, Israel Vasiliy V. Rosen, M.Sc., ZBM Laboratory icpaes@gmail.com, www.rosen.r8.org 1
  2. 2. 1. Introduction After Jones and Case, 1990 2
  3. 3. Introduction What do we analyze when we are analyzing plants?  Essential elements (major elements and micronutrients)  Toxic elements 3
  4. 4. Introduction The role of chemical elements in plants (adopted from Munson R., 1997, and Macnicol R., 1984) Essential Major Micronutrients Toxic Carbon (C) Boron (B), Silver (Ag) Oxygen (O) Chlorine (Cl) Aluminium (Al) Hydrogen (H) Copper (Cu) Arsenic (As) Iron (Fe) Barium (Ba) Nitrogen (N) Manganese (Mn) Berillium (Be) Phosphorus (P) Molybdenum (Mo) Cadmium (Cd) Potassium (K) Zinc (Zn) Mercury (Hg) Nickel (Ni) Lead (Pb) Sodium (Na) Cobalt (Co) Lithium (Li) Silica (Si) Chromium (Cr) Calcium (Ca) Selenium (Se) And all Magnesium (Mg) Vanadium (V) micronutrients at Sulfur (S) critical concentration 4
  5. 5. Introduction The levels of major elements and micronutrients in mature leaf tissue (after Munson R., 1997) 5
  6. 6. Introduction Concentration Units Major Elements % of dry weight grams per kilogram (g/kg) Micronutrients and Toxic Elements parts per million (ppm) = 10-6 = mg/kg parts per billion (ppb) = 10-9 = µg/kg 6
  7. 7. 2. AnalyticalChemistry Basics Qualitative and quantitative analysis Calibration and matrix Limit of Detection and Limit of Quantitation Accuracy and Precision 7
  8. 8. Analytical Chemistry Basics Qualitative and Quantitative Analysis ICP : ATOMIC EMISSION SPECTROMETRY AS QUALITATIVE ANALYSIS Is there the analyte in the sample? Qualitative Analysis If yes, which one? ICP: ATOMIC EMISSION SPECTROMETRY AS QUANTITATIVE ANALYSIS How much analyte is Quantitative Unknown Sample there? Analysis 8
  9. 9. Analytical Chemistry Basics Calibration Curve The calibration curve is a plot of detector response as a function of concentration (after Munson R., 1997) 9
  10. 10. Analytical Chemistry Basics Matrix Cd, 1 mg/L, in weak acid Cd, 1 mg/L, in base Analyte concentrations are equal, but intensities are different 10
  11. 11. Analytical Chemistry Basics Limit of Limit of Detection Quantitation LOD is the concentration at which LOQ is the lowest concentration at we can decide whether an element which a measurement is is present or not (Thomsen, 2003) quantitatively meaningful (Mitra, 2003) LOQ = 10*SDblank LOD = 3*SDblank or LOQ = 3.3*LOD 11
  12. 12. Analytical Chemistry Basics Accuracy and Precision Accuracy is how close a Precision is how close the measured value is to measured values are to each the actual (true) value. other. after http://www.mathsisfun.com12
  13. 13. 3. Plant Samples Pretreatment Sampling Procedure Decontamination Drying Grinding 13
  14. 14. Plant Samples Pretreatment Sampling Procedure What to sample? Mature leaves exposed to full sunlight just below the growing tip on main branches or stems are usually preferred (Jones B., 2003) How much material to sample? Depending on plant and investigation goal – usually tens (20-100) leaves or small plants 14
  15. 15. Plant Samples Pretreatment Sampling Procedure What DO NOT sample? After Jones B., 2003 15
  16. 16. Plant Samples Pretreatment Decontamination Contaminants Washing procedure Soil and dust particles: Fe, Al, Si  Tap waterand Mg. Calcareous soils – Ca.  Detergent solution , non-phosphate Liquide fungicides – Cu. (0.1 to 0.3%) Nutrition solution (fertilizer) –  Weak acid (HNO3 1%) – optionalNPK, essential elements.  Deionized water Investigator’s fingers - Cl 16
  17. 17. Plant Samples Pretreatment Drying  Put washed fresh samples in paper bag (or envelope). Do not use plastic bag since plastic retains moisture, thus accelerating respiration and decay.  Refrigerate (4-5º C) or air-dry the fresh samples if delivery time to laboratory is more than 12 h. Fresh plant samples should be dried at 65-80º C in a ventilated oven at least 24 h (usually 2-3 days) to stop the enzymatic activity. Higher drying temperature can affect the dry weight. 17
  18. 18. Plant Samples Pretreatment Grinding:particle size reduction  Different types of mills are available: Jaw, Rotor, Cutting, Knife, Mortar, Discs, Planetary Ball mills.  Material used: stainless steel, Zr2O, agate, porcelain.  Possible contaminants: Fe, Zn, Al, Na . Planetary Ball Mill Rotor Mill 18
  19. 19. Plant Samples Pretreatment Grinding: particle size units 19
  20. 20. 4. SamplePreparationTechniquesDry AshingWet Ashing, Microwave-assisted acid digestion 20
  21. 21. Sample Preparation Techniques Dry Ashing Analytes: B, Ca, Cu, Fe, Mg, Mn, P (but wet ashing is more recommended), K, Na, Zn. Procedure: 500 mg of dry sample digested in porcelain crucible in muffleoven during 4-6 h at 500º C . The ash dissolved in 1 N HCl. Element determination: AAS, ICP-AES, UV-VIS (B, P). Possible problems: easily volatilized elements are lost (Cl, S, As, Hg, Se);boron (B) may be also volatilized; insoluble silicates are formed and decreasedrecovery of other constituents, mainly trace elements; ashing temperature higherthan 500º C may decrease recovery of Al, B, Cu, Fe, K, Mn. After Miller, 1998 21
  22. 22. Sample Preparation Techniques Dry Ashing: Tips and Tricks If an ashing aid is needed, add either 5 mL HNO3, or 5 mL 7% Mg(NO3)2*6H2Oprior to muffel digestion. Dry on a hotplate and then digest. To prevent Cl loss do the following: mix the sample with lime (CaO, ¼ of thesample weight) and deionized water to make a thin paste. Dry the mixture, digest at500º C, dissolve ash with HNO3 or H2SO4 (not HCl !!!) The following acid mixtures may be used for ash dissolution: 300 mL HCl and100 mL HNO3 in 1000 mL deionized water; Aqua Regia (concentrated HNO3 :HCL1:3), HNO3 alone (less corrosive for for metal parts of analytical instruments). After Piper, 1950; Jones, 2001; personal experience 22
  23. 23. Sample Preparation Techniques Wet Ashing Analytes: B (teflon vessels only), Ca, Cu, Fe, Mg, Mn, Mo, P, K, Se, Na, S, Zn,trace elements. Procedure: 500 mg of dry sample digested with some combination of fouracids: HNO3, HCl, H2SO4 and HClO4, with optional addition of H2O2. Digestionis carried out in beakers on hot plate, in glass tubes on block, in open or closedteflon vessels in microwave oven. Element determination: AAS, ICP-AES, UV-VIS ( P, S). Possible problems: HClO4 may react with organic material and result in anexplosion; in low Ca tissues CaSO4 may precipitate when H2SO4 is used;contamination with B and Si when glass digestion tubes are used; contaminationwith elements adsorbed by teflon. After Piper, 1950; Jones, 2001; Miller, 23 1998; personal experience
  24. 24. Sample Preparation Techniques Wet Ashing: Instruments Digestion Block Microwave Laboratory Oven “Ethos 1”Teflon Vessel with Tº and pressure control 24
  25. 25. Sample Preparation Techniques Wet Ashing: Tips and Tricks  Samples with added acid(s) should be predigested at room temperature overnight to avoid violent reaction at the start of heating. This is especially important for closed microwave-assisted digestion.  H2O2 often contains Sn (tin) as a stabilizer. Do not use H2O2 if Sn is analyte.  Wet ashing on block has a high throughput, but closed vessel microwave-assisted digestion demonstrates less element loss and contaminations, and it is less time- consuming.  Increase sample weight for the determination of trace metals (Cd, Cr, Ba etc) to 1 g. Add internal standard (element that does not exist in your samples, Y or Sc) at the start of digestion to control preparation process quality. After Jones, 2001; Miller, 1998; personal experience 25
  26. 26. 5. Instrumentationused in plantanalysis X-ray fluorescence spectroscopy (XRF) Atomic absorption spectroscopy (AA) Flame Emission Spectrometry (Flame Photometry) ICP-AES/MS UV-VIS Spectrophotometry Elemental Analyzer, Chloride Analyzer, Ion Selective Electrodes etc. 26
  27. 27. Instrumentation XRF: X-Ray Fluorescence Spectroscopy Principle: Excitation of the sample by an X-ray source,secondary radiation measurement. Elements: with atomic number >8. LOD: 100 mg/kg for major elements (light) and 1 mg/kgfor traces (heavy). Sample Preparation: drying, fine grinding and pressing. Advantages: simple sample preparation; low cost;portable instrument. Disadvantages: spectral interferences; method is matrix-dependent. 27
  28. 28. Instrumentation AAS: Atomic Absorption Spectroscopy Principle: quantifies the absorption of ground state atoms in thegaseous phase; the analyte concentration is determined by optics fromthe amount of light absorption.Elements: all the metals. LOD: some µg/L (ppb), less than 1 ppb – with graphite furnace. Sample Preparation: dry and wet digestion methods. Advantages: highly specific for an element; minimum spectralinterferences; low-cost gases used (air+acetylene). Disadvantages: ionization enhancement of the signal for elementseasily ionized when operating in the absorption mode, especially Na andK; matrix interferences caused by viscosity or specific gravitydifferences between sample and reference standard; elements analyzedone at a time. 28
  29. 29. Instrumentation Flame Emission Spectroscopy (Flame Photometry) Principle: excitation of ground-state atoms by propane-butane flame (2000-3000 ºC), electron loss by analyte atom,when electron is recaptured, emission light of characteristicwavelength is emitted. Elements: Na and K; Li, Rb, Cs, Ca. LOD: about 0.1-0.5 mg/L. Sample Preparation: dry and wet digestion methods. Advantages: simple, quick and inexpensive analysis; widedynamic range (0-100 mg/L); ideal for elements with lowexcitation potential (Na and K) Disadvantages: only some elements may be determined;elements analyzed one at a time. 29
  30. 30. InstrumentationICP-AES: Inductively Coupled Plasma Atomic Emission Spectrometry Principle: electrons of excited atoms return to their ground-state and emit electromagneticradiation (light) at the wavelengths that are characteristic of the atoms that are excited. Argonplasma is the source of excitation (about 10 000 K). Elements: all the elements except gases and some non-metals (C, N, F, O, H). LOD: some µg/L (ppb), less than 1 ppb – with MS detector (ICP-MS technology). Sample Preparation: dry and wet digestion methods. Advantages: minimum chemical interferences; four to six orders of magnitude in linearityof intensity versus concentration; multielement capabilities; rapid analysis; accurate andprecise analysis; detection limits equal to or better than AAS for many elements. Disadvantages: occurrence of spectral interferences; use of argon gas which can beexpensive; instrument is relatively expensive to purchase. 30
  31. 31. Instrumentation UV-VIS Spectrophotometry Principle Instrument bApplications: Kjeldal digestion for total N: determination of NH4 and P in digestate; Mo and B after dry or wet ashing;NO3 in water extracts;Metals: Cu, Fe, Mg, Mn and Zn determination. 31
  32. 32. Instrumentation Just a few words about…. Elemental Analyzer Chloride Analyzer Ion-selective ElectrodeElements: C,H,N,S,O. Elements: Cl Elements: K+, Cl-, NO3-Digests finely grinded dry Titrates Cl- with Ag2+. Measures an electricalsamples. Readout range: 10-999 potential on the ion mg Cl/L exchanger that is selective to analyte ion. 32
  33. 33. Last but not least… First Law of Laboratory Work:Hot glass looks exactly the same as cold glass Thank you for your attention 33