This lecture is based on previously read lecture "Plant Mineral Analysis", 2012. Some new points were added, especially in LOD/LOQ section. The internal standard calculation was explained. The lecture was presented in the frame of International Course "Crop Production under Saline Stress As A Result Of Climatic Changes", The Faculty of Agriculture, The Hebrew University of Jerusalem.

1. 1
The Hebrew University of Jerusalem
The Faculty of Agricultural, Food and Environmental Quality Sciences
Rehovot, Israel
Vasiliy V. Rosen, Ph.D., ZBM Laboratory
icpaes@gmail.com
Elemental Analysis
of Plant Material

2. After Jones, 2001
1. Introduction
2

3. What do we analyze when
we are analyzing plants?
 Essential elements (major elements
and micronutrients)
 Toxic elements
Introduction
3

4. Essential
ToxicMajor Micronutrients
Carbon (C)
Oxygen (O)
Hydrogen (H)
Nitrogen (N)
Phosphorus (P)
Potassium (K)
Sodium (Na)
Silica (Si)
Calcium (Ca)
Magnesium (Mg)
Sulfur (S)
Boron (B),
Chlorine (Cl)
Copper (Cu)
Iron (Fe)
Manganese (Mn)
Molybdenum (Mo)
Zinc (Zn)
Nickel (Ni)
Cobalt (Co)
Chromium (Cr)
Selenium (Se)
Vanadium (V)
Silver (Ag)
Aluminium (Al)
Arsenic (As)
Barium (Ba)
Berillium (Be)
Cadmium (Cd)
Mercury (Hg)
Lead (Pb)
Lithium (Li)
And all
micronutrients at
critical
concentration
The role of chemical elements in plants
(adopted from Munson R., 1997, and Macnicol R., 1985)
Introduction
4

5. The levels of major elements and micronutrients in mature
leaf tissue (after Munson R., 1997)
Introduction
5
Parts per million = mg/kg
Percent

6. Introduction
Concentration Units
Major Elements
% of dry weight
gram per kilogram (g/kg)
Micronutrients and Toxic Elements
6

7. 2. Analytical
Chemistry Basics
Qualitative and quantitative analysis
Calibration and matrix
Limit of Detection and Limit of Quantitation
Accuracy and Precision
7

8. Analytical Chemistry Basics
Qualitative and
Quantitative Analysis
Is there the analyte in the
sample?
If yes, which one?
Qualitative
Analysis
How much analyte is
there?
Quantitative
Analysis
ICP: ATOMIC EMISSION SPECTROMETRY
AS QUANTITATIVE ANALYSIS
Unknown Sample
8
ICP : ATOMIC EMISSION SPECTROMETRY
AS QUALITATIVE ANALYSIS

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. 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. Analytical Chemistry Basics
Limit of
Detection
Limit of
Quantitation
LOD is the concentration at which
we can decide whether an element
is present or not (Thomsen, 2003)
LOQ is the lowest concentration at
which a measurement is
quantitatively meaningful (Mitra,
2003)
LOD = 3*SDblank LOQ = 10*SDblank
LOQ = 3.3*LOD
or
11
SD (or δ) – Standard Deviation

12. Analytical Chemistry Basics
Limit of
Detection
Limit of
Quantitation
LOD is the concentration at which
we can decide whether an element
is present or not (Thomsen, 2003)
LOQ is the lowest concentration at
which a measurement is quantitatively
meaningful (Mitra, 2003)
FDA, Elemental Analysis
Manual, 2014
Method LOQ!!!
Analytical Solution Detection Limit
12

13. LOD and LOQ determination on ICP-OES
Observed LOD = <5 ppb
Observed LOQ = ≈ 10 ppb
Analytical Chemistry Basics
13

14. Analytical Chemistry Basics
Accuracy and Precision
Accuracy is how close a
measured value is to
the actual (true) value.
Precision is how close the
measured values are to each
other.
after http://www.mathsisfun.com 14

15. 3. Plant Samples
Pretreatment
Sampling Procedure
Decontamination
Drying
Grinding
15

16. 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., 2001)
How much material to sample?
Depending on plant and
investigation goal – usually tens
(20-100) leaves or small plants
16

17. Plant Samples Pretreatment
Sampling Procedure
What DO NOT sample?
After Jones B., 2001
17

18. Plant Samples Pretreatment
Decontamination
 Tap water
 Detergent solution , non-phosphate
(0.1 to 0.3%)
 Weak acid (HNO3 1%) – optional
 Deionized water
 Soil and dust particles: Fe, Al, Si
and Mg. Calcareous soils – Ca.
 Liquide fungicides – Cu.
 Nutrition solution (fertilizer) –
NPK, essential elements.
 Investigator’s fingers - Cl
Contaminants Washing procedure
18

19. 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.
19

20. 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 20

21. Plant Samples Pretreatment
Grinding: particle size units
21
Mesh is the number of openings in one inch of screen
If sample weight > 0.5 g – use 20 mesh sieve
If sample weight < 0.5 g – use 40 mesh sieve

22. 4. Sample
Preparation
Techniques
Dry Ashing
Wet Digestion
Microwave-assisted acid digestion
22

23. 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 muffle
oven 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 decreased
recovery of other constituents, mainly trace elements; ashing temperature higher
than 500º C may decrease recovery of Al, B, Cu, Fe, K, Mn.
After Miller, 1998
23

24. 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*6H2O
prior 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 the
sample weight) and deionized water to make a thin paste. Dry the mixture, digest at
500º C, dissolve ash with HNO3 or H2SO4 (not HCl !!!)
 The following acid mixtures may be used for ash dissolution: 300 mL HCl and
100 mL HNO3 in 1000 mL deionized water; Aqua Regia (concentrated HNO3 :HCL
1:3), HNO3 alone (less corrosive for metal parts of analytical instruments).
After Piper, 1950; Jones, 2001; personal experience
24

25. Sample Preparation Techniques
Wet Digestion
 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 four
acids: HNO3, HCl, H2SO4 and HClO4, with optional addition of H2O2. Digestion
is carried out in beakers on hot plate, in glass tubes on block, in open or closed
teflon vessels in microwave oven.
 Element determination: AAS, ICP-AES, UV-VIS ( P, S).
 Possible problems: HClO4 may react with organic material and result in an
explosion; in low Ca tissues CaSO4 may precipitate when H2SO4 is used;
contamination with B and Si when glass digestion tubes are used; contamination
with elements adsorbed by teflon.
After Piper, 1950; Jones, 2001; Miller,
1998; personal experience
25

26. Sample Preparation Techniques
Wet Digestion: Instruments
Digestion Block
(open digestion)
Microwave Laboratory
Ovens (closed digestion)
26
Ethos I,
Milestone,
Italy
Ultrawave,
Milestone,
Italy
Discover,
CEM, USA

27. Sample Preparation Techniques
Wet Digestion: 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 may contain Sn (tin) as a stabilizer. Do not use H2O2 if Sn is analyte.
 Wet digestion 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
27

28. Sample Preparation Techniques
Wet Digestion: Tips and Tricks
Internal standard technique
28

29. 5. Instrumentation
used in plant
analysisX-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.
29

30. 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/kg
for traces (heavy).
 Sample Preparation: drying, fine grinding and pressing.
 Advantages: simple sample preparation; low cost;
portable instrument.
 Disadvantages: spectral interferences; method is matrix-
dependent.
30

31. Instrumentation
AAS: Atomic Absorption Spectroscopy
 Principle: quantifies the absorption of ground state atoms in the
gaseous phase; the analyte concentration is determined by optics from
the 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 spectral
interferences; low-cost gases used (air+acetylene).
 Disadvantages: ionization enhancement of the signal for elements
easily ionized when operating in the absorption mode, especially Na and
K; matrix interferences caused by viscosity or specific gravity
differences between sample and reference standard; elements analyzed
one at a time.
31

32. 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 characteristic
wavelength 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; wide
dynamic range (0-100 mg/L); ideal for elements with low
excitation potential (Na and K)
 Disadvantages: only some elements may be determined;
elements analyzed one at a time.
32

33. Instrumentation
ICP-AES: Inductively Coupled Plasma Atomic Emission
Spectrometry
 Principle: electrons of excited atoms return to their ground-state and emit electromagnetic
radiation (light) at the wavelengths that are characteristic of the atoms that are excited. Argon
plasma is the source of excitation (about 10 000 K).
 Elements: all the elements except gases and some non-metals (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 linearity
of intensity versus concentration; multielement capabilities; rapid analysis; accurate and
precise analysis; detection limits equal to or better than AAS for many elements.
 Disadvantages: occurrence of spectral interferences; use of argon gas which can be
expensive; instrument is relatively expensive to purchase.
33

34. Instrumentation
UV-VIS Spectrophotometry
Principle
Instrument
Applications:
 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.
34
b

35. Instrumentation
Just a few words about….
Elemental Analyzer
Elements: C,H,N,S,O.
Digests finely grinded dry
samples.
Chloride Analyzer
Elements: Cl
Titrates Cl- with Ag2+.
Readout range: 10-999
mg Cl/L
Ion-selective Electrode
Elements: K+, Cl-, NO3-
Measures an electrical
potential on the ion
exchanger that is selective
to analyte ion.
35

36. Thank you for your attention!
36
Questions?