Plant Mineral

Analysis

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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

After Jones and Case, 1990

1. Introduction

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What do we analyze when

we are analyzing plants?

Essential elements (major elements

and micronutrients)

Toxic elements

Introduction

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Essential

Toxic

Major 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., 1984)

Introduction

4

The levels of major elements and micronutrients in mature

leaf tissue (after Munson R., 1997)

Introduction

5

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

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2. Analytical

Chemistry Basics

Qualitative and quantitative analysis

Calibration and matrix

Limit of Detection and Limit of Quantitation

Accuracy and Precision

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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

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ICP : ATOMIC EMISSION SPECTROMETRY

AS QUALITATIVE ANALYSIS

Analytical Chemistry Basics

Calibration Curve

The calibration curve is a plot of detector response as a function of concentration

(after Munson R., 1997)

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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

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

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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 12

3. Plant Samples

Pretreatment

Sampling Procedure

Decontamination

Drying

Grinding

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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

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Plant Samples Pretreatment

Sampling Procedure

What DO NOT sample?

After Jones B., 2003

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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

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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.

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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

Plant Samples Pretreatment

Grinding: particle size units

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4. Sample

Preparation

Techniques Dry Ashing

Wet Ashing, Microwave-assisted acid digestion

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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

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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 for metal parts of analytical instruments).

After Piper, 1950; Jones, 2001; personal experience 22

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 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 23

Sample Preparation Techniques

Wet Ashing: Instruments Digestion Block

Microwave Laboratory

Oven “Ethos 1”

Teflon Vessel with Tº and pressure control

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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

5. Instrumentation

used in plant

analysis 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

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.

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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.

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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.

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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 (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 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.

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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.

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b

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.

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Last but not least…

Thank you for your attention

First Law of Laboratory Work:

Hot glass looks exactly the same as cold glass

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