department of agriculture and ecology

U N I V E R S I T Y O F C O P E N H A G E N

Recent developments in plant tissue analysis

Kristian Holst Laursen

Department of Agriculture and Ecology


Plant tissue analysisy

C• Plant physiology

C

OS

MoB

Zn

Cu

M

Cl

?

p y gy– Nutrient functionality– Nutritional status

• Environmental science

H

P

N

K

Mg

NiMn

CaFe

? Environmental science– Toxicology screening

• Molecular biologyGenetically modified plants– Genetically modified plants

• Food quality– Nutritional value

A th ti it t ti– Authenticity testing


Plant tissue analysis in the 18th century

Dry ashing

G

y g

Ti i GravimetryTitrimetry

N K C P

Drawing by Carl Friedrich Wilhelm Trautshold, 1842

Na, K, Ca, P


Plant tissue analysis anno 2009Plant tissue analysis anno 2009

Single-element analysis Multi-elemental analysis

• Test kits (N, P, K), colorimetric or titrimetric

• Neutron activation

• X-ray fluorescence• Spectrophotometry

• Enzyme assays (Fe, Cu)

ay uo esce ce

• LIBS

IR MS (N C S H O)• Spectroscopy

– Fluorescence (Mn)– Near infrared reflectance (N)

Atomic absorption

• IR-MS (N, C, S, H, O)

• ICP spectrometry/spectroscopy– ICP-OES/AES

– Atomic absorption – ICP-MS– LA-ICP-MS– MC-ICP-MS– HR-ICP-MS


Inductively Coupled Plasma-Mass Spectrometry

• Multi-elemental (>70 elements)• Large dynamic range (6-7 orders of

magnitude)magnitude)• High sensitivity (ppt range)• Labour consuming• Expensive

Nebulization Desolvation Vaporization Atomization Ionization Mass analysis

Liquidor

solid sample

Aerosol Particle Molecule Atom Ion Mass spectrum

Nebulizer/Laser Plasma Mass Spectrometer

Adjusted from: ICP-MS, Inductively Coupled Plasma Mass Spectrometry, A primer, Agilent Technologies


ICP-MS based plant tissue analysis

Sampling

Decontamination

Dryingy g

Homogenization

Digestion

ICP

Data Handling

Interpretation


Quantification methods IxQuantification methods

Accuracy >90%Several elementsTime consuming100%

IsotopeAccuracy ~70%Many elements

Acc

urac

y

Isotope Dilution

yFastFull-Q

Semi-Q

Periodic table

Cx

15-20 elements in 3-4 min

A

0%

Periodic table

H

He

Li

Be B

C

N

O

F

Ne

Na

Mg Al

Si

P

S

Cl

Ar

K

Ca

Sc

Ti

V

Cr

Mn

Fe

Co

Ni

Cu

Zn

Ga

Ge

As

Se

Br

Kr

Rb Sr Y Zr Nb Mo Tc Ru Rh Pd Ag Cd In Sn Sb Te I XeRb

Sr

Y

Zr

Nb

Mo

Tc

Ru

Rh

Pd

Ag

Cd

In

Sn

Sb

Te

I

Xe

Cs

Ba

La

Hf

Ta

W

Re

Os

Ir

Pt

Au

Hg

Tl

Pb

Bi

Po

At

Rn

Fr

Ra

Ac

Rf

Db

Sg

Bh

Hs

Mt

Uun

Uuu

Uub

Lanthanider Ce

Pr

Nd

Pm

Sm

Eu

Gd

Tb

Dy

Ho

Er

Tm

Yb

Lu

Actinider Th

Pa

U

Np

Pu

Am

Cm

Bk

Cf

Es

Fm

Md

No

Lr


Principle of semi-quantitative analysisPrinciple of semi quantitative analysis

I

I ix

iy

x

yy

EE

yxfRF

IC

).(

R f t

Periodic table

>70 elements in 60 seconds

30RFx = Ix/Cx

Response factorH

He

Li

Be B

C

N

O

F

Ne

Na

Mg Al

Si

P

S

Cl

Ar

K Ca Sc Ti V Cr Mn Fe Co Ni Cu Zn Ga Ge As Se Br Kr

on p

oten

tial (

eV)

15

20

25

H

He

NO

F

Ne

Ar

Kr

C

x x x Rb

Sr

Y

Zr

Nb

Mo

Tc

Ru

Rh

Pd

Ag

Cd

In

Sn

Sb

Te

I

Xe

Cs

Ba

La

Hf

Ta

W

Re

Os

Ir

Pt

Au

Hg

Tl

Pb

Bi

Po

At

Rn

Fr

Ra

Ac

Rf

Db

Sg

Bh

Hs

Mt

Uun

Uuu

Uub

1'st

ioni

zatio

0

5

10

H

Li

BeB

C

O

Na

Mg

Al

Si

PS

Cl

K

CaScTiVCrMnFeCoNiCu

Zn

Ga

Ge

AsSe

Br

Rb

SrYZrNbMoTcRuRh

PdAg

Cd

In

SnSbTe

I

Xe

CsBaLaCePrNdPmSmEuGdTbDyHoErTmYbLuHf

TaWReOsIrPtAu

Hg

TlPbBi

PoAtRn

Fr

RaAcThPaUNpPuAmCmBkCfEsFmMdNo

Lanthanider Ce

Pr

Nd

Pm

Sm

Eu

Gd

Tb

Dy

Ho

Er

Tm

Yb

Lu

Actinider Th

Pa

U

Np

Pu

Am

Cm

Bk

Cf

Es

Fm

Md

No

Lr

Atomic number

0 20 40 60 80 100 1200


10000 AK

S

10000 BApple leaves Durum wheat

titat

ive

(g

g-1 D

M)

100

1000

P

Al

B

Fe

Zn

Mg

Mn

Ca

S

titat

ive (

g g-1

DM

)

100

1000

FeK

Mg

Mn

Sem

i-qua

nt

1

10 Cu

NiNa

Sem

i-qua

n

1

10 CuAlBa

Zn

Sr

SeMo

FeC 10000 D

Full-quantitative (g g-1 DM)

0.1 1 10 100 1000 100000.1


0.1 1 10 100 1000 100000.1

LeekCabbage

ve (

g g-1

DM

)

100

Mo B

Ni

Fe

Zn

ve (

g g-1

DM

)100

1000

10000

Fe

DP

Ca

Sr

LeekCabbage

Sem

i-qua

ntita

tiv

10

Cd

Cu

BaSe

mi-q

uant

itativ

1

10 CuZn

MnSr

Cd

Cr


1 10 1001


0.1 1 10 100 1000 100000.1

MoCd


Equimolar std (4 μg g-1 dry matter)(

g g-1

DM

)

1000

10000

P

KMg

Ca

Non-equimolar standard

Sem

i-qua

ntita

tive

10

100

P

Cu

Fe

Zn

Mn

Ref. mat. StdK 16100 24000

Ca 15260 12000


1 10 100 1000 100001

Mg 2710 2400

P 1590 2400

Fe 83 60

M 54 4810000Ca

Mn 54 48

Zn 13 24

Cu 6 6

ve (

g g-1

DM

)

1000

10000

P

KMg

#Concentration in μg g-1 dry matter

Sem

i-qua

ntita

tiv

10

100 Fe

Zn Mn

#Concentration in μg g dry matter


1 10 100 1000 100001

Cu


-MagnesiumDiagnosing nutrient -Magnesiumdeficiencies

-CopperCopper

-Manganese


M i C M-Magnesium -Copper -Manganese

2500Full-Q

16Full-Q

70Full-Q

dry

mat

ter) 2000

Semi-Q

dry

mat

ter)

10

12

14 Semi-Q

dry

mat

ter)

50

60Semi-Q

tratio

n (

g g-1

d

1000

1500

tratio

n (

g g-1

d

6

8

10

ntra

tion

(g

g-1 d

30

40

Con

cen

500 Con

cen

2

4

Con

cen

10

20

Control -Mg0

Control -Cu0

Control -Mn0


Multi-elemental fingerprinting of plant tissueMulti elemental fingerprinting of plant tissueMass scan 7Li – 238U

Background subtracted spectrumg p

1e+9

1e+10

MgK

ount

s s-1

)

1e+6

1e+7

1e+8Mg

P

S

Cl

Mn

FeCu

Zn

n in

tens

ity (c

o

1e+3

1e+4

1e+5B

CaFe

Ni

Mo

Ion

1e+0

1e+1

1e+2

Mass (m/z)

0 50 100 150 200 2501e-1


Multi-elemental fingerprinting of rice by semi-tit ti ICP MS d h t iquantitative ICP-MS and chemometrics

• Discrimination of 3 lowland rice genotypes2 high quality genotypes and 1 traditional genotype– 2 high quality genotypes and 1 traditional genotype

– No visible differences in the harvested product– Grain yield, aromatic and nutritional difference

Periodic table

H

He

Li

Be B

C

N

O

F

Ne

Na

Mg Al

Si

P

S

Cl

Ar

K Ca Sc Ti V Cr Mn Fe Co Ni Cu Zn Ga Ge As Se Br KrK

Ca

Sc

Ti

V

Cr

Mn

Fe

Co

Ni

Cu

Zn

Ga

Ge

As

Se

Br

Kr

Rb

Sr

Y

Zr

Nb

Mo

Tc

Ru

Rh

Pd

Ag

Cd

In

Sn

Sb

Te

I

Xe

Cs

Ba

La

Hf

Ta

W

Re

Os

Ir

Pt

Au

Hg

Tl

Pb

Bi

Po

At

Rn

Fr

Ra

Ac

Rf

Db

Sg

Bh

Hs

Mt

Uun

Uuu

Uub

Lanthanider Ce

Pr

Nd

Pm

Sm

Eu

Gd

Tb

Dy

Ho

Er

Tm

Yb

Lu

Actinider Th

Pa

U

Np

Pu

Am

Cm

Bk

Cf

Es

Fm

Md

No

Lr


Multi-elemental fingerprinting of rice by semi-tit ti ICP MS d h t iquantitative ICP-MS and chemometrics

M)

1000

10000

Genotype AGenotype BGenotype C

A

entra

tion

g g-1

DM

1

10

100

Genotype C

10000

Tiss

ue c

onc

0 0001

0.001

0.01

0.1High quality rice Traditional riceFull-Q

n

g g-1

DM

)

10

100

1000

100000.0001B

Tiss

ue c

once

ntra

tion

0.01

0.1

1

Semi-Q

Mass (m/z)

0 20 40 60 80 100 120 140 160 180 200 220

T

0.0001

0.001


Multi-elemental fingerprinting of rice by semi-tit ti ICP MS d h t i

4

6Genotype AGenotype BGenotype C

A

quantitative ICP-MS and chemometrics0.4

B

PC2

-2

0

2

PC2

-0.2

0.0

0.2

MP

S

Ca

Fe ZnSr

Mo

PC1

-6 -4 -2 0 2 4 6-6

-4

PC1

-0.2 0.0 0.2 0.4

-0.4

MgKVMn

Ni

Cu

SeCd

2

4

6A

0.2

0.4B Mg

PS

Ti

Co As

Se RbSn

Cs

B

PC

2

-4

-2

0

Genotype AGenotype B

PC

2

-0.2

0.0Cl

KCa

Sc

VCr

Mn FeCu

Zn

GaBr

Se Rb

Sr

MoCd

In Sn

BaHg

Pb

PC1

-6 -4 -2 0 2 4 6-6

Genotype BGenotype C

Laursen et al. 2009, Journal of Analytical Atomic SpectrometryPC1

-0.3 -0.2 -0.1 0.0 0.1 0.2 0.3-0.4


High-throughput digestion

HNO3 and H2O2

24 hours

6G t AA B

24 hours

2

4

Genotype AGenotype BGenotype C

0.2

0.4 Na

Cl Ca

Fe

ZnAs

Br

Rb

Sr

Cs

B

PC2

-2

0

PC2

-0.2

0.0

Mg

Al

PS

K

Ca

TiCr

Mn

Co

Ni

ZnsRb

ZrMo

In

Sn

Dy

HgPb

Bi

PC1

-8 -6 -4 -2 0 2 4 6 8-6

-4

PC1

-0.4 -0.2 0.0 0.2 0.4

-0.4

MnCu

Laursen et al. 2009, Journal of Analytical Atomic Spectrometry


Full- or semi-quantitative ICP-MS analysis

Full-quantitative Semi-quantitative

q y

Full quantitative Semi quantitative

Elemental coverage + ++

Sample screening ++

Sample comparison + ++

High accuracy ++ +

Authenticity + ++Authenticity

High-throughput ++


Thank you for your attention!

Acknowledgements S H t dSøren HustedThomas H. Hansen Daniel P. PerssonJan K. SchjoerringSid l Bi k l d S h idtSidsel Birkelund SchmidtBente BroengBente Postvang

department of agriculture and ecology

Documents