organic metal species in the speciation vs.trace analysis … · 2005-08-25 · m easur m nt (q ,to...

1

Hyphenated Techniques in Environmental Speciation Analysis

Joanna SZPUNAR

CNRS UMR 5034, Hélioparc,64053 Pau, FRANCEhttp://www.univ-pau.fr/GCABI

Paris

Pau

Centre of Excellence in Environmental Analysis and Monitoring (CEEAM)« New Horizons and Challenges in Environmental Analysis and Monitoring »

18- 29.08.2003, Gdansk, POLAND

Chemical speciesspecific form of an element defined as to nuclear composition, electronic or oxidation state, and/or complex or molecular structure

Speciation analysisanalytical activities of identifying and/or measuring the quantities of one or more individual chemical species in a sample

Speciation of an element; speciationdistribution of an element amongst defined chemical species in asystem

IUPAC guidelines for terms related to chemical speciation and fractionation of elements

D.M. Templeton, F. Ariese, R. Cornelis, L.G. Danielsson, H. Muntau, H.P. Van Leeuven, R. Lobinski Pure Appl. Chem., 2000, 72, 1453-1470

Organic metal species in the environment

MenSn(3-n)+, BunSn(3-n)+, PhnSn(3-n)+

Me2Hg, MeHg+

MenEtmPb(4-m-n)+

Alkylmetal contaminants and pollutants

Natural metabolites with a covalent carbon-metal(loid) bond (As, Se, Sb)

arsenobetaine, arsenosugarsselenoaminoacids, selenopeptides

Gene metabolites:metallothioneins (Cd, Cu, Zn)

Metalloenzymes (Zn, Mo, Co)

Carbohydrate and transport proteins (Pb, Sr, Ba, Ca, Mg)

Metal coordination complexes

Enzyme metabolites: phytochelatins, citrate, nicotianamine (Cd, Cu, Zn, Ni)

Speciation vs. trace analysis

Hg

Pb

Sn

Elemental trace analysis

Hg

PbSn

Total tin

Sn

Pb

Hg

Elemental speciation

2 4 6 8 10

MBT

TPT

DBTMPhT

TBTTeBT

TPhT

DPhT

SnEt4

0102030405060

Inte

nsit

y

min

Sign

al i

nten

sity

4 6 8 10 12

MBT

DBT

TBTTeBT (I.S.)

DPhTMPhT

Retention time, min

TPhT

Non-specific detection(FID)

Element-specific detection303.4 nm Sn-channel

Why atomic spectrometry?

Electrospray MSMALDI MS

Isotope intensitymeasurement (Q, TOF, SF)

Isotope ratio measurement

(TOF, MC)

Isotopedilutionanalysis

Tracer studies

FT ICR CID MS

(QqQ, QqTOF) Ion Trap MS

postsource decay

AffinityHPLC

Electrochromatography

Gel electrophoresis

SDS-PAGE

Isoelectric focusing

Size-exclusion

Ion-exchange

Reversed-phase

CZE

MEKCCEC

Separation ICP MS detection

Identification

2

Study of arsenic metabolism in marine life by multidimensional HPLC with the parallel ICP MS and electrospray MS detection

Case studies

Advances in analytical monitoring for environmental organometallic pollutants



Heavy metal resistance mechanisms in plants

Volatile organometall(oid) species in the environment

Mercury and organomercurialsmercury enters the environment as a result of natural geologicalactivity and man-made pollution (agricultural: fungicides and seed preservatives, industrial: caustic soda production, amalgamation, pulp and paper preservatives, pharmaceuticals, electrical instruments)highly toxic to the central nervous systemtoxicity is highly species dependent (organic > metalic > ionic)special concern: seafood

Lead and organoleadsthe most abundant heavy metal in natureenters the environment as a result of combustion of leaded gasoline, from storage batteries, anticorrosion products, pigments and paints, mining and smelting activitieshighly toxic - cumulative poison, accumulates in soft tissuesbioavailability depends on speciation

Tin and organotins

Preservatifs for timber and wood, TBT

PVC stabilizers (thermal and UV) DMT, DBT and DOcT

Catalysts in the production of foams, silicons and otherindustrial processes,MBT, DBT

Antifouling paints TBT, TPhT, TCyT

Fungicides in crop (potato, celery, coffee, rice) protection, TPhT, TCyT

Protection of textile, paper and leather, TBT

General formulae: RnSnX(4-n)

R=organic group (butyl-, octyl-, cyclohexyl-, phenyl-)X=halogen, hydroxide, acetate

Biological effects observed:

Shell malformation of oysters - the disturbance of the calcium metabolismImposex - in marine snails females develop male sexual characteristics

Regulations: France (1982), UK (1987), USA (1988), Canada (1989), Switzerlandand Germany (1990)

GC with atomic spectrometric detection in elemental speciation analysis

limited to volatile and thermostable analytesderivatization is often requireda run usually takes 15 - 20 min

high separation efficiency: capillary GC offers 100 000 TPshighly compatible with AAS, AFS, MIP AES and ICP MSinterface is relatively easy to develop and constructa versatile commercial instrument exists

Speciation of organolead in rain waterby GC - MIP AES

3

GC-ICP MS for environmental analysis

Inte

nsit

y (75

As,

126 X

e,) c

ps

0

1

2

3

4

5

6

7

0 50 100 150 200 250 300

0

1

2

3

Retention time, s

Intensity ( 13C), cps

x103 x105

13C

75As

Me3As

Et3As

Ph3As

Tolerance to matrix

MBT

TPrT

DBT

MPhT

TBT

DPhT

TPhT

0 2 4 6 8 10 12

Inte

nsit

y, c

ps

1.0

0.5

0

Retention time, min

x103

Sensitivity

B. Bouyssiere, J. Szpunar, R. Lobinski, GC with ICP MS detection, Spectrochim. Acta, 2002, 57B, 805-828

Isotope ratios

Retention time, s

208Pb

206Pb207Pb

204Pb

Microwave assisted extraction

focused microwave field

magnetronmicrowaveobturator

samplein solution

waveguide

condenserFrequency: 2450 MHzPower : 20 to 200 W

Microwave-assisted hydrolysis of biomaterials

microwave assistedNaOH hydrolysis

TMAH hydrolysis

HCl-MeOH leaching

TMAH hydrolysis

acetic acid leaching

enzymic hydrolysis

5 10 15 20 25Time of solubilization, h

1

Microwave assisted acetic acid leaching

Supercritical fluid extraction

Tropolone extraction(hexane-ethyl acetate)

Tropolone extraction (benzene)

Acetic acid leaching

0 50 100 150 200 250Quantitative TBT recovery, min

Microwave-assisted leaching from sediment

Speciation analysis of organotin in sediment by GC-MIP AED after microwave-assisted extraction

4 6 8 10 12 14

0

10

20

30

40

50

60

MBT

DBT

TBT

TeBT (I.S.)

DPhTTPhT

MPhT

Retention time, min

Sign

al in

tens

ity,

em

issi

on u

nits

Concentration, µg/gNRCC PACS-1

certified foundMBT 0.28 + 0.17 0.76 + 0.05DBT 1.16 + 0.18 1.01 + 0.06TBT 1.27 + 0.22 1.19 + 0.08BCR 462

certified foundMBT (12 - 244) 175 + 15DBT 128 + 16 122 + 6TBT 70 + 14 61 + 7

BP-5 column (30m x 0.32 mm i.d.)

J. Szpunar, V. Schmitt, J.L. Monod, R. Lobinski, JAAS, 1996, 11, 193 - 199.

Multicapillary GC with ICP MS detectionImproved efficiency:shorter column, faster separation

Increased flow rates (up to 200 ml min-1):improved compatibility with atomic spectrometry

Isothermal separation:simplified equipment

Retention time, s0

0.2

0.4

0.6

0.8

1.0x104

0

1

2

3

4

5x105

1

3

0 60 120

Resp

onse

(Hg ,

Sn)

, cp s Response (Pb), cps

2

4

5 6

7 8

9 10

11

1 - MeEtHg2,4,9,10 - unidentified 3 - Et2Hg5 - Et4Sn

6 - Et4Pb7 - BuSnEt38 - Bu2SnEt211 - Bu3SnEt

~ 3 mm

A. Wasik, I. Rodriguez, R. Lobinski, Spectrochim. Acta, 1998, 53B, 867 - 879.

Cooling chamber

Cooling gas (N2)

3-way valve

Cut-off valve

Splitter

Dewar with liquid nitrogenInside coil made of copper

T-pipe

Trap temperaturecontrol system

Valve temperaturecontrol system

Purge and dryinggas inlet

Drying gas outlet

Purg

e ga

s

Carrier gas inlet

Sampleinlet

Nafion drier

Drying gas

Waste

Purge vesselwith sample

Oven temperaturecontrol system

AutomatedSpeciation Analyser

1996-2000

Detector

0 10 20 30 40 50 60Retention time, s

0

2

6

MeHgEt

HgEt2

4

µg/g as Hg TORT-1 DORM-1Certified 0.13 + 0.01 0.73 + 0.06Found 0.12 + 0.003 0.74 + 0.05

Sign

al in

tens

ity,

cps

8x104

Determination of MeHg+ in biological CRMs by purge-and-trap MC GC - ICP MS

4

Monitoring speciation of environmental pollutants: trends

Simplicity:one-step sample preparation

Speed:microwave-assisted chemistry, multicapillary GC

Downsizing and automation:purge, trap, flash evaporation onto a microcolumn

Choosing the right marketing targets:mercury speciation in the environment


Case studies





O

HO OH

OOH

As

O

CH3

H3CR

RA SO3HB OHC OSO3HD OPO3HCH2CHOHCHOH

O

HO OH

As

O

CH3

H3CR

Mr R391 OCH2CHNH2CH2SO3H268 OCH3418 OCH2(CHOH)4CH2OH355 OCOCNHCH2COOH342 OCH2CHOHCOOH371 - adenine

Speciation of As in marine life

DMATMAs+

Arsenobetaine

Arsenocholine

(CH3)2AsO(OH)(CH3)4As+

(CH3)3As-CH2-COOH(CH3)3As-CH2CH2OH

MMA CH3AsO(OH)2

(AsIII) OH-As(OH)2(AsV) O=As(OH)3

variations of retention times as a function of column lifetime and history because of the matrix effect

unavailability of calibration standards for most of organoarsenicals in marine life

Anion-exchange HPLC - ICP MS of oyster tissue

HPLC pump

column

injector

ICP MS

HPLC eluent(0.7-1.2 ml/min)

Injector

Plasma gas(Ar)

Auxiliary gas (Ar)

Torch

Spray chamber

Cross-flow nebulizer

Nebulizer gas (Ar)

Drain

coil

As(III)

Arsenobetaine

DMAAs(V)

? ? ? ? ??

Need for identification!

0

0.5

1.0

1.5

2.0

2.5

0 5 10 15 20Retention time, min

Sign

al in

tens

ity,

cps

x104

1 23

4

5 67

8 9 10

ES ionization suppression by the matrixmultidimensional chromatographyuse of volatile buffers

Problems in ES MS for identification of organoarsenic compounds

Insufficient sensitivityuse latest generation of MS instruments

Identification of an arsenic peak (monoisotopic) in a complex mass spectrum

fragmentation of all the peaks (rapidity of the CID mass spectrum acquisition required) looking for As characteristic fragmentsthe use of high res instrument (reflectron TOF, FT ICR)

Identification of the compoundMS/MS and ion trap MSn

5

Effect of sample preparation on the quality of ES mass spectra

High resolution electrospray MS using a time-of-flight mass analyser

Parameters of mass analysisresolution > 10 000mass accuracy below 10 ppm

Correction of the initial energy spread by:time delayed extraction of ionsthe use of an ion reflectron

DetectoreV = Ec = 0.5 mv2

Field-free drift region+++

+++++

+++ ++

V++

++ +++

+

+

Acceleration region

+25 kV 0 kV

Precise molecular mass determination of organoarsenic species by reflectron

TOF MS

328 330 3320123

329.

0460

331.1

342

330.

0488

x104

100 150 200 250 300 350 400 450 5000

1

2 114.

0383

185.

0934

135.

0241

162.

0912

121.0

850

329.

0460

190.

1213

236.

0441

86.0

566

351.0

181

309.

1454

420.

1260

458.

0840

x105

442.

1116

Mr theoretical: 329.0575Mr experimental: 329.0460Error: +0.015 (35 ppm):

C10H21O7As

the current is converted by FT into orbital frequencies of the ions which correspond to their mass-to-charge ratios

time

10 msec

Parameters of mass analysis:resolution > 200 000mass accuracy below 0.2 ppm

4000 3000 2000 1000

the cyclotron motion of ions is excited by a RF field to generate a time dependent current

Principle of FT ICR MS

ions entering a chamber are trapped by a powerful magnetic field in circular orbits with a frequency independent of their velocity

Determination of empiric formula of arsenic species via accurate Mrdetermination by FT ICR MS

C10H22O7As1

Courtesy of S. Pergantis, University of Creta

Ion of interest

experimental mass: 329.05767theoretical mass: 329.05760

error : + 0.00007 (0.22 ppm)

Structural characterization by tandem MS

Tandem-in-space MS

QCollision cell

Massseparation

Detector

Massseparation

Fragmentation

Pusher

QqTOF

QqQTandem-in-time MS

Accumulation Nonprecursor ions ejected

Collision Product ionsejected

and detected

Ion trap and FT ICR

6

MS/MSFragment

Masscalculated

Massexperimental

Differenceppm

329.0575 329.038 -59.57237 237.0104 236.989 -96.46195 194.9999 194.986 -73.3597 97.0142 97.010 -45.36

average -68.68

O

OH OH

O OH

OHAs

O

CH3

CH3

237

+H+

329

195165

165

195237 329

100 150 200 250 300

7

5

3

1

x104

97

Inte

nsit

y, c

ps QqQ

80 100 120 140 160 180 200 220 240 260 280 300 320 3400

2

4

6

8 97.0

098

329.

0379

236.

9828

194.

9739

164.

9624

x103 QqTOF

Structural characterization of organo-arsenic compounds by tandem-in-space MS

Collisionally induced dissociation (CID) tandem mass spectrum of m/z 329.05767obtained using sustained off-resonance

irradiation (SORI)

O

OH OH

O OH

OHAs

O

CH3

CH3

237

+H+

329

195165

Comprehensive characterization of an oyster tissue candidate CRM by multidimensional chromatography

A B

Size-exclusion

0

2

4

6

8

0 20 40 60 80

x104

No. Fractions

Sign

al I

nten

sity

/cps

Anion-exchange

0

2

4

6

0 100 200 300 400 500B1

B2

B4

x105

0

0.5

1.0

1.5

2.0

0 100 200 300 400 500

A1

A2

A3x10 5

B3

0 5 10 15 20 25 30

A1.1

A1.2

A1.3

A1.4A1.5

A1.6

012345 x103

Retention time/mins

Cation-exchange

00.5

1.01.52.02.5 x104

0 5 10 15

B1.1

B1.2

0 5 10 15 20 25 30 35

x105

0

1

2

3

4

5

AsC+TMAs+DMAsF

As-sug B

AsB

DMA

As-sug D

AsVAsBCH2

Retention time/mins

Sign

al I

nten

sity

/cps

Anion-exchange HPLC ICP MS of oyster tissue

AsB

DMA sug B

sug D

AsV

cations

AsBCH2

AsC+

TMAs+DMAsF

unknown


Case studies





Resistance mechanisms in plants exposed to heavy metal stress

Avoidance - preventing toxic ions to reach their target sites- reduction and volatilization into the atmosphere (e.g. Se, Hg)- formation of insoluble sulfites, carbonates, phosphates- bioinduction of specific ligands (e.g. phytochelatins)- complexation with ligands already present in the organism

Tolerance to metal ions that entered the organism

Characteristics of PC:General structure: (γ-Glu-Cys)n-Gly (n=2-11)Heavy-metal-complexing peptidesInduction by a large variety of metals (Cd, Pb, Zn...)Occurrence of iso-phytochelatins in plants Example:

[Cd3(PC3)4] complex

7

Sign

al in

tens

ity ,

cps

0 5 10 15 20 25 30Time, min

0,4

0,8

1,2

1,6x 105

standards

PC4

PC3

PC2

sample

Speciation of cadmium in soybean (Glycine Max) extract CZE - ES MS of Glycine Max extract

x 107

Migration time, min5 10 15 20 25

Inte

nsit

y, c

ps

2

4

6

8

Capillary:95 cm x 75 µm (i.d) x 150 µm (o.d)Electrolyte:acetate buffer 5 mM, pH 4Make-up flow rate: 2 µl/minMake-up composition:60% acetic acid 0.1M + 30% MeOH + 10 % H2OVoltage: 25 kV (current: 7 µA)Pressure: 0.4 psiInjection: 5 psi / 4 s

CZE-ES MS of soya extractx 107

Migration time, min5 10 15 20 25

Inte

nsit

y, c

ps

2

4

6

8

desGly PC2

iso PC2 (βAla)

desGly PC3

iso PC3 (βAla)

iso PC4 (βAla)

PC3PC4

15.36 min554.1

1

3x10 6

iso PC2(βAla)

x 106

17.11 min 786.3

400 500 600 700 800 900 1000m/z, u

0.5

1.5

iso PC3 (βAla)

12.64 min 772.1

1

3x 105

PC3

Peptide sequencing by electrospray MS/MS

Detection

Q1

Collision cellpressurized

Q3Q2

Massseparation

MassseparationFragmentation

K. Biemann, Ann. Rev. Biochem., 61, 977-1010 (1992)

Identification of the signal at 786 u by ESI MS/MS

(γGlu-Cys)3 (βAla)

iso-PC3 (βAla)

0 100 200 300 400 500 600 700 800m/z

Sign

al in

tens

ity

(TIC

)

786

657

554

425

322

193

768697594

465

362

233

γGluγGluγGlu

γGluγGlu

CysCys

CysCys βAla

1

2

3

4

x 104

y fragments

b fragments

Phytochelatins (PCs) (γGlu-Cys)n-Gly (n = 2 - 11)

(γ-Glu-Cys)nGly

PCn iso-PCn(β-Ala) des-GlyPCn iso-PCn (Ser) iso-PCn(Glu) iso-PCn (Gln)

(γ-Glu-Cys)n

Samples studied:rice (Oryza sativa)soya (Glycine max)maize (varieties: zwerge, boss, consul, ganz, cluth)horseradish

(γ-Glu-Cys)n β-Ala (γ-Glu-Cys)n (γ-Glu-Cys)nSer (γ-Glu-Cys)nGlu (γ-Glu-Cys)nGln

8

ConclusionsGC - ICP MS and HPLC - ICP MS appear as routine analytical techniques for environmental speciation analysis provided that target analytes are well defined

ICP MS is a valuable tool for HPLC screening biological extracts for unknown species and monitoring the efficiency of multidimensional purification protocols

ES MS appears as the best tool for the identification on unknownspecies but its successful use is still delicate at the sub-ppm levels because of the matrix effects

The mass accuracy of reflectron TOF MS and FT ICR offers the possibility of empiric formula determination which is the first step towards the indentification of unknown species

Q TOF, triple quad or ion trap MSn deliver vital structural informationthat cannot be obtained by any other method.

Paris

Pau

http://www.univ-pau.fr/GCABI

Ph.D. Students:Guillaume Ballihaut Pierre GustiLaurent Ouerdane Aleksandra PolatajkoRafal Ruzik

Permanents:Brice BouyssiereRyszard LobinskiFlorence PannierMartine Potin-GautierDirk SchaumlöffelJoanna SzpunarVeronique Vacchina

Post-docs:Oscar Bonilla PalaciosJorge Ruiz Encinar

Visiting Ph.D. Students:Vanesa Diaz Huerta

Lusi Fernandez SanchezKatarzyna Krupczynska

Tomasz Welerowicz

M.Sc. Students:Barbara Banas

Anna StopikowskaFormer co-workers:

Shona McSheehyIsaac Rodriguez

Corinne CasiotHubert Chassaigne

Aleksei MakarovAndrzej Wasik

Pawel PohlSandra Mounicou

Magda Sliwka-KaszynskaKasia Polec

organic metal species in the speciation vs.trace analysis … · 2005-08-25 · m easur m nt (q ,to...

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