Roberto MORABITOENEA - PROT

morabito@casaccia.enea.it

FONTI DI ERRORE NELL’ANALISI DI SPECIAZIONE

ISA 2002Le Spettroscopie nel Controllo Ambientale

Val Masino 29 aprile – 1 maggio 2002

IUPAC DEFINITIONS« Speciation »

(Chemical) Species: Specific chemical form of an element, defined according to its molecular,

complex, electronic or nuclear structure

Speciation analysis: Measurement of the amountof one or several chemical individual species

in a sample

Speciation of an element: Distributionof chemical species of an element in a system

SPECIATION

SPECIATION

CHEMICAL SPECIES

OXIDAT. STATES

Mainly: As, Cr, Sb, Se

LOW MOLECULAR WEIGHT

Mainly: Hg, Pb, Sn, (As, Se)

MACROMOLECULES

e.g.: PORPHYRINES,THIONEINES, etc.

* EXTRACTABLE

* DIALIZABLE

* OTHERSMainly: Zn, Cd, Cu, Pb

Ni, Mn, Fe, Cr

OPERATIONALLY

DEFINED

SPECIATION

ASSOCIATION

FORMS

ORGANOMETALLIC

COMPOUNDS

DETERMINATION OF CHEMICALFORMS OF ELEMENTS

Critical steps

Sampling,preservation(representative

sample)

Pre-treatment,conditioning

(storage, drying,homogenisation)

Chemicaltreatment

(extraction,derivatisation,

purification, etc.)

Separation(chromatography)

Detection,calibration

(detector, calibrants)

UNCERTAINITY OF ENVIRONMENTAL MEASUREMENTS

UT = (U2Sampling + U2

Storage + U2Treatment + U2

Analysis + …)1/2

SAMPLING STRATEGIES

CHOICE OF SAMPLES

COLLECTION OF SAMPLES

NUMBER OF SAMPLES

SPATIA L VARIABILITY

TEMPORAL VARIABILITY

Filtered water - Particulate matter Undisturbed surface sediments Biota

Sampling devices Sampling vessels

Representativeness of sampling point Representativeness of sampling area

Microlayer Interface water-sediment Vertical stratification Enclosed and flushed areas Dockyard activities

Tides - Seasons

Dep

t h

Typical Tributyltin concentration profile in the sediments

Resuspension

Reduced direct inputs

Degassing

SAMPLE STORAGEAdsorption on the container wallsContamination from the container wallsStability

SAMPLE TREATMENTContaminationLossesChanges in speciationLow extraction efficiencyLow derivatization yields

SAMPLE ANALYSISInterferentsMatrix effectsCalibrationBlanksAnalytical staff expertise

SOURCES OF ERRORS

WATER SAMPLESTemperaturepHType of containers

BIOLOGICAL AND SEDIMENT SAMPLESTemperatureHumidityType of containers

PARAMETERS CONTROLLING THE STABILITYOF CHEMICAL SPECIES DURING STORAGE

SELECTION OF METHODSFOR SPECIATION ANALYSIS

Stabilisation / Storage (examples)«  Preserver the original speciation »

STABILISATION- Acidification (waters) -

- Lyophilisation (biota, sediments, etc.) -- Oven-drying (sediments) -

- Pasteurisation (sediments) -- Gamma-irradiation (biota, sediments,waters) -

STORAGE

- Storage at ambient temperature for MeHg -- Storage at 4°C for trimethyllead -

- Freezing recommended for butyl- and phenyl-tins (biota, sediments) -

SELECTION OF METHODSFOR SPECIATION ANALYSIS

Extraction (examples)

ACIDSAcetic acid (butyltins)

Sulphuric acid, distillation (MeHg)Nitric acid/micro-wave (MeHg)

MIXTURE ACID/ORGANIC SOLVENTHydrochloric acid / toluene (MeHg, butyltins)

Hydrochloric acid / methanol (butyltins) Sulphuric acid / toluene (MeHg)

ORGANIC SOLVENTSToluene (MeHg)

Methanol / hexane (butyltins)Tropolone / pentane (butyltins)

OTHERSCO2 / methanol, supercritical fluid (MeHg)

CO2 / tropolone, supercritical fluid (butyltins) Protease / lipase (butyltins)TMAH (butyltins, IAL, TAL))

COMPARISON OF 12 SELECTED EXTRACTION METHODS FOR THE DETERMINATION OF ORGANOTINS IN BIOLOGICAL SAMPLES

•SAME SAMPLE (BCR 477)

•SAME OPERATOR

•SAME ANALYTICAL STEPS AFTER EXTRACTION

THE PERFORMANCE OF THE OVERALL METHOD CAN BE STRICTLY RELATED TO THE EFFICIENCY

OF THE EXTRACTION METHOD APPLIED

General conditions adopted in the considered extraction

methods

Methods Extracting solvent

Complexing agent

Acid Extraction technique

1 Methanol Tropolone HCl Ultrasonically shaken 2 Methanol Tropolone - Ultrasonically shaken 3 Methanol - - Ultrasonically shaken 4 Hexane Tropolone HCl Ultrasonically shaken 5 Toluene Tropolone - Ultrasonically shaken 6 Dichloromethane Tropolone HBr Mechanically shaken 7 Pentane Tropolone HBr Mechanically shaken 8 Hexane Tropolone - Refluxed 9 Dichloromethane Tropolone - Refluxed 10 Hexane - - Soxhlet extraction 11 Methanol - HCl Microwave oven 12 Methanol - HAcO Microwave oven

SELECTION OF METHODSFOR SPECIATION ANALYSIS

Derivatisation (examples)« Transformation of a given compound

to enable a better separation »

HYDRIDE GENERATION (NaBH4)RnSn(4-n)+ -------- RnSnH(4-n) + H2

with n = 1,2,3 (R= methyl, ethyl or butyl)ETHYLATION (NaBEt4)

PROPYLATION (NaBPr4)PHENYLATION (NaBPh4)

GRIGNARD REACTIONS- Methylation - Ethylation

- Propylation- Butylation- Pentylation- Hexylation

TBT DBT MBT MPhT DPhT TPhT0

20

40

60

80

100

120 %

Volatilization lossesEvaporation down to dryness

Hexyl

Pentyl

Propyl

Ethyl

Methyl

TBT DBT MBT MPhT DPhT TPhT0

20

40

60

80

100

120

%

Volatilization lossesEvaporation down to 0.3 mL

Hexyl

Pentyl

Propyl

Ethyl

Methyl

SELECTION OF METHODSFOR SPECIATION ANALYSIS

Separation (examples)« necessary step, considering that the determination of different

chemical species, in general, cannot be performed with a sufficient selectivity (e.g. selective detection by AAS) »

GAS CHROMATOGRAPHY- Packed column -

- Capillary column -

HIGH PERFORMANCELIQUID CHROMATOGRAPHY (HPLC)

OTHERS- Cryoegenic trapping (cold trap) -

- Capillary electrophoresis -- Ion exchange microcolumns -

SELECTION OF METHODSFOR SPECIATION ANALYSIS

Detection (examples)

ELEMENT SPECIFIC- Atomic absorption spectrometry -

(electrothermal, flame, quartz furnace)

MULTI-ELEMENT- Inductively coupled plasma atomic emission spectrometry (ICP-AES) -

- Inductively coupled plasma mass spectrometry (ICP-MS) -

SPECIFIC OF ONE GIVEN SPECIES- Voltammetry -

NON SPECIFIC- Flame ionisation detection (FID) -

- Flame photometric detection (FPD) -- Electron capture detection (ECD) -

CALIBRATION- Stoichiometry, purity, primary and internal standards, calibration method (e.g. standard additions)

Analitycal methods for the determination of mercury compounds in environmental samples

SEPARATIONAND

DETECTIONCHROMATOGRAPHICNON-CHROMATOGRAPHIC

GC•Packed column•Capillary column

HPLC

•Selective liquid-liquid extraction •Selective reduction by SnCl2.

CV-AAS

ECD (only for GC separation)MIP-AES, ICP-AESCV-AASICP-MS(MS)

Analitycal methods used for the determination of mercury compounds in

environmental samples

EXTRACTIONAcid digestion and extraction in toluene

CLEAN UPBack extraction in aqueous cysteine solution

DERIVATIZATIONAqueous derivatization with NaBPh4

GC-MS DETERMINATION

GC-MS characterization of phenylic derivatives of mercury compounds and instrumental parameters

MeHgPhEtHgPh

PhHgPh

ORGANOTIN ANALYSIS BY:

GC-FPD GC-MS

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.000

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TIC: 2107C03A.D

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Abundance

TIC: 0901009.D

TPrT

TBT

DBT

MBT

DPhT

MPhT

TPhT

Inorganic Sn

DETERMINATION OF CHEMICALFORMS OF ELEMENTS

Sources of errorsCompromise: To preserve the original « speciation »

while permitting the analysis

• Sampling: contamination, losses, alteration of speciation• Storage: contamination, losses, stability of species• Extraction: contamination, incomplete recovery, degradation of chemical species• Derivatisation: inhibition, degradation of chemical species• Separation: peak overlap, degradation• Detection: interferences• Calibration: stability of species in solution, purity

MEASUREMENT QUALITY

CONTROL

GLP

SOP’s

Staff Training

Instrument Control

Calibrants and Blank Control

MEASUREMENT QUALITY

ASSESSMENT

Independent methods

Spiking experiments

Reference materials

Interlaboratory studies

Statistic analysis

TOOLS FOR QUALITY ASSURANCE

EUROPEAN PROJECTSFOR SPECIATION ANALYSIS

ORGANOTIN COMPOUNDSIN THE ENVIRONMENT

(1988-2002)

JUSTIFICATIONToxicity of tributyltin and triphenyltin to

marine biota (fish larvae, molluscs)EVALUATION OF THE STATE-OF-THE-ART

DEVELOPMENT OF NEW METHODSPRODUCTION OF CRMS

PARTICIPANTSCo-ordinations by the University of Pau, the University of

Plymouth, ENEA and the Free University of AmsterdamCollaborations with the Joint Research Centres (Ispra and Geel) and the CID-CSIC (Barcelona)

23 laboratories from 9 EU countriesTwo bursaries

INTERLABORATORY STUDIES

Improvement schemes(method performance studies)

The principle followed is similar to a method validation, i.e.analyses of increasingly complex

matrices to detect possible errors atthe different analytical steps.

These schemes have a pedagogic goal andinvolve technical meetings in the frame of

which participants may exchange theirexperience and discuss weak points of

their method(s) as well as source(s) of error(s).

S1

S2

M1

M2

Simple solutions(pure substances

or mixtures of substances)

Solutions simulating the matrixor extracts (spiked or not)

« Matrix » reference materialsimilar to the sample,spiked with a known

amount of the analyte

« Matrix » reference materialwith a composition close

to the sample composition

DETECTIONDERIVATISATION

CLEAN-UPSEPARATION

EXTRACTIONMINERALISATION

OVERALLMETHOD

Test material – TBT spiked sedimentCoordination BCR

Preparation JRC of Ispra (IT)

Collection Lake Maggiore (IT)

Drying Air drying then oven drying (120 °C)

Sieving 90 m mesh size

Sterilization Not performed

Homogeneization mixer

Storage Brown glass bottles +4 °C

Humidity < 4%

Certified species None

Participation 19 laboratories from 7 Countries

RM 424Coordination University of Plymouth (UK)

Preparation University of Pau (F) and IRMM (B)

Collection Sado estuary (PT)

Drying Air drying then oven drying (80 °C)

Sieving 75 m mesh size

Sterilization 120 °C for 1h

Homogeneization mixer

Storage Brown glass bottles +4 °C

Humidity < 3%

Certified species None

Certification 18 laboratories from 8 Countries

BCR 477Coordination ENEA (IT)

Preparation ENEA (IT) and JRC of Ispra (IT)

Collection La Spezia harbour (Italy)

Drying Freeze drying

Sieving 125 m mesh size

Sterilization Not performed

Homogeneization Mixer (15 days)

Storage Brown glass bottles –20 °C

Humidity < 5%

Certified species TBT, DBT, MBT

Certification 16 laboratories from 7 Countries

BCR 477 - TBT, DBT AND MBT IN MUSSEL TISSUE

Compound

Certified

value

Uncertainity

Unit

Set of

accepted results

TBT

DBT

MBT

2.20

1.54

1.50

0.19

0.12

0.27

g/g

g/g

g/g

18

15

8

BCR 710Coordination ENEA (IT)

Preparation JRC of Ispra (IT) and CNRS (FR)

Collection Arcachon Bay (France)

Drying Freeze drying

Sieving 125 m mesh size

Sterilization Not performed

Homogeneization Mixer (15 days)

Storage Brown glass bottles –20 °C

Humidity < 5%

Certified species TBT, DBT, CH3Hg, AsBet

Certification 23 laboratories from 11 Countries

BCR 710 – As, Hg and Sn species in oyster tissue

Compound Certified value

Standard deviation

Unit Set of accepted results

TBT 133 25 g/kg 10

DBT 82 17 g/kg 9

AsBet 33 5 mg/kg 8

CH3Hg 115 8 g/kg 12

EUROPEAN PROJECTSFOR SPECIATION ANALYSIS

ORGANOTIN COMPOUNDSIN THE ENVIRONMENT

(1988-2001)

WORK PROGRAMME• Two trials (solutions, spiked sediment): 1988-90

• Two coastal sediment certifications: 1990-93• Certification of a mussel CRM: 1994-97

• Certification of a freshwater sediment CRM: 1997-99• Certification of an oyster CRM: 1999-2001

RESULTS• Detailed studies of derivatisation methods

• Systematic studies of extraction recoveries• Development of methods (SFE, HPLC-ID-ICPMS)• One non-certified (harbour) sediment (RM 424)

• Two sediment CRMs (BCR 462 - BCR 646)• One mussel CRM (BCR 477)• One oyster CRM (BCR 710)

MTBT (sediment)in µg/kg

GC-FPD

GC-FPD

GC-FPD

GC-FPD

GC-MS

GC-MS

GC-MIP-AES

HPLC-ICPMS

15.0 25.0 35.0

CRMs AVAILABLE FOR ORGANOTIN SPECIATION ANALYSIS

• SEDIMENTS

– PACS 2 (TBT and DBT, NRCC)

– RM 424 (TBT, BCR)

– BCR 462R (TBT and DBT, BCR)

– BCR 646 (butyl- and phenyl-tins, BCR)

• FISH– NIES 11 (TBT and TPhT,

NIES)

• MUSSELS– BCR 477 (butyltins, BCR)

• OYSTERS – BCR 710 (TBT and DBT,

BCR) (Mulspot Project at the certification stage)

CRMs AVAILABLE FOR METHYLMERCURY SPECIATION ANALYSIS

• SEDIMENTS– IAEA 356

– BCR 580

• CRUSTACEANS– NRCC / TORT-2

– NRCC / LUTS-1

• OYSTERS– BCR 710 (Mulspot Project at the

certification stage)

• ALGAE– IAEA 140

• FISH– NRCC / DOLT-2

– NRCC / DORM-2

– BCR 463

– BCR 464

• MUSSELS– NIST 1974a

– NIST 2974

– NIST 2976

– NIST 2977

CRMs AVAILABLE FOR As, Cr, and Pb SPECIATION ANALYSIS

MATERIAL SPECIES PRODUCER/CAT.

Dogfish muscle Arsenobetaine, (CH3)4As

NRCC / DORM-2

Tuna fish Arsenobetaine, DMA

BCR 627

Oyster tissue Arsenobetaine BCR 710

Freeze-dried water CrIII, CrVI BCR 544

Urban dust Trimethyllead BCR 605

THE REGULATORY FRAME

EC DirectivesMarine pollution, Conventions (North Sea, Mediterranean Sea,

Rhine),Groundwater Directive

Mention: « elements and their compounds »Consequences

•Chemical species are generally not determined by control laboratories•Limited knowledge of their toxic impact and occurrence (studies needed)

•Analytical methods often at the prototype stage (still few commercialised instruments)• Insufficient transfer of knowledge

Needs• Precise the chemical forms to be analysed (according to their toxicity)

• Make the techniques accessible to routine laboratories• Develop multi-disciplinary synergies

• Educate legislators !