Post on 20-Jan-2016
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
1000
2000
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7000
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10000
11000
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13000
14000
15000
16000
17000
18000
19000
20000
21000
22000
23000
24000
25000
26000
Time-->
Abundance
TIC: 2107C03A.D
8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.000
10000
20000
30000
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60000
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Time-->
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 !