Welcome to Welcome to Analysis/fractionation and Analysis/fractionation and

speciation of water samples speciation of water samples Module 19; KJM_MENA4010Module 19; KJM_MENA4010

ModuleModule plan, Week 1 plan, Week 1See: See:

http://www.uio.no/studier/emner/matnat/kjemi/KJM-http://www.uio.no/studier/emner/matnat/kjemi/KJM-MENA4010/h09/moduler/MENA4010/h09/moduler/analyser_fraksjonering_vannprover.xmlanalyser_fraksjonering_vannprover.xml

Thursday October 8th;Thursday October 8th; LectureLecture, Auditorium 3, hr. 08:15 – 10:00 , Auditorium 3, hr. 08:15 – 10:00

• Difference between total analysis, fractionation and speciesDifference between total analysis, fractionation and species• The significance of species activities rather than total concentration The significance of species activities rather than total concentration

in terms of mobility and toxicityin terms of mobility and toxicity• Chemical analytical speciation and fractionation methods Chemical analytical speciation and fractionation methods • Water sampling from different compartments Water sampling from different compartments

of the environmentof the environment• Sampling strategies for environmental samplesSampling strategies for environmental samples

Field workField work, Vansjø, hr. 10:00 ~ 17:00, Vansjø, hr. 10:00 ~ 17:00• Visit to Morsa – Vansjø watershedVisit to Morsa – Vansjø watershed• Sampling of water samples from Dalen watershed and tributaries to Sampling of water samples from Dalen watershed and tributaries to

Vansjø, and Vansjø lakeVansjø, and Vansjø lake

ModuleModule plan, Week 1 plan, Week 1 Friday, October 9th;Friday, October 9th;

LectureLecture, Auditorium 3, hr. 08:15 – 09:00 , Auditorium 3, hr. 08:15 – 09:00 • Important species in natural water samplesImportant species in natural water samples• Central equilibriums in natural water samples Central equilibriums in natural water samples • Concentrations and activitiesConcentrations and activities

LabworkLabwork, hr. 09:15 ~ 17:00, hr. 09:15 ~ 17:00• Sample preparationSample preparation

Filtration, UV oxidationFiltration, UV oxidation• Analysis of: Analysis of:

pH, Conductivity, UV/VIS absorbency, Alkalinity, Al fractionspH, Conductivity, UV/VIS absorbency, Alkalinity, Al fractions• Presentation of instruments:Presentation of instruments:

Major Anions and Cations on IC, TOC, ICP Major Anions and Cations on IC, TOC, ICP

Module plan, Week 2Module plan, Week 2

Thursday, October 15th;Thursday, October 15th; LectureLecture, Auditorium 2, hr. 09:15 – 11:00, Auditorium 2, hr. 09:15 – 11:00

• Challenges with simultaneous equilibriumChallenges with simultaneous equilibrium• Speciation programs (MINEQL)Speciation programs (MINEQL)

PC labPC lab, hr. 11:15 – 16:00, hr. 11:15 – 16:00• Practice in using MINEQLPractice in using MINEQL

Friday, October 16th Friday, October 16th Independent report writing Independent report writing

Difference between Difference between total analysis, total analysis,

fractionation and fractionation and speciationspeciationKJM MENA 4010KJM MENA 4010

Module 19Module 19

What is speciation and fractionation?What is speciation and fractionation?

SpeciationSpeciation**

SpecificSpecific form of an element defined as to form of an element defined as to electronic or oxidation state, electronic or oxidation state, complex or molecular structure complex or molecular structure and isotopic compositionand isotopic composition

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

FractionationFractionation**

Process of classification of an analyte or a Process of classification of an analyte or a

group of analytesgroup of analytes from a certain sample from a certain sample according to physical (e.g., size, solubility) according to physical (e.g., size, solubility) or chemical (e.g., bonding, reactivity) or chemical (e.g., bonding, reactivity) propertiesproperties

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

Why is chemical speciation Why is chemical speciation important?important?

MobilityMobility and solubility of a compound depend on and solubility of a compound depend on in which form it can exist in solutionin which form it can exist in solution

Fe(III) is insoluble, while Fe(II) is very solubleFe(III) is insoluble, while Fe(II) is very soluble The The bioavailabilitybioavailability of metals and their of metals and their

physiological and toxicological effects physiological and toxicological effects depend on the actual species present depend on the actual species present – not on the total concentration– not on the total concentration

Examples: Examples: • Cr (VI) more toxic than Cr (III)Cr (VI) more toxic than Cr (III)• Organometallic Hg, Pb, and Sn are Organometallic Hg, Pb, and Sn are moremore toxic than inorganic toxic than inorganic

Methylmercury (CHMethylmercury (CH33HgHg++) readily passes through cell walls. It is far more ) readily passes through cell walls. It is far more toxic than inorganic formstoxic than inorganic forms

• Organometallic Al, As and Cu are Organometallic Al, As and Cu are lessless toxic than inorganic forms toxic than inorganic forms Inorganic Al are more toxic to aquatic organism Inorganic Al are more toxic to aquatic organism

than Al bound to organic ligand than Al bound to organic ligand Copper toxicity correlates with free Cu-ion conc.Copper toxicity correlates with free Cu-ion conc.

- has reduced toxicity in the presence of organic matter- has reduced toxicity in the presence of organic matter

AluminumAluminum The environmental and biological effects The environmental and biological effects

of Al are associated with the forms present of Al are associated with the forms present in aquatic systemin aquatic system

In aquatic systems, Al exists mainly as:In aquatic systems, Al exists mainly as:• free aqueous Alfree aqueous Al3+3+ , AlOH , AlOH2+2+ , Al(OH) , Al(OH)++, Al(OH), Al(OH)33 and Al(OH) and Al(OH)4 –4 –

• AlFAlF2+2+ , AlF , AlF22++, AlF, AlF33, ,

• monomeric SOmonomeric SO442–2– complexes, Al-Org complexes, Al-Org

Al speciation depend on soln. pH & conc. of ligandAl speciation depend on soln. pH & conc. of ligand

Toxicity:Toxicity:• AlAl3+3+, AlOH, AlOH2+2+, Al(OH), Al(OH)++ ((more toxicmore toxic))• Al-F and Al-Org Al-F and Al-Org ((less toxicless toxic))

pH dependence on Al speciationpH dependence on Al speciationMineql calculationsMineql calculations

Tot [Al] = 1E-5 M

[SO42-] = 1E-4 M

[F-] = 1E-6 M

G.W Brummer In the : Bernhard M, Brickman FE, Sadler PJ (eds) The importance of chemical “speciation” in environmental process. Springer, Berlin Heidelberg New York, 1986, p 170

Mobility of metals in soil and soil solution

pH

The significance of species activities The significance of species activities in terms of in terms of mobilitymobility

The distribution of an element among different species The distribution of an element among different species profoundly affects its profoundly affects its transporttransport by determining such by determining such properties as: properties as: solubility and diffusion coefficientsolubility and diffusion coefficient

Inorganic complexesInorganic complexes• Formation of hydroxides Formation of hydroxides

is often a key determinant of element solubilityis often a key determinant of element solubility • Inorganic ligands Inorganic ligands

E.g. NiClE.g. NiCl22 and NiSO and NiSO44 are water soluble are water soluble while NiO and Niwhile NiO and Ni33SS22 are highly insoluble in water are highly insoluble in water

Charge and oxidation statesCharge and oxidation states• Profoundly affect mobilityProfoundly affect mobility

E.g. The Fe(II) ion is soluble, E.g. The Fe(II) ion is soluble, whereas Fe(III) is more prone whereas Fe(III) is more prone to hydrolysis and subsequent precipitationto hydrolysis and subsequent precipitation

Organic complexesOrganic complexes• Macromolecular compounds and complexes Macromolecular compounds and complexes

Dissolved natural organic matter (DNOM) Dissolved natural organic matter (DNOM) complex heavy metals and complex heavy metals and sorb organic micro pollutants sorb organic micro pollutants enhancing thereby solubility and mobilityenhancing thereby solubility and mobility

The significance of species activities The significance of species activities in terms of in terms of toxicitytoxicity

The distribution of an element among different species The distribution of an element among different species profoundly affects its profoundly affects its bioavailabilitybioavailability by determining such properties as: by determining such properties as: Charge and distribution coefficientCharge and distribution coefficient

Charge and oxidation statesCharge and oxidation states• Profoundly affect toxicityProfoundly affect toxicity

E.g. Cr(III) is an essential element, but Cr(VI) is genotoxic and carcinogenic E.g. Cr(III) is an essential element, but Cr(VI) is genotoxic and carcinogenic Toxic effect of As and its compound decreases in sequence As (III)>As (V) Toxic effect of As and its compound decreases in sequence As (III)>As (V)

Inorganic compoundsInorganic compounds• Aqueous speciesAqueous species

Heavy metals (CuHeavy metals (Cu2+2+, Cd, Cd2+2+, Zn, Zn2+2+, Pb, Pb2+2+) are commonly more toxic in their aqueous form) are commonly more toxic in their aqueous form• Inorganic complexesInorganic complexes

E.g. Transient polymeric aluminum-hydroxo complexes (Al(OH)E.g. Transient polymeric aluminum-hydroxo complexes (Al(OH)33) with high toxicity) with high toxicity

Organic complexesOrganic complexes• Organometallic compoundsOrganometallic compounds

Hydrophobicity (KHydrophobicity (KOWOW) and volatility (V) and volatility (VPP) are important) are important Bioaccumulation in fatty tissues and penetration of membrane barriers Bioaccumulation in fatty tissues and penetration of membrane barriers

• E.g. MeHgE.g. MeHg• Macromolecular compounds and complexesMacromolecular compounds and complexes

Heavy metals and organic micro pollutants bound to DNOM Heavy metals and organic micro pollutants bound to DNOM are generally concidered less toxic. are generally concidered less toxic.

Effect of a pollutantEffect of a pollutant Determined by its concentration and Determined by its concentration and

physical, chemical and biological characteristics physical, chemical and biological characteristics of the:of the:

• PollutantPollutant Solubility in water and Solubility in water and

organic solventorganic solvent• Bio-accumulability (KBio-accumulability (Kowow))• Bio-magnification Bio-magnification

Degradability, persistence (tDegradability, persistence (t½½)) Organic complexability (KOrganic complexability (Kexex)) Density Density

• RecipientRecipient Stagnant conditions (redox)Stagnant conditions (redox) Hardness (Ca+Mg)Hardness (Ca+Mg) pH (speciation)pH (speciation) Humic contentHumic content

Bio-magnificaton

Total Total analysisanalysis

Most standard chemical analytical Most standard chemical analytical methods determine the methods determine the total amount (component) total amount (component) of an element in the sampleof an element in the sample AAS, ICP and ICAAS, ICP and IC The sample is typically digested The sample is typically digested

where all analyte is transferred to its where all analyte is transferred to its aqueous form prior to analysisaqueous form prior to analysis• X-Me X-Me Me(HMe(H22O)O)2,4 or 62,4 or 6

n+n+

Chemical analytical Chemical analytical speciationspeciation methods methods Isotopic composition

Mass spectrometry (MS) (e.g. 14C/12C) Charge and oxidation states

A. Selective organic complexation with spectrophotometric detection

Separation with HPLC, detection with e.g. ICP Inorganic compounds and complexes

B. Potentiometric determination of the activity of Free aqueous species (e.g. ion selective electrodes for Free F, Ca)

Organic complexes C. Anodic stripping voltametry on electroactive species

Organometallic compounds D. Separation with GC or HPLC, detection with e.g. ICP

Macromolecular compounds and complexes Size exclusion, ion-exchange, affinity and reversed phase

chromatography

Speciation

A. SpectroscopyA. Spectroscopy

Less application because of Less application because of low sensitivitylow sensitivity

Oxy-anions of Mn, Cr Oxy-anions of Mn, Cr absorb UV/VISabsorb UV/VIS

Proxy for Humic substances Proxy for Humic substances Many species can be determined by Many species can be determined by

addition of selective color forming addition of selective color forming reagentsreagents

Molecular Absorption spectrophotometry Molecular Absorption spectrophotometry (MAS)(MAS)

• For metal species using chromogenic For metal species using chromogenic reagentsreagents

Al-8HQAl-8HQ

Species/element specific techniquesSpecies/element specific techniques

Speciation

MnO4-

B & C. Electro analytical techniquesB & C. Electro analytical techniques

Ion selective Electrode (ISE)Ion selective Electrode (ISE) Detection of free-aqua ions (FDetection of free-aqua ions (F--, Cu, Cu2+2+, Cd, Cd2+2+, Pb, Pb2+2+)) Low sensitivity (µmol/L –mmol/L)Low sensitivity (µmol/L –mmol/L) Less applicable for the speciation of metalsLess applicable for the speciation of metals E.g. Fluoride Selective electrode - Al speciation E.g. Fluoride Selective electrode - Al speciation

Polarographic and Voltametric methodsPolarographic and Voltametric methodsDifferential Pulse Polarography (DPP)Differential Pulse Polarography (DPP)

Gives separated signals for free-metal ion and Gives separated signals for free-metal ion and metal complexmetal complex

DC- polarographyDC- polarography Can be used to distinguish between red-ox statesCan be used to distinguish between red-ox states

Species/element specific techniquesSpecies/element specific techniques

Speciation

D. Separation and detection techniques D. Separation and detection techniques

Separation Separation ChromatographyChromatography

Liquid chromatography/HPLCLiquid chromatography/HPLC• Size, affinity to mobile/stationary phaseSize, affinity to mobile/stationary phase

Gas chromatography (GC)Gas chromatography (GC)• VolatilityVolatility

Detection techniques Detection techniques Non specific techniquesNon specific techniques

HPLC: Photometer, Refractometer, Diode-array, Electrochemical HPLC: Photometer, Refractometer, Diode-array, Electrochemical and Conductivity and Conductivity

GC:Termal Conductivity Detector (TCD), GC:Termal Conductivity Detector (TCD), Flame Ionization DetectorFlame Ionization Detector (FID) (FID) Element specificElement specific

FAAS , GFAAS, ICP-AES, ICP-MSFAAS , GFAAS, ICP-AES, ICP-MS

Species/element specific techniquesSpecies/element specific techniques

Speciation

SeparationSeparation- Liquid chromatography-HPLC- Liquid chromatography-HPLC

Samples introduced to chromatographic column Samples introduced to chromatographic column of stationary phase (SP)of stationary phase (SP)

Mobile phase (MP) pumped through columnMobile phase (MP) pumped through column Separation based Separation based

on the interaction on the interaction of the analyte with of the analyte with the the SPSP and and MPMP

Separated speciation:Separated speciation: inorganic species, inorganic species,

such as cations, such as cations, anions (IC)anions (IC)

metal complexesmetal complexes organometallic organometallic

compounds compounds

Speciation

SeparationSeparation- Gas chromatography (GC)- Gas chromatography (GC)

Need to be Volatile and thermal stable Need to be Volatile and thermal stable compoundscompounds

Organometallic species Organometallic species Volatile species :Volatile species :

• MeMe22Hg, MeHg, Me22 Se, Me Se, Me44Sn, MeSn, Me33Sb, Tetraalkylated Sb, Tetraalkylated leadlead

Non-volatile species after derivatizationNon-volatile species after derivatization

Speciation

Problems with analytical speciationProblems with analytical speciation

Often, chemical species present in a given Often, chemical species present in a given sample are sample are not stablenot stable enough to be determined enough to be determined as suchas such During the measurement process the partitioning of During the measurement process the partitioning of

the element among its species may be changedthe element among its species may be changed• This behavior can be caused by intrinsic properties of This behavior can be caused by intrinsic properties of

measurement methods that affect the equilibrium between measurement methods that affect the equilibrium between speciesspecies

For example a change in pH necessitated by the analytical For example a change in pH necessitated by the analytical procedure procedure

DetectionDetection Limit (DL) problems Limit (DL) problems When you split an analyte at low concentration then When you split an analyte at low concentration then

each specie concentration may fall below DLeach specie concentration may fall below DL

Solution: Solution: Chemical analytical Chemical analytical fractionationfractionation

Isolate various classes of species of an element Isolate various classes of species of an element and determine the sum of its concentrations in each class and determine the sum of its concentrations in each class

In some instances, fractionation may be refined by In some instances, fractionation may be refined by supplementary speciation analysis. supplementary speciation analysis.

With further analyses and/or calculations the inorganic fraction With further analyses and/or calculations the inorganic fraction can be subdivided into individual species. can be subdivided into individual species.

Fractionation

Based on Based on By means ofBy means ofSizeSize Filtration, size-exclusion chromatographyFiltration, size-exclusion chromatography

AffinityAffinity ChromatographyChromatography

SolubilitySolubilityExtractionExtraction

HydrophobicityHydrophobicity

ChargeCharge Ion-exchangeIon-exchange

ReactivityReactivity Complexation to complexbinderComplexation to complexbinder

ExtractionExtraction Water sampleWater sample

Solvent extractionSolvent extraction Solid Phase Extraction (SPE)Solid Phase Extraction (SPE)

• Ion exchange resins Ion exchange resins

Soil sampleSoil sample Leaching methodLeaching method

• Sonication, stirring, shaking or Sonication, stirring, shaking or soxhlet with organic solventsoxhlet with organic solvent

Sequential Extraction (Tessier) Sequential Extraction (Tessier) Supercritical Fluid Extraction (SFE)Supercritical Fluid Extraction (SFE)

Fractionation

CO2

Retains:Retains: Labile free aqueous metal ionsLabile free aqueous metal ions Labile inorganic complexesLabile inorganic complexes Labile metal organic complexLabile metal organic complex

Eluted:Eluted: Non-labile metal complexes Non-labile metal complexes

(Strong complexes)(Strong complexes)

Ion exchange resins Ion exchange resins for fractionation of metals in waterfor fractionation of metals in water

Fractionation

Example;Example;Al fractionationAl fractionation

Fractionation of Fractionation of Monomeric aluminiumMonomeric aluminium from polymeric forms from polymeric forms is accomplished by is accomplished by 20 sec. complexation 20 sec. complexation with 8-hydroxyquinoline at pH 8.3 with subsequent with 8-hydroxyquinoline at pH 8.3 with subsequent extraction into MIBK organic phaseextraction into MIBK organic phase

Organic bound monomeric aluminiumOrganic bound monomeric aluminium is separated from is separated from inorganic aluminium (mainly labile) by trapping the latter inorganic aluminium (mainly labile) by trapping the latter fraction on an Amberlight IR-120 ion exchange columnfraction on an Amberlight IR-120 ion exchange column

The Al concentrations in the organic extracts are The Al concentrations in the organic extracts are determined photometrically determined photometrically

Chemical analytical fractionation; Chemical analytical fractionation; ShortcomingsShortcomings

The discrimination The discrimination inherent in the inherent in the method can be more method can be more or less selective but it or less selective but it is not absoluteis not absolute Small variations in the Small variations in the

methodical cutoff may methodical cutoff may cause significant cause significant variations in the outputvariations in the output

I.e. Operationally I.e. Operationally defineddefined

Small variations in the cutoff will often give large variations in the results

Sampling Chemical SpeciationTechniques

Fractionation Techniques

Element specific detection Techniques

Summary

Sample prep.

Liquid phase• Organic extraction• Solid Phase ExtractionSolid phase (soil)• Leaching• Sequential extraction

Sample prep.

Separation• HPLC• GC

• GFAAS• ICP-AES • ICP-MS

ISEMASMS

Water sampling from different Water sampling from different compartments of the environmentcompartments of the environment

Sampling and sample preparationSampling and sample preparation

SoilSoil Sample genetic horizonsSample genetic horizons DryingDrying StorageStorage Sieving (2 mm)Sieving (2 mm)

Soil waterSoil water Samples from different soil Samples from different soil

genetic horizonsgenetic horizons Conservation (biocide, cooling)Conservation (biocide, cooling) Filtration (through 0.45µm filter)Filtration (through 0.45µm filter)

Sample conservationSample conservation Content of labile substances in water sample can be altered due to the Content of labile substances in water sample can be altered due to the

chemical, physical, and biological reactions during storage chemical, physical, and biological reactions during storage Nutrients: PONutrients: PO44

3-3-, NH, NH44++, NO, NO33

--, Silicates, Silicates Volatile: HCOVolatile: HCO33

--, - pH at circum neutral pH , - pH at circum neutral pH Best is to analyze immediately upon arrival at labBest is to analyze immediately upon arrival at lab Reduce biological activity by:Reduce biological activity by:

Refrigeration –Freezing (hysteresis effect)Refrigeration –Freezing (hysteresis effect) Store in darkStore in dark Add Biocide HgClAdd Biocide HgCl22, Azid (may release nutrients), Azid (may release nutrients) Filtering through 0,2um filterFiltering through 0,2um filter Acid HAcid H22SOSO44

Trace metal conservation : Acidification to pH<2 with HNOTrace metal conservation : Acidification to pH<2 with HNO33 Special: Hg with ClSpecial: Hg with Cl--

The procedure The procedure should notshould not disturb disturb the chemical equilibrium between the chemical equilibrium between the different forms of the elements the different forms of the elements that exist in a given matrixthat exist in a given matrix

Species transformations stimulated by Species transformations stimulated by change in:change in: Temperature, light, pH, redox Temperature, light, pH, redox

(in case of liquid samples)(in case of liquid samples)

Problem associated with sampling , storage and sample preparation for speciation/fractionation

Water sampling equipmentWater sampling equipment DepositionDeposition

Bulk precipitationBulk precipitation Wet onlyWet only Canopy throughfallCanopy throughfall Ground vegetation Ground vegetation

throughfallthroughfall Soil waterSoil water

Percolation lysimeterPercolation lysimeter Suction lysimeterSuction lysimeter

RunnoffRunnoff V-notch weirV-notch weir

Sampling strategySampling strategy Large spatial and Large spatial and

temporal variationtemporal variation

Worst case or Worst case or representativerepresentative Spatial variation:Spatial variation:

• Regional or HotspotsRegional or Hotspots

Temporal variationTemporal variation Seasonal & ClimateSeasonal & Climate Runoff concentrations of

both solutes and suspended material show large variations both over time and as a function of discharge.

Discharge and concentration measured continuously in runoff water from a small catchment in Norway and N Sweden

Deelstra et al., 1998, Sampling technique and strategy. In: Measuring runoff and nutrient losses from agriculturalland in Nordic countries. TemaNord, Nordic Council of Ministers, 1998:575

Point sampling strategiesPoint sampling strategies

Different water sampling strategies depending on the objectives of the measurements. Study processes

• E.g. event studies Study of total loss

• Flux dependent sampling Study of chemical and/or biological conditions

• Time averaged sampling

Point sampling strategiesPoint sampling strategies Point sampling with variable time interval – Episode studies

Rainfall - and snow melt events, which often lead to high soil and nutrient losses, influence to a high degree the sampling frequency.

• Calculations of soil and nutrient losses based on this method are biased. Point sampling with fixed time intervals

The accuracy of the result is strongly dependent on the sampling frequency Volume proportional point sampling

Point sampling is triggered each time a certain volume of water has passed the monitoring station. In general, load estimates based on this system leads to an improvement

Flow proportional composite water sampling An alternative to point sampling systems is volume proportional mixed water

samples. In this case a small water sample is taken, each time a preset volume of water has passed the monitoring station

Combined sampling Sampling systems might be combined so as best to suit its purpose.

• It is assumed that the chemical concentration of runoff water during low flow periods can be considered constant

Integrated Integrated monitoringmonitoring