Difference between Difference between total analysis, total analysis,

fractionation and fractionation and speciationspeciationKJM MEF 4010KJM MEF 4010

Module 19Module 19

ModuleModule plan, Week 1 plan, Week 1 Thursday February 15th;Thursday February 15th;

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 in terms of The significance of species activities rather than total concentration in terms of

mobility and toxicitymobility and toxicity• Chemical analytical speciation and fractionation (Al) methods Chemical analytical speciation and fractionation (Al) methods • Water sampling from different compartments of the environmentWater sampling from different compartments of 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 forest streams, tributeries to Vansjø and VansjøSampling of water samples from forest streams, tributeries to Vansjø and Vansjø

Friday, February 16th;Friday, February 16th; 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 ~ 17:00Analysis of: Analysis of: pH, Alkalinity, TOC, pH, Alkalinity, TOC, Major Anions and Cations on IC, Al-fractions and P-fractionsMajor Anions and Cations on IC, Al-fractions and P-fractions

Module plan, Week 2Module plan, Week 2

Thursday, February 22th;Thursday, February 22th; LectureLecture, Auditorium 3, hr. 09:15 – 11:00, Auditorium 3, 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, February 23rd Friday, February 23rd Individual report writing Individual report writing

Total Total analysisanalysis

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

analyte is transferred to its aqueous form analyte is transferred to its aqueous form • Me(HMe(H22O)O)2,4 or 62,4 or 6

n+ n+ prior to analysisprior to analysis

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 isotopic composition, electronic or oxidation isotopic composition, electronic or oxidation state, and/or complex or molecular structure. state, and/or complex or molecular structure. ” ”

* 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) properties.” properties.” ““Chemical Fractionation = Chemical Fractionation =

Classification according to chemical properties.”Classification according to chemical properties.”

* 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

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: • Methylmercury (CHMethylmercury (CH33HgHg++) is kinetically inert, and readily passes through ) is kinetically inert, and readily passes through

cell walls. It is far more toxic than inorganic formscell walls. It is far more toxic than inorganic forms• Organometallic Hg, Pb, and Sn are more toxic than inorganicOrganometallic Hg, Pb, and Sn are more toxic than inorganic• Organometallic As are less toxic than inorganicOrganometallic As are less toxic than inorganic• Copper toxicity correlates with free Cu-ion conc.- has reduced toxicity in Copper toxicity correlates with free Cu-ion conc.- has reduced toxicity in

the presence of organic matterthe presence of organic matter• Cr (VI) more toxic than Cr (III)Cr (VI) more toxic than Cr (III)• Inorganic Al are more toxic to aquatic organism than Al bound to organic Inorganic Al are more toxic to aquatic organism than Al bound to organic

ligand ligand

AluminumAluminum Toxicity of Al, and its environmental and Toxicity of Al, and its environmental and

biological effect are associated with the forms biological effect are associated with the forms present in aquatic systempresent in aquatic system

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

• AlF AlF 2+2+ , AlF , AlF +2+2, AlF, AlF33, , • monomeric SOmonomeric SO44 2–2– 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:• Al Al 3+3+, AlOH , AlOH 2+2+, Al(OH), Al(OH)++ ((more toxicmore toxic))• Al-F and Al-Org Al-F and Al-Org ((less toxicless toxic))

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

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

The distribution of an element among different compounds The distribution of an element among different compounds profoundly affects its transport by determining such properties profoundly affects its transport by determining such properties as: charge, solubility, and diffusion coefficientas: charge, solubility, and diffusion coefficient

Electronic and oxidation statesElectronic and oxidation states• Profoundly affect mobilityProfoundly affect mobility

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

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

waterwater Inorganic complexesInorganic complexes

• Formation of hydroxides is often a key determinant of element solubilityFormation of hydroxides is often a key determinant of element solubility Organic complexesOrganic complexes

• Organometallic compoundsOrganometallic compounds E.g. BiomethylationE.g. Biomethylation of Hg by S reducing bacteria in soil is believed to be a of Hg by S reducing bacteria in soil is believed to be a

main cause for increased conc. of Hg in surface waters in Scandinavia. main cause for increased conc. of Hg in surface waters in Scandinavia. • Macromolecular compounds and complexes Macromolecular compounds and complexes

Dissolved natural organic matter (DNOM) complex heavy metals and sorb Dissolved natural organic matter (DNOM) complex heavy metals and sorb organic micro pollutants enhancing thereby solubility and mobilityorganic micro pollutants enhancing 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 compounds profoundly The distribution of an element among different compounds profoundly affects its bioavailability by determining such properties as: affects its bioavailability by determining such properties as: Charge, solubility, and diffusion coefficientCharge, solubility, and diffusion coefficient

Isotopic compositionIsotopic composition• Not very essential for toxicityNot very essential for toxicity

Electronic and oxidation statesElectronic and oxidation states• Profoundly affect toxicityProfoundly affect toxicity

E.g. Cr(III) may be an essential element, but Cr(VI) is genotoxic and carcinogenic E.g. Cr(III) may be 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 E.g. Occupational exposure to Ni and its compounds. NiE.g. Occupational exposure to Ni and its compounds. Ni33SS22 is a potent carcinogen is a potent carcinogen

Inorganic complexesInorganic complexes E.g. Transient polymeric aluminum-hydroxo complexes with high toxicityE.g. Transient polymeric aluminum-hydroxo complexes with high toxicity

Organic complexesOrganic complexes• Organometallic compoundsOrganometallic compounds

Hydrophobicity and volatility are importantHydrophobicity and volatility are important Bioaccumulation in fatty tissues and penetration of membrane barriersBioaccumulation 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 are generally concidered less Heavy metals and organic micro pollutants bound to DNOM are generally concidered less toxic. toxic.

Some metals of “environmental” concernSome metals of “environmental” concern

Effect of a pollutantEffect of a pollutant Determined by its concentration and physical, chemical and Determined by its concentration and physical, chemical and

biological characteristics of thebiological characteristics of the• PollutantPollutant

Solubility in water and Solubility in water and organic solventorganic solvent

• Bio-accumulability (KBio-accumulability (Kowow) ) Degradability, persistence (tDegradability, persistence (t½½)) Organic complexability (KOrganic complexability (Kexex)) Density Density

• RecipientRecipient Stagnant conditions (thermocline)Stagnant conditions (thermocline) Hardness (Ca+Mg)Hardness (Ca+Mg) pH (speciation)pH (speciation) Dissolved Organic Carbon (DOC)Dissolved Organic Carbon (DOC)

Chemical analytical Chemical analytical speciationspeciation methods methods Isotopic compositionIsotopic composition

Mass spectrometry (MS) (e.g. Mass spectrometry (MS) (e.g. 1414C/C/1212C)C) Electronic and oxidation states Electronic and oxidation states

A. Selective organic complexation with A. Selective organic complexation with spectrophotometric detectionspectrophotometric detection

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

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

Organic complexes Organic complexes C. Anodic stripping voltametry on electroactive speciesC. Anodic stripping voltametry on electroactive species

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

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

chromatographychromatography (e.g. P and Al fractionation)(e.g. P and Al fractionation)

Speciation

A. SpectroscopyA. Spectroscopy

Less application because of low sensitivityLess application because of low sensitivity Oxy-anions of Mn, Cr absorb UV/vis Oxy-anions of Mn, Cr absorb UV/vis Many species can be determined by addition of Many species can be determined by addition of

selective color forming reagentsselective color forming reagents Molecular Absorption spectrophotometry (MAS)Molecular Absorption spectrophotometry (MAS)

• For metal species using chromogenic reagentsFor metal species using chromogenic reagents Al-8HQAl-8HQ

Species/element specific techniquesSpecies/element specific techniques

Speciation

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 Element specific techniquesElement specific techniques

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

GC:TCD, FIDGC:TCD, FID Element specific: FAAS , GFAAS, ICP-AES, ICP-MSElement specific: FAAS , GFAAS, ICP-AES, ICP-MS

Species/element specific techniquesSpecies/element specific techniques

Speciation

Liquid chromatography-HPLCLiquid chromatography-HPLC Samples introduce to chromatographic column of Samples introduce to chromatographic column of

stationary phase (SP)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

Speciation of mercury in soil and sediment Speciation of mercury in soil and sediment by selective solvent and acid extraction by selective solvent and acid extraction **

MP: Methanol+2-

mercaptoethanol+NH3AC

SP: C-18 column

*Y. Han , H. M. Kingston , H. M. Boylan, G. M. M. Rahman, S. Shah, R. C. Richter, D. D. Link, S. Bhandari, Anal. Bioanal. Chem.,2003, 375,428.

Gas chromatographyGas chromatography

SeparationSeparation Volatile and thermal stabilityVolatile and thermal stability

Species to be analyzed organometallic Species to be analyzed organometallic Volatile species :-MeVolatile species :-Me22Hg, MeHg, Me22 Se, Me Se, Me44Sn, Sn,

MeMe33SbSbTetraalkylated leadTetraalkylated lead

Non-volatile species after derivatization:Non-volatile species after derivatization:• Different methods..Different methods..

Speciation

GC-ICP-TOF-MS for the speciation analysis of GC-ICP-TOF-MS for the speciation analysis of organo-lead compounds in environmental water organo-lead compounds in environmental water samples*samples*

Toxicity increases with increase the number of alkyl Toxicity increases with increase the number of alkyl group i.e speciation of organolead cpds necessary group i.e speciation of organolead cpds necessary

In aqueous solution ionic …Derivitization NaEtIn aqueous solution ionic …Derivitization NaEt44

GC-oven program 60GC-oven program 6000C to 200C to 20000C at 30 C at 30 00C minC min-1-1

* M. Heisterkamp, F. C. Adams, Fresenius J. Anal. Chem.,2001, 370,597

ProblemProblem

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, for example, This behavior can be caused by, for example,

a change in pH necessitated by the analytical procedure, or a change in pH necessitated by the analytical procedure, or by intrinsic properties of measurement methods that affect by intrinsic properties of measurement methods that affect the equilibrium between speciesthe equilibrium between species

DetectionDetection limit problems limit problems

Solution: Solution: Chemical analytical Chemical analytical fractionationfractionation

Isolate various classes of species of an element and Isolate various classes of species of an element and determine the sum of its concentrations in each class 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 calculations the inorganic fraction With further analyses and 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 ionexchangeionexchange

reactivityreactivity Complexation to complexbinderComplexation to complexbinder

ExtractionExtraction 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)

Water sampleWater sample Solvent extractionSolvent extraction Ion exchange resinsIon exchange resins Solid Phase Extraction (SPE)Solid Phase Extraction (SPE)

Fractionation

Retains:Retains: Free metal ionsFree metal ions Labile metal organic complexLabile metal organic complex Labile inorganic complexesLabile inorganic complexes

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

(Strong complexes)(Strong complexes)

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

Fractionation

Example;Example;Al fractionationAl fractionation

Fractionation of Fractionation of monomeric aluminium monomeric 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 aluminium is separated from Organic bound monomeric aluminium 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

Operationally definedOperationally defined

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• Extraction• Ion exchange resinsSolid phase (soil)• Sequential extraction• Leaching

Sample prep.

Separation• HPLC• GC

• GFAAS• ICP-AES • ICP-MS

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) Grinding/HomogenizingGrinding/Homogenizing

Soil waterSoil water Samples at different depth (5-40cm)Samples at different depth (5-40cm) ConservationConservation Filtration (through 0.45µm filter)Filtration (through 0.45µm filter)

The procedure The procedure should notshould not disturb disturb the the chemical equilibrium between the different chemical equilibrium between the different forms of the element that exist in a given forms of the element that exist in a given matrixmatrix

Species transformations stimulated by Species transformations stimulated by change in:change in: Temperature, light, pH (in case of liquid Temperature, light, pH (in case of liquid

samples)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

The Vansjø ProblemThe Vansjø Problem

In 1964 the level of nutrients in In 1964 the level of nutrients in the water course was at the water course was at average level with an evenly average level with an evenly distributed algae growth distributed algae growth

Ten years later the water Ten years later the water course was starting to become course was starting to become eutrophic with increased algae eutrophic with increased algae growth and an almost doubling growth and an almost doubling of the levels of nutrients as of the levels of nutrients as well as blossoming of well as blossoming of bluegreen alga. bluegreen alga.

Bluegreen alga produce toxins Bluegreen alga produce toxins A number of remedial actions A number of remedial actions

have not given the required have not given the required effect. effect.

WatershedWatershed

698 km698 km22

Forest and agricultural Forest and agricultural landland

8 communes8 communes Below the marine limitBelow the marine limit Drinking water source for Drinking water source for

60.000 people60.000 people

Sampling strategySampling strategy Spatial and temporal Spatial and temporal

variationvariation Worst case or Worst case or

representativerepresentative Regional variation: Regional variation:

Regional or HotspotsRegional or Hotspots Temporal variation: Temporal variation:

Climate (e.g. runoff Climate (e.g. runoff intensity)intensity)

Temporal variationTemporal variation Runoff concentrations of

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

Water sampling routines should be utilised which best can handle these variations.

Discharge and nitrate concentration measured continuously in runoff water from a small agricultural catchment in Norway

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 Study of chemical and/or biological conditions

Point sampling strategiesPoint sampling strategies Point sampling with variable time interval

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 much influenced by 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