Determination of organic

pollutants in environmental

samples

Helena ProsenFaculty of Chemistry and Chemical Technology

University of Ljubljana

Role of chemical analysis in environmental

chemistry

determination of pollutant concentration in different environmental compartments

global monitoring of pollutant spreading

isolation and identification of the degradation and transformation products

elucidation of the degradation mechanisms and intermediates

monitoring of pollutant effects on the organisms via determination of concentration in tissues

monitoring of success of pollutant removal strategies

Steps of analytical method in the

environmental analysis

sampling and preparation of the representative sample

sample preparation, pollutant preconcentration

choice and application of the suitable technique for the pollutant determination

evaluation of the results

Sampling

is done in the field – any chance of field measurements?

sampling plan: where and when, how many sample aliquots?

static or dynamic system?

representative sample: spatial and temporal variability

sampling “by grid” or random (!) sampling

other factors: weather conditions, traffic conditions, sampling in ecological and other disasters

if the concentration depends on the depth (soil, static surface water bodies etc.): sampling by layers

absolutely haphazard, single sampling suitable only for the thoroughly homogenous system (!) or for a preliminary analysis

grab sample: sampled at a certain site at certain time

composite sample: made of several grab samples; more representative, but information on variability, concentration trends etc. lost

How many samples should be analysed?

random sampling of a few samples

analyte determination: what is the standard deviation s?

number of samples N that should be sampled and analysed to obtain the results within the expected confidence limits ±e: 2

e

stN

t... Student factor from the statistical tables at the chosen confidence level (usually 95 %) and random degrees of freedom, that are adjusted accordingly

continuous monitoring by suitable sensors (gases CO, NOx, SO2, certain pollutants, total organic carbon in water etc.) –most effective to discover sudden concentration changes

Transport and storing of samples

In the unsuitably transported or stored sample, chemical composition can change due to:

evaporation of volatile components: low T < 4 oC, freezing, no shaking during the transport

gas absorption from the air - O2 causes oxidation, analyte precipitation: no pouring, low pH<2 (HNO3)

photoreactions on exposure to light: brown bottles, storage in dark

biological transformation caused by microorganisms: low T, low or high pH, preservative addition (!)

interaction of analytes with the container walls (e.g. adsorption of hydrophobic analytes on plastic container walls)

dissolution of container components in the sample (e.g. plastic containers: phtalic acid esters)

Preparation of subsamples

reducing the size of sample (subsample):

gases, liquids: well mixed, sampling of smaller part

solids: (grinding, sifting), mixing, sampling of smaller part by quartering

after the suitable preparation (grinding, sifting): test sample, from which (once or several times) a suitable quantity for the analysis is taken

test sample is further prepared for the analysis: dissolution, digestion, extraction, distillation...

Sample preparation before the analysis

Aims:

direct analysis of the analyte in the sample rarely possible

sample too complex: other sample components (matrix) interfere with the analysis

concentration of analyte in the sample too low, should be isolated and preconcentrated

Sample preparation techniques

Volatile analytes (Tbp <150 oC)

headspace (HS) analysis: analysis of the gaseous phase above the liquid/solid sample

gaseous sample

liquid or solid sample heated

Sample in closed container (e.g. vial):

gaseous phase sampled by gas-tight

syringe: static HS (SHS) analysis

sampled by microextraction fiber (SPME)

Sample purged with inert gas:

(N2, He, Ar) and analytes subsequently trapped in cryotrap, liquid trap or sorbent trap: dynamic HS (DHS) analysis or purge-and-trap

adsorption on sorbent trap (sorbent in metal or glass tubes): active sampling by suction of gaseous sample through the trap (usually field sampling)

various sorbents for different analytes; important parameter break-through volume (in L of sample per g of sorbent)

advantages: simple to use, possible field sampling, interferents (e.g. H2O) poorly adsorbed; possible to derivatize the analytes at the same time

disadvantages: sample contamination due to sorbent components or reactions with it; not all analytes are equally sorbed/desorbed (errors in ratio determination in the actual sample)

Sorbent Analytes Desorption

Tenax non-polar VOC (Tbp 40-200 oC) thermal

carbon molecular sieve hydrocarbons C2-C5 thermal

active carbon medium-to-low volatility compounds solvents

polyuretane foam medium volatility compounds solvents

activated SiO2 amines, polar compounds solvents

graphitized carbon hydrocarbons C4-C12, high-molecular compounds (PCB)

thermal;solvents

XAD-2 medium volatility compounds, PAH solvents

Desorption:

thermal: tube with sorbent heated to high temperature (200-500 oC) and purged with gas, which flows to cooled trap (cooled GC injector, GC column at low temperature). Analytes subsequently determined by gas chromatography.

with solvents: sorbent washed with a suitable solvent to dissolve the analytes; then concentrated and analyzed by GC.

Preconcentration with cryotrap:

all compounds below certain Tbp are concentrated

desorption: thermal or with solvents

Preconcentration in absorbing liquid (impinger): analyte solution in

solvent

inert gas

sample flowmeter

cryotrap

Preconcentration and (semi-)quantitative analysis:

for medium concentrations (e.g. organic compounds up to 50 mL/m3)

colorimetric indicator tubes (Dräger, Kittagawa)

especially for occupational environment

Source: publiclab.org

Volatile and non-volatile organic analytes in

liquid samples

solvent extraction (SE)

solid-phase extraction (SPE)

solid-phase microextraction (SPME)

recovery (): ratio between the quantity of the analyte in the extract and total quantity present in the sample (0-1 or 0-100 %)

Solvent extraction, SE,

or liquid-liquid extraction, LLE

solvent is immiscible with the sample

analyte is distributed between two phases: depends on the analyte and solvent properties (“like dissolves like”), temperature, pH, ionic strength etc.

extraction more efficient by using small solvent volumes several times than by using high solvent volume once

expected recoveries 80-100 %

preconcentration: solvent can be subsequently evaporated (volatility!)

0

1

2

3

4

5

6

7

8

9

10

0 2 4 6 8 10

nx

n/x

0 x

10

0%

n... number of extraction repeats

xn/x0 =q... ratio of analyte concentrations in the sample after (xn) and before (x0) n extraction repeats

n

d VKV

Vq

21

1

V1… volume of phase 1 (sample)

V2… volume of phase 2 (solvent)

Kd… distribution coefficient

Solvent selection

Solvent Dielectric constant

UV cut-off at 1 AU (nm)

Tbp (oC) Viscosity (cP)

Water solubility (% v/v)

Acetic acid 6.30 230 118 1.26 100

Acetone 20.70 330 56 0.32 100

Acetonitrile 37.50 190 82 0.37 100

Benzene 2.27 280 80 0.65 0.18

Chloroform 4.81 245 61 0.57 0.82

Cyclohexane 2.02 200 81 1.00 0.01

Dichloromethane 8.93 235 41 0.44 1.6

Dimethylformamide 36.70 268 155 0.92 100

Dimethylsulfoxide 4.70 268 189 2.00 100

Dioxane 2.25 215 101 1.54 100

Ethanol 25.80 210 78 1.20 100

Ethylacetate 6.02 260 77 0.45 8.7

Diethylether 4.33 220 35 0.32 6.89

n-Heptane 1.92 200 98 0.39 0.0003

n-Hexane 1.88 200 69 0.33 0.001

Methanol 32.70 205 65 0.60 100

n-Propanol 20.33 210 97 2.27 100

Tetrahydrofuran 7.58 215 65 0.55 100

Toluene 2.38 285 111 0.59 0.05

Water 81.10 200 100 1.00 100

Solid-phase extraction, SPE

liquid sample is forced to pass through solid sorbent: analytes are retained, then desorbed with a suitable solvent

diminished solvent volume, automatization possible

effectiveness depends on sorbent choice, desorption solvent, pH, ionic strength, sample volume etc.

sorbents in the form of cartridges or disks

possible in off-line or on-line configuration

recoveries 80-100 %

extract can be further concentrated by solvent evaporation

S P E

o f f - l i n e o n - l i n e

d i s k c a r t r i d g e

disk cartridge

low volumecartridges/precolumns

direct coupling to aseparation method

(LC, GC)

sorbent particle -loaded membranesof PTFE (flexible)

or glass fibre (rigid)

sorbent particles(40-60 m)

packed in an opensyringe barrel

or similar

m

stacked disksin an open

syringe barrel

Off-line SPE

On-line SPE

a a n n a a l l y y t tsample sample

mobile phase mobile phase

Sample loading Back-flush elution

Sorbent selection

SAMPLE ANALYTE SORBENT

Aqueoussolution

Organicsolution

high polarity

low polarity

neutral

ionized

neutral

ionized

cationic

anionic

strong

strong

weak

weak

RP/NP

RP/IE

NP

RP

WCX

SCX

AMINO

SAX

Solid-phase microextraction, SPME

sorbent / viscous liquid phase on 1 cm quartz glass fiber

fiber exposed to gaseous sample or immersed in liquid sample

equilibrium between analyte on fiber and in sample reached after certain (equilibration) time

analyte thermally desorbed in the gas chromatograph injector or with solvent (for HPLC - unusual)

only up to 10 % of analyte from sample sorbed on fiber (low capacity)

Volatile and non-volatile organic analytes in

solid samples

Soxhlet extraction (SOX)

ultrasound extraction (USE)

microwave extraction (MWE)

pressurized liquid extraction (PLE)

supercritical fluid extraction (SFE)

matrix solid-phase dispersion (MSPD)

Extraction in Soxhlet apparatus (SOX)

high quantity of sample, cheap (?)

high volume of solvent (0,3-0,5 L)

time-consuming: 24-48 h

extract should be further cleaned and concentrated

not suitable for thermally unstable analytes

Ultrasound extraction (USE)

US bath, US probe

cheap, fast

high volume of solvent

extract should be further cleaned and concentrated

locally increased T makes it unsuitable for thermally labile analytes

Microwave extraction (MWE)

fast, automated

solvent should absorb microwaves

high temperature and pressure (closed system) or at atmospheric pressure (open system)

comparatively expensive equipment

Safety disk

Seal cap

Outer cap

Rupture disk

Inner liner

Outer case

Support plate

microwaveirradiation

Pressurized liquid extraction (PLE)

commercial instrument ASE® (accelerated solvent extraction)

high pressure, T > Tbp

(solvent)

fast, automated

extracts should be cleaned

very expensive equipment

Supercritical fluid extraction (SFE)

CO2 + polar modifiers up to 20 %

non-toxic

non-flammable

easy removal of the solvent

selectivity achieved by adjusting conditions (T, p)

labour intensive technique

very expensive equipment

1. Supercritical fluid reservoir2. Oven3. Extraction cell4. Restrictor5. Collector

Matrix solid-phase dispersion, MSPD

sorbent

sampleblending of

sample with sorbent

empty syringe column

fritfrit

sample/sorbentblend is compressed

solvent

eluate

Analytical techniques in environmental

analysis

electrochemical: potentiometry (specific indicator electrodes), polarography (direct, anode stripping)

spectroscopic: atomic and molecular spectroscopy (UV-vis), infrared (IR) spectroscopy, fluorescence spectroscopy, X-ray fluorescence

separation: chromatography, electromigration

mass spectrometry (MS)

Potentiometry

pH of liquid and solid samples (in suspension)

measurement of ORP (oxidation-reduction potential)

specific indicator electrodes for the determination of NH3, CO2, F, S2, Pb2+ etc.

less common, but specific electrodes exist for organic analytes

Source: http://chemwiki.ucdavis.edu/

Voltammetry

anode stripping polarography (ASP): determination of several inorganic cations at low concentrations possible

amperometry: O2 (Clark) sensor (BOD5 determination)

combined with specific biorecognition elements also for organic analytes (pesticides, phenols etc.)

Guard

cathode

Gold cathode

Anode

Glass-covered cathode wire

Silicon rubber or Teflon membrane

Source: http://www.edaphic.com.au

Conductometry

determination of conductivity of liquid samples

electronic noses

Source: Electrochemistry Encyclopedia, http://knowledge.electrochem.org

Molecular absorption spectrophotometry

organic species with chromophores, derivatization

absorbance measurement in the 200-800 nm range

poor selectivity

Source: http://elchem.kaist.ac.kr

Fluorescence spectrometry

analyte absorbs light at exc

and emits light at em

em > exc

very specific determination for fluorescent compounds; derivatization; quenchers

organic pollutants: PAHs, some pesticides etc.

Source: http://chemwiki.ucdavis.edu/

IR spectroscopy

radiation absorption in the mm-mm wavelength range (vibration and rotation energy levels in the molecule): specific absorption bands for the functional groups

qualitative analysis: which analyte?

quantitative analysis: % transmittance, less useful

Source: Pejcic et al., Sensors

2009, 9(8), 6232-6253

Gas chromatography

useful for volatile and thermally stable compounds

detectors: flame-ionization d. (FID), electrone-capture d. (ECD), thermal conductivity d. (TCD), photoionization d. (PID), mass-spectrometric d. (MSD)

Source: http://www.chromedia.org

High-performance liquid chromatography

applicable to separation of most organic compounds

detectors: spectrophotometric d. (UV-Vis), refractory index d. (RID), fluorescence d. (FLD), mass-spectrometric d. (MSD)

ion chromatography: for ionized species only, e.g. organic acid anions; detectors: conductivity d., electrochemical d.

Source: http://www.kutztown.edu

Mass spectrometry

Processes:

ionization of analytes in ion source: ions of molecules and molecular parts (fragments) in gaseous phase

separation of ions in gaseous phase in mass analyzer by their m/z

detection of ions

detector signal manipulation: mass spectrum

Types of ionization:

electron ionization (EI): repeatable mass spectra

atmospheric pressure ionizations (APCI, ESI): compatible with liquid chromatography

EI mass spectrum of atrazine

50 100 150 200

200

215

58173

4368

132 158122104 14527 93 18783

Sig

nal

m/z

Sensors and field assays

aims: fast preliminary detection of problematic analytes; also for lay use

special sample preparation not needed or very simple

calibrated to instrumental analytical methods

positive results should always be checked – frequent “false positives” because of interferents

sensors: biosensors, electrochemical, optic (spectroscopic)

field assays: colorimetric,

immunoassays (immobilized

antibodies)

direct enzyme immunoassay, EIA

indirect EIA

Special determinations: aerosols (PM)

particulate matter is sampled by pumping through filters, which later desorbed

fractionation by size: usually PM10 filters (particles <10 mm) or PM2,5 (particles <2,5 mm)

mass concentration of particles in air determined by weighing of the filters (in mg/m3)

materials: glass, quartz glass, paper, polymers – chosen with respect to analytical and sample preparation method (digestion, extraction...)

Special determinations: general parameters of

water quality

concentration of organic compounds in waste or surface waters important as they act as food source for natural aerobic microorganisms, causing eventual decrease in oxygen concentration

Several methods:

total organic carbon, TOC

biochemical oxygen demand (BOD5)

chemical oxygen demand (COD)

permanganate index (PI)

Total organic carbon (TOC) analyzer

Source:

www.ankersmid.co.ro

Evaluation of analytical results

Measurement uncertainty (u) is an interval within which a true value is expected.

Random or indeterminate error is an always present scattering of results both in (+) AND (-) direction around certain value (average), consequence of measurement imprecision.

Systematic or determinate error is a displacement of measurement result in (+) OR (-) direction with respect to true value; repeatable.

Random error – precision, repeatability

Results symmetrically spread around average value (usually arithmetic average):

N

x

x

N

i

i 1

• spread of results around average value given by standard deviation (s):

• relative standard deviation, RSD:

• RSD in % is coefficient of variation, CV

1

1

2

N

xx

s

N

i

i

x

sRSD

Systematic error - accuracy

For a known true value, difference of the analytical result tested by null hypothesis and Student (t) test.

Reasons for systematic error:

methodological: wrong method, non-linear signal, incorrect sample preparation, interferents, incorrect sample storage, incorrect sampling etc.

instrumental: incorrectly calibrated instruments

personal: omission of the prescribed procedure, incorrectly prepared or outdated reagents, incorrect calculations etc.

Acceptability of measurement error depends

on the analyte concentration

Analyte concentration Acceptable error as CV

10 - 100 % 0,1 %

1 - 10 % 0,5 %

0,1 - 1 % 0,5 - 1 %

0,01 - 0,1 % 1 - 2 %

0,0001 - 0,01 % 5 %

10–7 - 10–4 % (ppm - ppb) 20 %

< 10–7 % (< ppb) 50 - 100 %

in the evaluation of measurement error (uncertainty) both random AND systematic error

Combined uncertainty of the result - uc(R)

random error

systematic error

...)()( 2

2

2

22

1

22

ba

N

i

i

i

Rc sb

Rs

a

Rxu

x

RsRu

...

baR E

b

RE

a

RE

„Fishbone“ diagram: all parameters contributing to the combined measurement uncertainty of the result

Quality assurance, QA, and quality control,

QC, of the results

aim of analytical method: implementation of the results

specification of the analytical method

accuracy of results checked

validation of analytical method

keeping documentation and laboratory control charts

use of standard procedures (standard operational procedure, SOP) and principles of good laboratory practice, GLP

Validation parameters

selectivity or specificity

linearity

precision: of instrument; repeatability; reproducibility

accuracy: determination of recovery

measurement range: linear, dynamic

limit of detection, LOD: at S/N = 3

limit of quantitation, LOQ: at S/N = 10

robustness