determination of organic pollutants in environmental · steps of analytical method in the...
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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