The Optimisation and Validation of a Method to Determine Organic
Pollutants in Water Samples
Speaker(s)/Author: C. Schoeman
Co-Author(s): S. Mnguni
Rand Water Analytical Services
PO Box 3526, Vereeniging,1930, South Africa
e-mail: cshoema@randwater.co.za
e-mail: smnguni@randwater.co.za
Phone: 016 430 8408 Fax: 016 455 2055
Abstract
Limited legislation for organic pollutants in SANS 241 necessitated a monitoring programme
for these compounds at Rand Water. Both target and screening analyses are necessary and
any extraction methodology should preferably suit both analyses and should be able to detect
compounds in the low ppt range. Also, catchment and treated waters must both be extracted
with this method.
The optimisation and validation of a Solid Phase Extraction (SPE) method is discussed. A
lack of reproducibility in SPE analyses (evidenced by an inconsistent recoveries of the
triazines and endrin) using a particular brand of SPE cartridges was observed and led to an
investigation of alternative SPE cartridges. At least one manufacturer has recognised these
inconsistencies in SPE cartridges and has revamped their entire production process in an
effort to rectify this.
Organic pollutants that were investigated include Pesticides, Polynuclear Aromatic
Hydrocarbons and Polychlorinated Biphenyls. Phthalates are excluded as the method cannot
yield clean blanks due to ubiquitous nature of these compounds. The extracts are analysed
using Gas Chromatography-Time of Flight Mass Spectrometry.
The SPE parameters that were investigated included the SPE phases themselves (traditional
silica C18 and divinylbenzene) from different suppliers, the SPE tube dimensions and the
desorption solvents.
The method is also used for screening of water samples it is necessary for desorption of the
compounds to be optimal and to achieve this different solvents are investigated.
The validation of the method is discussed and parameters that will be included are the Limits
of Detection and Quantitation; Accuracy and Bias; Uncertainty of the method; and the
robustness of the method. Validations for methods operating in the low ng/L range are a
challenge and the difficulties encountered are discussed.
This work is on-going and as existing SPE phases are improved and new phases are
developed further investigations into their effectiveness in our application will be
investigated.
1. Introduction
The determination of organic pollutants in water samples is a priority for any water authority
and limited legislation for these compounds in SANS 241 necessitates some form of in-house
monitoring programme. Both target and screening analyses are necessary and any extraction
methodology should preferably suit both analyses and should be able to detect compounds in
the low ppt range. Also, catchment and treated waters must both be extracted with this
method.
The optimisation and validation of a SPE method is discussed. A lack of reproducibility in
SPE analyses (evidenced by an inconsistent recoveries of, amongst others, triazines and
endrin) using a particular brand of SPE cartridges was observed. This led to an investigation
of alternative SPE cartridges. At least one manufacturer has recognised these inconsistencies
in SPE cartridges and has revamped their entire production process in an effort to rectify this.
Organic pollutants include Pesticides, PAHs and PCBs. Phthalates are excluded as the
method cannot yield clean blanks due to ubiquitous nature of these compounds. The extracts
are analysed using GC-TOFMS.
The SPE parameters that were investigated included the SPE phases themselves (traditional
silica C18 and divinylbenzene); the SPE tube dimensions; and the desorption solvents. SPE
cartridges from 5 different manufacturers were evaluated and phases included two
divinylbenzene and three C18 SPE’s. The dimensions of the SPE tubes are also considered to
be particularly important as low volumes of solvent are preferred for desorption of
compounds from the SPE cartridges. Narrower SPE tubes are better suited to a more
thorough interaction between the solvent and the trapping material. The disadvantage of such
SPE tubes is that they contain less material and their trapping capacity can be exceeded. Flow
rates are also reduced because of the smaller diameters of these SPE cartridges and this will
be particularly evident in raw water samples.
Because the method is also used for screening of water samples it is necessary for desorption
to be optimal for as many compound classes as possible and to achieve this different solvents
are investigated. The solvents that were investigated were toluene, ethyl acetate and
dichloromethane.
The validation of the method is discussed and parameters that will be included are the Limits
of Detection and Quantitation; Accuracy and Bias; Uncertainty of the method; and the
robustness of the method. Validations for methods operating in the low ng/L range are a
challenge and the difficulties encountered are discussed.
This work is on-going and as existing SPE phases are improved and new phases are
developed further investigations into their effectiveness in our application will be
investigated.
2. Experimental
2.1. Reagents and SPE Cartridges
2.1.1 Gas required for GC-TOFMS, helium of 99,9999%.
2.1.2 SPE Cartridges
2.1.2.1 Supplier A divinylbenzene 60 mg/3 ml
2.1.2.2 Supplier A divinylbenzene 200 mg/6 ml
2.1.2.3 Supplier B divinylbenzene (Plexa) 60 mg/1 ml
2.1.2.4 Supplier B C18 Silica (Nexus) 60 mg/3 ml
2.1.2.5 Supplier C C18 Silica 200 mg/6 ml
2.1.3 Ethyl acetate, dichloromethane, methanol, and acetone (GPR).
2.1.4 Conc. Phosphoric Acid (GPR).
2.1.5 De-ionised water. Method blanks must be run with every set of samples to
ensure that this water is free of organic compounds that may interfere with the
analysis.
2.1.6 Sodium Sulphate cartridges.
2.2. Instrumentation
2.2.1
2.2.2
2.2.3
Agilent20 place SPE manifold, vacuum pump
Techne Sample Concentrator
Agilent 7890 Gas Chromatograph-LECO Pegasus HT Time Of Flight Mass
Spectrometer.
2.3 Abbreviations
SPE
GC-TOFMS
GC
PAHs
PCBs
µl
°C
=
=
=
=
=
=
=
Solid Phase Extraction
Gas Chromatography-Time Of Flight Mass Spectrometry
Gas Chromatography
Polynuclear Aromatic Hydrocarbons
Polychlorinated Biphenyls
Microliters
Degrees Centigrade
2.4. Extraction Procedure
2.4.1
2.4.2
2.4.3
SPE cartridges are conditioned sequentially with dichloromethane, ethyl
acetate, methanol and de-ionised water (containing 10 ml methanol and 1 ml
phosphoric acid per litre) taking care not to allow the cartridges to dry
between eluents.
10ml Methanol is added to samples which are then eluted through the
cartridges at a flow rate not exceeding 10ml/min. Once the sample have eluted
the cartridge they are allowed to dry under vacuum for about 10 minutes.
Sodium Sulphate cartridges are placed below the SPE cartridges to remove
any water. Adsorbed compounds are desorbed from the cartridges by passing a
series of solvents through the cartridges under a gentle vacuum. Solvents are
collected in a GC vial. The solvents used are
(2.4.4
toluene)
ethyl acetate
dichloromethane
The extracts are then gently blown with nitrogen down to about 150 µl at 40
40°C and made up 200 µl with ethyl acetate.
This extract is then analysed on the GC-TOFMS.
2.5 Comparisons
Comparison of different capacity/size divinylbenzene SPEs from the same supplier (Supplier
A) were compared. The initial inconsistent results using a divinylbenzene 200 mg/6 ml SPE
and low desorption solvent volumes (500 µl) indicated that SPE cartridges of smaller
dimensions (60 mg/3 ml) were more suitable and all further work was undertaken using the
smaller SPE cartridges.
2.5.1
Evaluation of different desorption solvents.
It was feared that adsorbed compounds of interest were remaining on the SPE
cartridges and an investigation into an addition rinse step using toluene was
carried out. The desorption is usually facilitated by sequential rinses of 2x 500
µl ethyl acetate followed by a 500 µl rinse with dichloromethane. The results
are shown in Table 1.
2.5.2
Comparison of Different Sorbents
The Divinylbenzene from two suppliers (Supplier A and B)were compared
and results are shown in Table 2. Also compared were a divinylbenzene and
C18 silica SPEs from the same supplier (Supplier B). Over seventy compounds
are determined using this method, only selected compounds are shown for
comparative purposes.
2.5.3
The Effect of Increasing the Desorption Rinse Volume
Rinse volumes were also varied to determine if increasing the rinses to 2x 2ml
of ethyl acetate followed by 2ml of dichloromethane would increase the
analyte recoveries.
Such desorptions necessitated desorption in test tubes, rather than the usual
desorption into GC vials.
3. Results and Discussion
Table 1. The Effect of Toluene on Recoveries
DVB, normal
DVB, plus
Compound
desorption
toluene
A-BHC
3.5
3.8
Hexachlorobenzene
5.6
5.2
Simazine
51.5
63.1
Atrazine
37.5
29.2
Propazine
33.8
46.4
B-BHC
17.9
20.0
Pentachlorophenol
44.1
27.1
G-BHC (lindane)
18.2
19.2
Results are expressed in ng/L.
DVB, normal
desorption
16.3
15.1
0.0
0.0
0.0
14.0
37.4
15.3
DVB, plus
toluene
17.2
21.5
0.0
0.0
0.0
16.8
0.0
16.4
The results indicate that the additional toluene desorption has not been particularly effective,
and in some cases, has in fact been detrimental. This, added to the fact that method blanks are
also more complex with the toluene, indicates that toluene should rather be omitted from the
desorption procedure. The toluene (results not shown) does enhance the recovery of the larger
PAHs but since these recoveries are already sufficient for the method requirements are not
worth the added effort of using the toluene with the other desorption solvents.
Table 2 Comparison of SPE Sorbents
Compound
Supplier A DVB Supplier B DVB Supplier B C18
A-BHC
21.4
3.5
16.3
Hexachlorobenzene
23.0
5.6
15.1
Simazine
57.7
51.5
0.0
Atrazine
31.6
37.5
0.0
Propazine
40.0
33.8
-18.8
B-BHC
17.1
17.9
14.0
Pentachlorophenol
35.6
44.1
37.4
G-BHC (lindane)
16.5
18.2
15.3
Results are expressed in ng/L.
The above results shown in Table 2 indicate, that using the parameters listed, the
divinylbenzene SPE is superior for the extraction of the compounds of interest. The C 18, for
many of the compounds, yields poor recoveries. Also the divinylbenzene SPEs from different
suppliers are also not the same and for certain compounds were markedly different.
Additional experiments that compared matrix-spiked calibration standards using the different
divinylbenzene SPEs further reinforce these observations (results not shown). The
compounds that are more difficult to extract display erratic calibration curves while those
compounds that are uncomplicated are unaffected indicating the extraction procedures
themselves are rugged and suitable.
Table 3. Recoveries using different rinse volumes
Compound Name (ug/L) GC vial Desorption Test tube desorption % improvement
a-BHC
82.1
83.3
2
HCB
60.9
99.7
64
Simazine
73.8
84.7
15
Atrazine
89.9
97.7
9
b-BHC
91.0
102.7
13
g-BHC (lindane)
81.4
80.7
-1
Results are expressed in ng/L and %.
The above results show that recoveries can be enhanced when larger volumes of rinse
solvents are used during desorptions. Once again, these better recoveries come at the expense
of more complex method blanks that result from the additional solvent volumes and
additional glassware.
Such recovery enhancements should only be used when the method detection limits cannot be
met using standard desorption procedures.
4. Validation Data
Table 4. Method Validation Statistics
Range,
F
COMPOUND
ng/L
Factor
A-BHC
0-100
984
Hexachlorobenzene 0-100
989
Simazine
0-100
3244
Atrazine
0-100
1945
Propazine
0-100
1064
B-BHC
0-100
1222
Pentachlorophenol 0-100
231
G-BHC (lindane)
0-100
1039
LOD,
ng/L
6
6
3
4
5
5
9
6
Table 5. Method Uncertainties
COMPOUND
Relative Expanded
A-BHC
Hexachlorobenzene
Simazine
Atrazine
Propazine
B-BHC
Pentachlorophenol
G-BHC (lindane)
LOQ,
ng/L
20
20
10
13
18
18
29
20
Precision,
%
7.0
5.7
3.2
2.0
1.7
2.5
13.4
16.0
Accuracy,
%
109.0
83.3
110.1
113.8
114.4
104.0
114.5
105.1
Bias,
%
9.0
-16.7
10.1
13.8
14.4
4.0
14.5
5.1
Uncertainty, ng/L
Uncertainty, %
Concentration
Uncertainty
20.7
24.6
13.4
11.7
13.9
15.5
51.0
26.0
44
33
44
46
46
42
46
42
9
8
6
5
6
6
23
11
5. Conclusions
The result indicated that variations between SPE cartridges do exist and that care should be
taken, for example, when switching between different brands and even between different
batches of the same cartridges from the same supplier.
It is critical that quality checks are carried regularly to ensure consistent and reliable results.
Acknowledgements
This study was sponsored by Rand Water as part of their strategic initiative towards the
monitoring of semi-volatile organic pollutants in water samples.