You are on page 1of 10

Journal Code

R C M

Article ID
Dispatch: 08.05.15
2 2 0 No. of Pages: 8

CE:
ME:

Research Article
Received: 10 February 2015

Revised: 27 April 2015

Accepted: 28 April 2015

Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2015, 29, 18


(wileyonlinelibrary.com) DOI: 10.1002/rcm.7220

Improvement of analytical method for chlorine dual-inlet isotope


ratio mass spectrometry of organochlorines
Tetyana Gilevska1, Natalija Ivdra1,2, Magali Bonifacie3 and Hans-Hermann Richnow1,2*
Department of Isotope Biogeochemistry, Helmholtz Centre for Environmental Research UFZ, Permoserstr. 15, D-04318
Leipzig, Germany
2
Isodetect GmbH Company for Isotope Monitoring, Deutscher Platz 5b, D-04103 Leipzig, Germany
3
Group of the Geochemistry of Stable Isotopes, The Institute of Earth Physics of Paris, Sorbonne Paris Cit, Universit Paris
Diderot, UMR 7154 CNRS, F-75005 Paris, France
1

RATIONALE: The development of compound-specic chlorine isotope analysis (Cl-CSIA) is hindered by the lack of

international organochlorine reference materials with isotopic compositions expressed in the 37Cl notation. Thus, a
reliable off-line analytical method is needed, allowing direct comparison of the 37Cl values of molecularly different
organic compounds with that of ocean-water chloride, to refer measurement results to a Standard Mean Ocean Chloride
(SMOC) scale.
METHODS: The analytical method includes sealed-tube combustion of organochlorines, and precipitation and
subsequent conversion of the formed inorganic chlorides into methyl chloride (CH3Cl) for the determination of 37Cl
values by Dual-Inlet Isotope Ratio Mass Spectrometry (DI-IRMS). A sample preparation step most sensitive to the
sample size dissolution of the inorganic copper chlorides formed by combustion of -HCH was identied.
RESULTS: Recovery of 94 5% of chlorine could be reached by applying determined optimal conditions for the
dissolution, implying good external precision of 37Cl values (0.18 0.03, 1, n = 3). Validation of the optimized
method by the analysis of the produced and initial CH3Cl sample with known 37Cl values vs SMOC resulted in a
difference of 0.11 0.04 (1), conrming the external precision and accuracy of the entire method.
CONCLUSIONS: The efciency of the sample preparation method for CH3Cl-DI-IRMS analysis is independent both of
the chemical structure of the chlorinated compound and of the amount of chlorine in the sample. This method has the
potential to be applied to a broad range of chlorinated organic compounds, e.g. reference material for the calibration
of methods for Cl-CSIA against SMOC. Copyright 2015 John Wiley & Sons, Ltd.

Organochlorine pollutants are of great interest to environmental scientists due to their persistence, bioaccumulation
and frequent toxicity.[1] Isotopic compositions analysis has
facilitated better understanding of the environmental fate of
organic chlorinated compounds (OCs) by tracing their sources
and transformation processes.[2,3] Although the determination
of chlorine stable isotope composition (37Cl values) has the
potential to help identify the sources and degradation of
OCs in the environment, its application is mainly limited by
the currently available analytical techniques. In contrast to
the number of efcient high-throughput methods developed
and routinely applied for compound-specic isotope analysis
(CSIA) of carbon, hydrogen, oxygen and nitrogen, methods
for the CSIA of chlorine in organochlorine compounds still
need to be improved for routine applications.[4,5] In the
existing analytical set-ups mixtures of organochlorine

*Correspondence to: H.-H. Richnow, Department of Isotope


Biogeochemistry, Helmholtz Centre for Environmental
Research UFZ, Permoserstr. 15, D-04318 Leipzig, Germany.
E-mail: hans.richnow@ufz.de

These authors contributed equally to this work

Rapid Commun. Mass Spectrom. 2015, 29, 18

compounds are generally separated by gas chromatography


(GC) and then transferred by a carrier gas either to a conversion unit for production of the HCl by high-temperature
combustion (GC-HTC) which is then used for the analysis of
chlorine isotopes,[6,7] or directly to the mass analyzer unit for
measurement of the chlorine isotope ratios.[8,9] Currently,
Isotope Ratio Mass Spectrometry (IRMS),[8,10] Quadrupole
Mass Spectrometry (qMS)[8,1113] and Multiple Collector
Inductively Coupled Plasma-source Mass Spectrometry
(MC-ICPMS)[9] are used as mass analyzers for the
determination of 37Cl values. The majority of the existing
Cl-CSIA methods demand comparison with molecularly
identical reference compounds to refer isotopic ratios of target
analytes to the Standard Mean Ocean Chloride (SMOC) scale,
and for the quality control following the principle of identical
treatment.[14] Thus far organic international standards for
CSIA of chlorine are not available. Therefore, the chlorine
isotope composition of reference materials must be previously
determined by off-line methods which allow direct referencing with ocean-water chloride samples.
Thermal-ionization mass spectrometry (TIMS) and dualinlet isotope ratio mass spectrometry (DI-IRMS) are the mass
spectrometric methods in current use for the determination of
37Cl values directly related to the SMOC scale. As reported

Copyright 2015 John Wiley & Sons, Ltd.

66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65

T. Gilevska et al.

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65

by Rosenbaum et al. for seawater samples,[15] DI-IRMS is best


suited for samples containing >10 mol Cl, allows 37Cl
values to be measured with a precision of 0.1 (2). TIMS
is more sensitive and applicable for samples containing
~0.10.3 mol of Cl with achievable uncertainties of 0.2.
However, these uncertainties do not directly refer to the
37Cl values of OCs since both methods require off-line
conversion of organochlorine compounds into inorganic
chloride, which then can be processed in parallel with
standard samples of ocean-water chloride for the analysis.
Prior to DI-IRMS analyses, chlorine from OCs is rst
S1 transformed into CuCl via combustion with CuO (Scheme 1,
path a)[16,17] or into NaCl by trapping Cl into a sodium
carbonate solution after an oxygen combustion bomb
(Scheme 1, path b).[18] Alternatively, organic chlorine can be
transformed into NaCl via reductive dehalogenation.[19,20]
The obtained inorganic chlorides are then converted into
AgCl (Scheme 1, paths c and d) and subsequently into CH3Cl
for DI-IRMS (Scheme 1, path e).[16] Alternatively, CuCl,
obtained in the combustion reaction, can be directly
converted into CH3Cl for 37Cl measurements by DI-IRMS
(Scheme 1, path f).[2123]
Different physical and chemical processes during the
conversion of OCs could generate isotopic fractionation,
which needs to be thoroughly quantied. It has been reported
that losses in the conversion of CuCl into CH3Cl and during
the purication of CH3Cl could bias the obtained 37Cl value,
leading to a correlation between the recovery of chlorine in the
sample preparation procedure and the accuracy of the 37Cl
values.[17,21] Many attempts have been made to optimize
different sample preparation steps in order to reach the
highest possible recoveries of chlorine and the reproducibility
of 37Cl values for the entire procedure.[17,22,23] However, no
clear relationship between losses at each separate conversion
step and changes in the isotopic compositions has been
reported up to now. In addition, all previously reported
methods were optimized for specic amounts of combusted
chlorinated compound, so the applicability and efciency of
these procedures for samples containing different amounts
of chlorine stayed unrevealed.

Scheme 1. Chlorine from OCs is rst transformed into CuCl


via combustion with CuO (path a)[16,17] or into NaCl by
trapping Cl into a sodium carbonate solution after an oxygen
combustion bomb (path b).[18] Alternatively, organic chlorine
can be transformed into NaCl via reductive dehalogenation.[19,20] The obtained inorganic chlorides are then
converted into AgCl (paths c and d) and subsequently into
CH3Cl for DI-IRMS (path e).[16] Alternatively, CuCl, obtained
in the combustion reaction, can be directly converted into
CH3Cl for 37Cl measurements by DI-IRMS (path f).[2123]

wileyonlinelibrary.com/journal/rcm

In this study we present a sample preparation method prior


to CH3Cl-DI-IRMS, which holds the potential to be used for
the determination of 37Cl values of organochlorines as
reference material for the calibration of the Cl CSIA methods
against SMOC. We selected off-line conversion of OCs into
CH3Cl via CuCl and AgCl, which allows one to obtain isotopic
precisions of 0.15 and high overall recoveries of chlorine
(>97%).[16,24] The rst step of the procedure combustion of
OCs to CuCl is applicable to a broad range of organic
contaminants from small molecules, such as chlorinated
methanes and ethenes,[21,23] to chemically persistent complex
molecules, such as chlorinated pesticides.[17,22]
We have chosen -hexachlorocyclohexane (-HCH, Lindane)
as a model compound for the optimization of the sample
preparation method. -HCH and other isomers of HCH were
used worldwide as agricultural insecticides until they were
banned or restricted to specic applications by the Stockholm
Convention on persistent organic pollutants.[25,26]
In the course of this study, we investigated in detail each
transformation from -HCH into CH3Cl to determine the
inuence on the accuracy of obtained 37Cl values of chlorine
recoveries at different sample preparation steps. The least
efcient steps, leading to changes in the isotopic compositions,
were identied and optimized. Our sample preparation
method is applicable for different sample sizes, as we propose
the optimal water/chlorine ratio for the dissolution step with
the possibility of recalculating the necessary volume of water
for each particular amount of chlorine. We applied the
optimized procedure to a CH3Cl sample with a known 37Cl
value relative to SMOC. Finally, we compared the obtained
37Cl value of the recovered CH3Cl and that of non-processed
CH3Cl to conrm the accuracy and precision of the optimized
method.

EXPERIMENTAL
Solvents and chemicals
Acetone (99.5%), potassium nitrate (KNO3; 99%), citric acid
monohydrate (C6H8O7*H2O; 99.5%), silver nitrate (AgNO3)
and copper(I) chloride (CuCl; 99.9%) were purchased from
Carl Roth (Karlsruhe, Germany). Ultra-high purity (uhp)water (resistivity of 18 M) was prepared with a Merck
Milli-Q A+ system from Millipore (Billerica, MA, USA).
-HCH (99.1%) was purchased from HiMEDIA (Mumbai,
India); CH3Cl (99.90%, #N30) was obtained from Air Liquide
(Paris, France); copper oxide (CuO; >98%) and potassium
phosphate dibasic dihydrate (Na2HPO4*2H2O; 98%) were
obtained from Sigma Aldrich, (Steinheim, Germany). Nitric
acid (HNO3; 69%) was purchased from Merck (Darmstadt,
Germany). Quartz glass (Schott) tubes (20 mm o.d.) were
prepared at the glass workshop of the Helmholtz Centre for
Environmental Research UFZ (Leipzig, Germany).
Initial procedure for conversion of -HCH into CH3Cl
-HCH was converted into CH3Cl prior to Cl DI-IRMS. We
up-scaled the method to 250 mol of Cl to ensure reliable
DI-IRMS analysis over all optimization steps and to
minimize the inuence of a blank on the chlorine sample.
The sample preparation procedure consisted of the following

Copyright 2015 John Wiley & Sons, Ltd.

Rapid Commun. Mass Spectrom. 2015, 29, 18

66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130

Chlorine stable isotope analyses of organic compound


steps: step 1: high-temperature combustion of -HCH to CuCl
in an evacuated sealed quartz ampule with excess of CuO
(Eqn. (1)); step 2: disproportionation of Cu(I)Cl to form
Cu(II)Cl2 and dissolution of the formed soluble CuCl2 in
water (hereafter called dissolution) (Eqn. (2)); step 3:
precipitation of chlorides as AgCl (Eqn. (3)); and step 4:
halogen exchange reaction of AgCl with an excess of CH3I to
form CH3Cl (Eqn. (4)). The obtained gaseous CH3Cl was then
chromatographically separated from excess CH3I and puried
before DI-IRMS at the Institut de Physique du globe de Paris
(IPGP Laboratoire de Gochimie des Isotopes Stables, Paris,
France) following the procedure routinely used to perform
DI 37Cl measurements (see section 2 of the Supporting
Information).[2732] In step 1 the ratio of CuO to the mass of
Cl in the sample was 200 mg CuO per 1 mg of Cl (that is
~7.1 mg for 1 mol of Cl); in accordance with Holmstrand
et al.,[17] the combustion temperature was 620 C[16,17] and
the volume of water for the dissolution process in step 2 was
25 mL.[16] The amounts of chemicals for the precipitation of
AgCl in step 3 were calculated proportionally to the amount
of Cl reported by Jendrzejewski et al.[16] (detailed amounts
and further experimental details of the procedure can be
found in section 1 of the Supporting Information).
step 1 :  HCH 24 CuO9 Cu2 O 6 CuCl
6 CO2 3 H2 O

(1)

step 2 : 2CuIClCu CuIICl2aq

(2)

step 3 : CuCl2aq 2 AgNO3aq CuNO3 2aq


2 AgCl

(3)

step 4 : AgCl CH3 Iexcess CH3 Cl AgI

(4)

complete dissolution of KNO3 and buffer reagents.


Afterwards, 3.75 mL of 1 M AgNO3 solution were added.
The solution was left in darkness to cool down for 1 h and
the suspension was ltered on a glass microber lter and
dried in the darkness at r.t. The obtained AgCl on the lter
was split into 24 subsamples to give approximately 50 mol
of chlorine on each part of the lter. Step 4: each lter part
was then placed in a borosilicate glass tube, excess of CH3I
(100 L) was added, the tube sealed and left at 80 C for 72 h
to form CH3Cl. CH3Cl was then separated from excess CH3I
by preparative gas chromatography using two Porapak-Q
lled columns and analyzed by DI-IRMS as described by
Eggenkamp[32] and Bonifacie et al.[31]
Dual-inlet isotope ratio mass spectrometry (DI-IRMS)
The 37Cl measurements of CH3Cl gas were performed using
a triple collector gas-source dual-inlet mass spectrometer
(Delta plus XP; Thermo Fisher Scientic, Bremen, Germany)
at IPGP. The 37Cl values were obtained by determining the
+
35 +
signal intensity of m/z 52 (CH37
3 Cl ) and m/z 50 (CH3 Cl )
using two different collectors with resistances of 1109
and 3108, respectively. One measurement consisted of a
series of 10 individual comparisons of the ratio 52/50 in the
sample CH3Cl to that of the CH3Cl gas used as a laboratory
standard. The reference gas is compared with CH3Cl
prepared from seawater chloride at least twice a day, and
typically following each 5 to 6 samples. This procedure
checks for instrumental drift during the day, and allows direct
referencing of the 37Cl values of unknown samples to the
SMOC scale.[2830] The chlorine isotope composition of each
produced CH3Cl sample was determined by DI-IRMS twice.
Gas chromatography/mass spectrometry (GC/MS)

Optimized procedure
The entire sample preparation procedure after optimization
consisted of the following steps: step 1: organic sample,
containing 250 2 mol of Cl was transferred to the quartz
ampule (pre-heated at 700 C for 1 h), containing 1.8 g of
CuO (pre-heated at 800 C for 1 h). The ampule was then
evacuated to ~1200 mbar, and sealed, while the lower part
was immersed in liquid nitrogen. The ampule was
afterwards heated in the furnace (with a temperature
increase of 5 C/min towards 620 C and kept at 620 C for
1 h), before being allowed to cool to room temperature (r.t.),
washed with 10% HNO3 and uhp-water. Step 2: the ampule
was broken and all solid residues together with quartz cullet
were transferred to a screw-capped bottle, containing 80 mL
uhp-water, followed by vortex-mixing for 1 min and 1 h of
sonication at 50 C. The solution was decanted from the solid
residues, ltered through a nylon lter (0.22 m) and the
ltration residues were rinsed with an additional 20 mL of
water to obtain a total volume of 100 mL for the rst extract
(H2O/Cl ratio 0.4 mL/mol). Subsequently, 80 mL of fresh
uhp-water were added to the solid residues, the dissolution
procedure was repeated, nished with washing of the
ltration residues with an additional 20 mL of water, and the
two extracts were combined to obtain 200 mL of the nal
solution. Step 3: 20 g of KNO3 and a pH 2.2 buffer (4.5 g of
citric acid*H2O + 140 mg of Na2HPO4*2H2O) in dry form were
added to the obtained CuCl2 solution and heated at 80 C until

Rapid Commun. Mass Spectrom. 2015, 29, 18

The concentration of the organic compounds was determined


by GC/MS to test the conversion of -HCH into copper
chloride. Details of the GC/MS method are available in
section 3 of the Supporting Information.
Ion chromatography (IC)
IC analyses of chlorine concentration were performed at the
Department of Analytical Chemistry at UFZ on a Dionex
ICS-2000 ion chromatograph (Thermo Fisher Scientic).
Detailed information can be found in section 4 of the
Supporting Information.
Chloride blanks
Chloride blanks were analyzed to investigate the total added
amount of chlorine at the dissolution step and through the
entire conversion of OCs into AgCl. Two experiments were
performed, using 1.8 g subsamples of commercially available
CuO. The rst subsample was suspended in 200 mL of uhpwater (as for two subsequent extractions of 250 mol of Cl).
The optimized dissolution procedure was then applied and
the concentration of the chlorine extracted from the mixture
with CuO was measured by IC. The second subsample of
CuO was heated in a sealed evacuated ampule and
subsequently extracted with 200 mL of uhp-water as
described above for the optimal procedure. A 1 mL aliquot
of the ltered solution was taken for the determination of

Copyright 2015 John Wiley & Sons, Ltd.

wileyonlinelibrary.com/journal/rcm

66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65

T. Gilevska et al.
chlorine concentration. The rest of the solution was subjected
to the AgCl precipitation procedure. The mass of the dried
AgCl precipitates and the chloride ion concentration in the
ltrate were determined. From these experiments the amount
of chlorine which may be added over all steps of the entire
procedure was determined.

RESULTS AND DISCUSSION


Chloride blanks
The chlorine concentration, measured by IC, in both prepared
blank solutions was determined as 0.11 mg/L, which
corresponded to 0.1% of the amount of Cl in the initial
organochlorine compound. Furthermore, there was no
measurable amount of AgCl recovered on the lter after the
precipitation procedure. Thus, the chloride blanks both of
the dissolution step (Eqn. (2)) and of the entire optimized
conversion procedure showed no signicant amount of
external chlorine.
Initial procedure: efciency and accuracy

reported by previous studies.[16,17] Such low recovery yields


of chlorine suggest that the methods previously developed
cannot be applied to the higher amount of -HCH without
adjustments. The measured 37Cl value of CH3Cl, produced
from -HCH by the initial procedure, was 1.15 0.34
(n = 2) (Table 1). -HCH is generally produced by the
chlorination of benzene with Cl2 gas, derived from brines,
with 37Cl values typically between 0.5 and 0.[30,33]
The signicantly lower 37Cl value of -HCH that we
observed in experiments with only 23% yield, than the
value of brine, could thus result from chlorine isotopic
fractionation due to preferential loss of 37Cl isotopes. In
addition, the external precision of 0.34 is much larger
than usually achieved for the sample preparation, i.e. from
seawater (0.08, 2), indicating that the sample
preparation procedure used was not optimal under the
applied conditions and for the amount of chlorine taken.
Thus, a revision of all conversion steps with respect to the
37Cl precision and accuracy was conducted, as presented
below.

Revision and optimization of sample preparation steps

The initial procedure for the conversion of -HCH into AgCl


showed low recovery yields of chlorine (23 3% (n = 2)
T1 calculated from the mass of precipitated AgCl) (Table 1). This
is in sharp contrast to the nearly complete conversion yields

The efciency of each individual step (Eqns. (1) to (4)) was


tested by determining the recovery yields (dened as the
recovered amount of Cl, measured by weighing AgCl
precipitates or determination of the chlorine concentration

Table 1. Optimization of sample preparation method with -HCH

Water ratio,
Chlorine
Cl value
(n = 2),
mL/mol Cl recoveries, %
37

Optimization parameters
Initial procedure

0.10

22.2
21.6

Sonication at the dissolution procedure


Vortex mixing for 1 min
Sonication for 2 h
Sonication for 1 day
Sonication for 2 days
Repeated extraction

0.06
0.06
0.06
0.06
0.10

1
32
38
34
57.1
44.9
31
49
53
69
73
76

Enhanced oxygen supply


Elevated temperature
Water volume

0.06
0.06
0.28
2.00
5.00

Conversion of -HCH under optimized conditions


1st extraction

0.40

2nd extraction

0.40

Combinedc

82
93
90
7
6
6
94 5

Average 37Cl
value from multiple
ampulesb,

1.39
0.91

1.15 0.34 (n = 2)

0.60
0.70

-0.63 0.10 (n = 2)

0.12
0.22

0.17 0.08 (n = 2)

0.29
0.25
0.25
0.66
0.89
1.22

0.26 0.02 (1, n = 3)


0.92 0.28 (1, n = 3)
0.18 0.03 (1, n = 3)

Difference between duplicate measurements of the same prepared CH3Cl sample was typically below 0.02.
external precision of sample preparation procedure from different subsamples of -HCH.
c
Combined extract represents the sum of recoveries from two subsequent extractions and calculated 37Cl values, based on
the mass balance.
b

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65

wileyonlinelibrary.com/journal/rcm

Copyright 2015 John Wiley & Sons, Ltd.

Rapid Commun. Mass Spectrom. 2015, 29, 18

66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130

Chlorine stable isotope analyses of organic compound


in the solution by HPLC/the expected amount of Cl). The
potential impact of these losses on the obtained 37Cl values
was then taken into consideration.
Step 4 transformation of AgCl, obtained from seawater
standard solutions, into CH3Cl (Eqn. (4), following exactly
the protocol of Jendrzejewski et al.[16] and routinely used in
IPGP[2830] (Supporting Information, section 2)) proved to
be complete (overall yields close to 100%, including the
purication of CH3Cl from CH3I by preparative GC) and thus
should not inuence the accuracy of the isotope values in the
investigated sequence of steps.
Therefore, for the further revision of the previously
suggested procedure and its adaptation for large samples of
-HCH, we took into account the following parameters: (1)
incomplete combustion of -HCH in step 1; (2) incomplete
precipitation of AgCl in step 3; (3) inefcient dissolution
process in step 2.
(1) Incomplete combustion of -HCH may lead to the
formation of partly dechlorinated organic by-products
associated with lower yields of obtained CuCl and
substantial changes in the 37Cl values of CH3Cl. We tested
the conversion of -HCH into CuCl by combusting -HCH
as described in the initial procedure. After reaction, the
ampule was slowly cooled down to +5 C, then cracked
and the copper oxide particles were dispersed in 10 mL of
acetone to dissolve any unreacted organic material
potentially remaining in the ampule. GC/MS analysis of
the residues after combustion showed that 0.01% of the
initially loaded compound was recovered, conrming
complete conversion of -HCH into inorganic reaction
products. No traces of other organic products of -HCH
degradation were detected. These results were in the good
agreement with reports on complete combustion for
DDTs[17] and polychlorinated hydrocarbons[16,34] and
conrm that the selected combustion conditions are
suitable for structurally different OCs.
(2) Efciency of the AgCl precipitation procedure (Eqn. (3))
was tested by applying it to a solution of NaCl in 25 mL
of water with chlorine ion concentration equal to the
CuCl concentration after 100% conversion of 250 mol
of Cl from -HCH. A test of the precipitation efciency
resulted in 36.6 mg of AgCl being precipitated from the
NaCl solution, corresponding to 102% (the efciency
higher than 100% could result from the uncertainty of
weighting NaCl and/or AgCl). This complete
precipitation of chloride is reinforced by the fact that no
traces of remaining dissolved chlorine were detected by
IC analysis of the ltrate after precipitation.
(3) Dissolution of CuCl. After conrmation that both the
combustion and the precipitation processes are complete
at selected conditions, both in agreement with previously
reported results for entire procedures,[16,17] we identied
that CuCl2 formation and dissolution is the least efcient
step of the conversion of OCs into AgCl. We tested
several modications of this particular step: (i)
sonication, (ii) repeated extraction, (iii) elevated
temperature, (iv) oxygen supply, and (v) water volume.
(i) As previously suggested by Jendrzejewski et al., insufcient
dissolution time may lead to incomplete extraction of
water-soluble chlorine species.[16] Thus, introduction of a

Rapid Commun. Mass Spectrom. 2015, 29, 18

sonication step allowed us to signicantly increase the


total recoveries of chlorine in the dissolution process and
to shorten the overall time needed for this step. We
determined the concentration of dissolved chlorine in the
solution after 1 min, 2 h, 1 day and nally after 2 days of
sonication, corresponding to 1, 32, 38 and 34% of the
initially loaded chlorine, respectively (Table 1). This test
suggests that increasing the sonication time does not
signicantly increase the chlorine recovery yields.
(ii) Subsequently, we tested if the yield of the dissolution
process can be increased by repeated extraction. After
combining two extracts and washings after 2 h of
sonication and subsequent ltration, 51% of the chlorine
was recovered in precipitated AgCl and then converted
into CH3Cl (Table 1). A 13% increase of the efciency in
the combined extract relative to the result achieved in the
previous experiment by a single extraction after 2 h of
sonication showed that it might be necessary to repeat
an extraction from the same solid residues after
combustion. DI-IRMS analysis of combined extracts
showed an elevated 37Cl value of 0.63 0.10 (n = 2)
compared with the value of 1.15 0.34 (n = 2) obtained
through the initial procedure and associated with more
than two times lower overall yields (23%). These results
suggest that the isotopically heavier fraction of chlorine
can still remain undissolved in the residues after an
incomplete dissolution process, leading to biased lower
37Cl values of extracted chlorine. This observation
conrms that it is crucially important to achieve complete
transformation of the dissolution process for the accurate
determination of the 37Cl values.
(iii) During the dissolution step (Eqn. (2)) the water-insoluble
by-product copper oxychloride (Cu3Cl2(OH)4) can be
formed by the oxidation of CuCl by atmospheric oxygen
in the presence of water (Eqn. (5)).
4 CuCl O2 2 H2 OCu3 Cl2 OH4 CuCl2aq

(5)

On the other hand, copper formed in the disproportionation


reaction at the dissolution step (Eqn. (2)) may be then
oxidized with oxygen, shifting the reaction (Eqn. (2))
equilibrium towards the formation of the desired product
(water-soluble CuCl2). Thus, by introducing a gentle oxygen
ow for 2 h into the CuOCuCl water suspension after
combustion, we tested whether an enhanced oxygen supply
affects the prevalence of the disproportionation process (Eqn.
(2)) over the competing oxidation reaction (Eqn. (5)) to obtain
maximum possible recoveries of chlorine. An enhanced
oxygen supply caused only insignicant improvement of
the efciency of dissolution, allowing us to recover 31% of
chlorine in the 15 mL of the extraction solution (Table 1).
Thus, the negative effects of the possible side-reaction (Eqn.
(5)) were negligible or compensated for by the improved
kinetics of the dissolution (Eqn. (2)) due to the better mixing
of the solution. Thus, an enhanced oxygen supply was not
considered as an optimization measure.
(iv) We tested the impact of elevated temperature on the
efciency of the dissolution process by heating the
suspension of CuOCuCl, formed in step 1, at 90 C for
2 h in a water bath. Formation of HCl in a further reaction
of the combustion products CuCl and water (Eqn. (6)),

Copyright 2015 John Wiley & Sons, Ltd.

wileyonlinelibrary.com/journal/rcm

66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65

T. Gilevska et al.

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65

followed by its volatilization, was reported by Holt and


Sturchio,[21] as probably being the major source of the
losses of chlorine, associated with more variable 37Cl
values.
2 CuCl H2 OCu2 O 2 HCl

(6)

To prevent any losses of chlorine in the form of HCl, the heating


was performed in screw-capped bottles. Elevated temperature
at the dissolution step allowed us to reach 49% conversion of
chlorine (compared with 34% conversion, obtained with the
same water/Cl ratio by 2 days of sonication at the room
temperature), showing the temperature-enhanced kinetics of
the dissolution process (Eqn. (2)). Thus, we used a temperature
of 50 C in the ultrasonic bath during the sonication for
further experiments.
(v) The most signicant effect on the efciency of the
dissolution process was caused by changing the volume
of water during extraction of the soluble chlorine species
(Eqn. (2)) after combustion. By increasing the water/Cl
ratio from 0.1 to 5 mL/mol 76% Cl recovery was reached.
The recovered Cl had a 37Cl value of 0.17 0.08 (n = 2),
that is 0.98 higher than the value of 1.15 0.34
obtained for the lowest recoveries of chlorine in the initial
procedure, and thus exhibiting the same trend towards
increasing 37Cl value with increasing chlorine recovery
yields. In an additional set of experiments with dissolution
of commercially available crystalline CuCl in different
volumes of uhp-water (ratio mL/mol Cl from 0.36 to
8.00, section 5, Supporting Information) we determined
the optimal water/Cl ratio as 0.4 mL/mol, resulting in
96% chlorine recovery (Supplementary Table S-1,
Supporting Information). Thus, a water/Cl ratio of
0.4 mL/mol was used for the further optimization steps.

37Cl value of 0.18 0.03 (n = 3) more accurately represents


the isotopic composition of the organochlorine compound
studied here. We hence conclude that incomplete chlorine
extraction leads to analytically biased too low 37Cl values of
the studied organochlorine compounds. Therefore, we
recommend applying repeated extractions to obtain complete
chlorine recovery and thus achieve accurate and precise
determinations of the Cl isotopic compositions of organochlorine compounds.
Conversion of CH3Cl under optimized conditions
The determined optimal conditions of the dissolution process
were incorporated into the full procedure for the conversion
of commercially available CH3Cl gas with known isotopic
composition (Table 2). The optimized conversion procedure
that we dened here allowed CH3Cl to be transformed into
water-soluble CuCl2 and back to CH3Cl with a slight off-set
of 0.11 0.04 (1, n = 3) (Table 2), which shows an
improvement in comparison with a decrease of 0.23
0.05 (1) for the entire sample preparation procedure
with 89% of recoveries reported by Holt et al.[21]
Isotopic precision of the method
The external precision of all the 37Cl values of seawater
independently prepared and analyzed over the course of this
study was 0.07 (standard deviation of 15 measurements);
the shift within 1 day was in the range from 0.02 to 0.07
(n = 2). The difference between duplicate DI-IRMS analyses
of two introduced subsamples of the same produced CH3Cl
was 0.02. The external precision of the method,
determined from different ampules with identical starting
material (-HCH), was 0.03% (1, n = 3) (Table 1). Similarly,
the precision of the 37Cl values, obtained through the

Table 2. Validation of the overall optimized procedure


with CH3Cl
Conversion of -HCH under optimized conditions
We applied the total set of optimized parameters (repeated
extraction by sonication for 2 h at 50 C with two times
100 mL of water, corresponding to a water/Cl ratio of
0.4 mL/mol) to prove the efciency of the full procedure
both in terms of complete chlorine recoveries and in the
reproducibility of the determined 37Cl values (Table 1). To
test if the remaining residues of chlorine species after a noncomplete dissolution process have the same chlorine isotopic
composition as initially dissolved CuCl2, fractions from two
repeated extractions were separately analyzed for chlorine
contents and 37Cl values. The optimized extraction
procedure was performed in triplicate from three combusted
subsamples of -HCH. Chlorine concentration and DI-IRMS
measurements showed that 88% of the chlorine, with a 37Cl
value of 0.26 0.02 (n = 3), can be recovered in the rst
extraction of CuCl2, but the second recovered fraction of
soluble chlorine salt (6%) was signicantly enriched in 37Cl,
exhibiting a 37Cl value of 0.92 0.28 (n = 3). We have
calculated the combined 37Cl value of the two extracts with
the 94 5% of recovered chlorine (sum of recoveries from 1st
and 2nd fractions), based on the mass balance. The obtained

wileyonlinelibrary.com/journal/rcm

After the entire sample preparation


procedure
37Cl
value of
commercial
CH3Cl,
1.06 0.02

37Cl
valuea
(n = 2),
1.18
1.13
1.20

Average
37Cl
value 1b
from 3
ampules,

37Clc,

1.17 0.04

0.11 0.04

Difference between duplicate measurements of the same


prepared CH3Cl sample, produced by subsequent
transformation of commercial CH3Cl to soluble CuCln,
precipitation of AgCl and transformation back to the
CH3Cl for the DI-IRMS analysis, was typically below 0.01.
b
External precision of sample preparation procedure from
different subsamples of CH3Cl.
c
Difference between 37Cl value of unprocessed CH3Cl and
that of CH3Cl after the entire sample preparation
procedure.

Copyright 2015 John Wiley & Sons, Ltd.

Rapid Commun. Mass Spectrom. 2015, 29, 18

66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
T2 81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130

Chlorine stable isotope analyses of organic compound


sample preparation procedure from CH3Cl, was 0.04
(1, n = 3) (Table 2). This external precision for OCs is of
the order of the uncertainty related to the preparation of
sea water standards (0.08, 2), when only inorganic
chloride is processed to CH3Cl.
Such a good external precision for the studied compounds
(-HCH and CH3Cl) proves the applicability of the optimized
method for the chlorine isotope analysis of organochlorines.

CONCLUSIONS
We present a method for the determination of the chlorine
stable isotope composition of -HCH by DI-IRMS in 37Cl
notation vs SMOC, using seawater for direct referencing. In
the course of this study the links between losses of chlorine at
every separate conversion step and accuracy of the measured
37Cl values were quantied. Dissolution of water-soluble
chlorine salts, formed by combustion of -HCH, was identied
to be the critical step, most sensitive to the amount of chlorine in
the sample and associated with signicant losses of chlorine.
Incomplete chlorine extraction at this dissolution step led to
inaccurate lower 37Cl values of the studied organochlorine
compound. Optimization of this step resulted in complete
chlorine recoveries through the entire sample preparation
procedure (94 5%) and more accurately determined the
isotopic composition of -HCH (0.18 0.03, 1), which
was enriched in 37Cl by 0.97 in comparison with the nonoptimized initial procedure. Thus, we recommend the
developed optimized chlorine extraction method for the
determination of the 37Cl compositions of organochlorine
compounds. The optimized sample preparation procedure
should be applicable for different amounts of chlorine in the
sample. Validation of the procedure with a CH3Cl sample with
known isotopic composition proved the method to be accurate
and precise and showed a total deviation of 37Cl results of less
than 0.11 0.04 (1, n = 3).
As the most critical step is not related to the oxidation of
the chlorinated compound and, thus, is independent of its
chemical structure, the presented optimized procedure holds
the potential for 37Cl determination of a broad range of
chlorinated organic compounds.

Acknowledgements
We gratefully acknowledge H. G. M. Eggenkamp, Thomas
Giunta, Gerard Bardoux and Michaela Wunderlich for
analytical support and helpful discussions on the improvement of applied methods, as well as Kristina Hitzfeld, Julian
Renpenning and Angela Woods for critical comments. We
acknowledge nancial support from the European Union
under FP7-People-ITN-2010 (Grant Agreement No. 264329).

REFERENCES
[1] A. Cincinelli, F. Pieri, Y. Zhang, M. Seed, K. C. Jones.
Compound specic isotope analysis (CSIA) for chlorine
and bromine: A review of techniques and applications to
elucidate environmental sources and processes. Environ.
Pollut. 2012, 169, 112.

Rapid Commun. Mass Spectrom. 2015, 29, 18

[2] Y. Abe, R. Aravena, J. Zop, B. Parker, D. Hunkeler.


Evaluating the fate of chlorinated ethenes in streambed
sediments by combining stable isotope, geochemical and
microbial methods. J. Contam. Hydrol. 2009, 107, 10.
[3] M. Blessing, T. C. Schmidt, R. Dinkel, S. B. Haderlein.
Delineation of multiple chlorinated ethene sources in an
industrialized area A forensic eld study using
compound-specic isotope analysis. Environ. Sci. Technol.
2009, 43, 2701.
[4] T. C. Schmidt, L. Zwank, M. Elsner, M. Berg, R. U.
Meckenstock, S. B. Haderlein. Compound-specic stable
isotope analysis of organic contaminants in natural
environments: a critical review of the state of the art,
prospects, and future challenges. Anal. Bioanal. Chem. 2004,
378, 283.
[5] M. Elsner, M. A. Jochmann, T. B. Hofstetter, D. Hunkeler, A.
Bernstein, T. C. Schmidt, A. Schimmelmann. Current
challenges in compound-specic stable isotope analysis of
environmental organic contaminants. Anal. Bioanal. Chem.
2012, 403, 2471.
[6] K. L. Hitzfeld, M. Gehre, H. H. Richnow. A novel online
approach to the determination of isotopic ratios for
organically bound chlorine, bromine and sulphur. Rapid
Commun. Mass Spectrom. 2011, 25, 3114.
[7] J. Renpenning, K. L. Hitzfeld, T. Gilevska, I. Nijenhuis, M.
Gehre, H. H. Richnow. Development and validation of an
universal interface for compound-specic stable isotope
analysis of chlorine (Cl-37/Cl-35) by GC-high-temperature
conversion (HTC)-MS/IRMS. Anal. Chem. 2015, 87, 2832.
[8] A. Bernstein, O. Shouakar-Stash, K. Ebert, C. Laskov, D.
Hunkeler, S. Jeannottat, K. Sakaguchi-Soder, J. Laaks, M.
A. Jochmann, S. Cretnik, J. Jager, S. B. Haderlein, T. C.
Schmidt, R. Aravena, M. Elsner. Compound-specic
chlorine isotope analysis: A comparison of gas
chromatography/isotope ratio mass spectrometry and gas
chromatography/quadrupole mass spectrometry methods
in an interlaboratory study. Anal. Chem. 2011, 83, 7624.
[9] M. R. M. D. Van Acker, A. Shahar, E. D. Young, M. L.
Coleman. GC/multiple collector-ICPMS method for
chlorine stable isotope analysis of chlorinated aliphatic
hydrocarbons. Anal. Chem. 2006, 78, 4663.
[10] O. Shouakar-Stash, R. J. Drimmie, M. Zhang, S. K. Frape.
Compound-specic chlorine isotope ratios of TCE, PCE
and DCE isomers by direct injection using CF-IRMS. Appl.
Geochem. 2006, 21, 766.
[11] K. Sakaguchi-Soder, J. Jager, H. Grund, F. Matthaus, C.
Schuth. Monitoring and evaluation of dechlorination
processes using compound-specic chlorine isotope
analysis. Rapid Commun. Mass Spectrom. 2007, 21, 3077.
[12] C. Aeppli, H. Holmstrand, P. Andersson, O. Gustafsson.
Direct compound-specic stable chlorine isotope analysis
of organic compounds with quadrupole GC/MS using
standard isotope bracketing. Anal. Chem. 2010, 82, 420.
[13] B. A. Jin, C. Laskov, M. Rolle, S. B. Haderlein. Chlorine
isotope analysis of organic contaminants using GC-qMS:
Method optimization and comparison of different
evaluation schemes. Environ. Sci. Technol. 2011, 45, 5279.
[14] R. A. Werner, W. A. Brand. Referencing strategies and
techniques in stable isotope ratio analysis. Rapid Commun.
Mass Spectrom. 2001, 15, 501.
[15] J. M. Rosenbaum, R. A. Cliff, M. L. Coleman. Chlorine stable
isotopes: A comparison of dual inlet and thermal ionization
mass spectrometric measurements. Anal. Chem. 2000, 72,
2261.
[16] N. Jendrzejewski, H. G. M. Eggenkamp, M. L. Coleman.
Sequential determination of chlorine and carbon isotopic
composition in single microliter samples of chlorinated
solvent. Anal. Chem. 1997, 69, 4259.

Copyright 2015 John Wiley & Sons, Ltd.

wileyonlinelibrary.com/journal/rcm

66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65

T. Gilevska et al.
[17] H. Holmstrand, P. Andersson, O. Gustafsson. Chlorine
isotope analysis of submicromole organochlorine samples
by sealed tube combustion and thermal ionization mass
spectrometry. Anal. Chem. 2004, 76, 2336.
[18] L. I. Wassenaar, G. Koehler. On-line technique for the
determination of the delta Cl-37 of inorganic and total organic
Cl in environmental samples. Anal. Chem. 2004, 76, 6384.
[19] M. Numata, N. Nakamura, T. Gamo. Precise measurement
of chlorine stable isotopic ratios by thermal ionization mass
spectrometry. Geochem. J. 2001, 35, 89.
[20] M. Numata, N. Nakamura, H. Koshikawa, Y. Terashima.
Chlorine stable isotope measurements of chlorinated
aliphatic hydrocarbons by thermal ionization mass
spectrometry. Anal. Chim. Acta 2002, 455, 1.
[21] B. D. Holt, N. C. Sturchio, T. A. Abrajano, L. J. Heraty.
Conversion of chlorinated volatile organic compounds to
carbon dioxide and methyl chloride for isotopic analysis of
carbon and chlorine. Anal. Chem. 1997, 69, 2727.
[22] C. M. Reddy, N. J. Drenzek, T. I. Eglinton, L. J. Heraty, N. C.
Sturchio, V. J. Shiner. Stable chlorine intramolecular kinetic
isotope effects from the abiotic dehydrochlorination of
DDT. Environ. Sci. Pollut. Res. 2002, 9, 183.
[23] O. Shouakar-Stash, S. K. Frape, R. J. Drimmie. Stable hydrogen,
carbon and chlorine isotope measurements of selected
chlorinated organic solvents. J. Contam. Hydrol. 2003, 60, 211.
[24] C. Aeppli, D. Bastviken, P. Andersson, O. Gustafsson.
Chlorine isotope effects and composition of naturally
produced organochlorines from chloroperoxidases, avindependent halogenases, and in forest soil. Environ. Sci.
Technol. 2013, 47, 6864.
[25] J. Vijgen, P. C. Abhilash, Y. F. Li, R. Lal, M. Forter, J. Torres, N.
Singh, M. Yunus, C. G. Tian, A. Schaffer, R. Weber.
Hexachlorocyclohexane (HCH) as new Stockholm Convention
POPs a global perspective on the management of Lindane
and its waste isomers. Environ. Sci. Pollut. Res. 2011, 18, 152.
[26] H. van den Berg. Global status of DDT and its alternatives
for use in vector control to prevent disease. Environ. Health
Perspect. 2009, 117, 1656.

[27] H. G. M. Eggenkamp, J. J. Middelburg, R. Kreulen.


Preferential diffusion of Cl-35 relative to Cl-37 in sediments
of Kau Bay, Halmahera, Indonesia. Chem. Geol. 1994, 116,
317.
[28] M. Bonifacie, C. Monnin, N. Jendrzejewski, P. Agrinier, M.
Javoy. Chlorine stable isotopic composition of basement
uids of the eastern ank of the Juan de Fuca Ridge (ODP
Leg 168). Earth Planet. Sci. Lett. 2007, 260, 10.
[29] M. Bonifacie, J. L. Charlou, N. Jendrzejewski, P. Agrinier, J.
P. Donval. Chlorine isotopic compositions of high
temperature hydrothermal vent uids over ridge axes.
Chem. Geol. 2005, 221, 279.
[30] A. Godon, N. Jendrzejewski, H. G. M. Eggenkamp, D. A.
Banks, M. Ader, M. L. Coleman, F. Pineau. A crosscalibration of chlorine isotopic measurements and
suitability of seawater as the international reference
material. Chem. Geol. 2004, 207, 1.
[31] M. Bonifacie, N. Jendrzejewski, P. Agrinier, M. Coleman, F.
Pineau, M. Javoy. Pyrohydrolysis-IRMS determination of
silicate chlorine stable isotope compositions. Application
to oceanic crust and meteorite samples. Chem. Geol. 2007,
242, 187.
[32] H. G. M. Eggenkamp, Utrecht University 1994.
[33] H. G. M. Eggenkamp, R. Kreulen, A. F. Koster van Groos.
Chlorine stable isotope fractionation in evaporites. Geochim.
Cosmochim. Acta 1995, 59, 5169.
[34] N. Jendrzejewski, H. G. M. Eggenkamp, M. L. Coleman.
Characterisation of chlorinated hydrocarbons from
chlorine and carbon isotopic compositions: scope of
application to environmental problems. Appl. Geochem.
2001, 16, 1021.

SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of this article at the publisher's website.

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65

wileyonlinelibrary.com/journal/rcm

Copyright 2015 John Wiley & Sons, Ltd.

Rapid Commun. Mass Spectrom. 2015, 29, 18

66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130

USING e-ANNOTATION TOOLS FOR ELECTRONIC PROOF CORRECTION


Required software to e-Annotate PDFs: Adobe Acrobat Professional or Adobe Reader (version 7.0 or
above). (Note that this document uses screenshots from Adobe Reader X)
The latest version of Acrobat Reader can be downloaded for free at: http://get.adobe.com/uk/reader/
Once you have Acrobat Reader open on your computer, click on the Comment tab at the right of the toolbar:

This will open up a panel down the right side of the document. The majority of
tools you will use for annotating your proof will be in the Annotations section,
pictured opposite. Weve picked out some of these tools below:

1. Replace (Ins) Tool for replacing text.

2. Strikethrough (Del) Tool for deleting text.

Strikes a line through text and opens up a text


box where replacement text can be entered.
How to use it

Strikes a red line through text that is to be


deleted.
How to use it

Highlight a word or sentence.

Highlight a word or sentence.

Click on the Replace (Ins) icon in the Annotations


section.

Click on the Strikethrough (Del) icon in the


Annotations section.

Type the replacement text into the blue box that


appears.

3. Add note to text Tool for highlighting a section


to be changed to bold or italic.

4. Add sticky note Tool for making notes at


specific points in the text.

Highlights text in yellow and opens up a text


box where comments can be entered.
How to use it

Marks a point in the proof where a comment


needs to be highlighted.
How to use it

Highlight the relevant section of text.

Click on the Add note to text icon in the


Annotations section.

Click on the Add sticky note icon in the


Annotations section.

Click at the point in the proof where the comment


should be inserted.

Type the comment into the yellow box that


appears.

Type instruction on what should be changed


regarding the text into the yellow box that
appears.

USING e-ANNOTATION TOOLS FOR ELECTRONIC PROOF CORRECTION

5. Attach File Tool for inserting large amounts of


text or replacement figures.

6. Add stamp Tool for approving a proof if no


corrections are required.

Inserts an icon linking to the attached file in the


appropriate pace in the text.
How to use it

Inserts a selected stamp onto an appropriate


place in the proof.
How to use it

Click on the Attach File icon in the Annotations


section.

Click on the Add stamp icon in the Annotations


section.

Click on the proof to where youd like the attached


file to be linked.

Select the file to be attached from your computer


or network.

Select the stamp you want to use. (The Approved


stamp is usually available directly in the menu that
appears).

Click on the proof where youd like the stamp to


appear. (Where a proof is to be approved as it is,
this would normally be on the first page).

Select the colour and type of icon that will appear


in the proof. Click OK.

7. Drawing Markups Tools for drawing shapes, lines and freeform


annotations on proofs and commenting on these marks.
Allows shapes, lines and freeform annotations to be drawn on proofs and for
comment to be made on these marks..

How to use it

Click on one of the shapes in the Drawing


Markups section.

Click on the proof at the relevant point and


draw the selected shape with the cursor.

To add a comment to the drawn shape,


move the cursor over the shape until an
arrowhead appears.

Double click on the shape and type any


text in the red box that appears.

For further information on how to annotate proofs, click on the Help menu to reveal a list of further options: