Proceedings of the Institute of Natural Sciences, Nihon University
No.48（2013）pp.231 − 242
Simultaneous Determination of 15 Halocarbons at Pico- to Nano - Mol
per Liter Levels in Water and Biological Samples Using Dynamic
Headspace Extraction and Gas Chromatography − Mass - Spectrometry
Gen TANIAI ＊ , Hiroshi ODA ＊＊ , Yuki YONEYAMA ＊ , Minami ABE ＊ , Takashi YAMAKOSHI ＊ ,
Keisuke AMBIRU ＊ , Michiko KURIHARA ＊＊＊ and Shinya HASHIMOTO ＊＊＊
（Accepted November 14, 2012）
Halocarbons emitted from ecosystems play key roles in the degradation of the tropospheric and stratospheric ozone.
In the estimation of the global budget, known sources of halocarbons have been considered insufficient with respect to
the global sink. Therefore, it remains necessar y to identify new sources of halocarbons. To date, however, ver y few
marine phytoplankton and bacteria have been studied. Research into halocarbon production from marine sources
traditionally relies on purge-and-trap techniques, but these cannot be used to measure halocarbons in foam-forming
samples (e.g., bacterial culture). An alternative method, an automated dynamic headspace (DHS) extraction technique
with the gas chromatography − mass spectrometr y (GC/MS) method, was applied to the simultaneous analysis of 15
−1
halocarbons in aqueous and bacterial samples at pico- to nano- mol L range. The reproducibilities (n = 10) of the
measurements were 24.7 % for CH 3Cl, 16.3 % for CH3Br, 10.5 % for CH 3I, and from 1.4 % to 8.8 % for the remaining
halocarbons. Linear regression of the standard solution prepared with bacterial culture medium indicated that this
method was successfully applied to the analysis of trace levels of halocarbons in bacterial culture. The DHS GC/MS
method potentially provides a sensitive means for tracing numerous natural sources of halocarbons from environmental
or biological samples.
Keywords: Dynamic headspace analysis (DHS), Gas chromatography-mass spectrometry (GC/MS),
Halocarbon, Bacteria
Introduction
sources [6, 7]. Therefore, it remains necessary to identify
Halocarbons are among the volatile organic compounds
new sources of halocarbons to gain a better understanding
(VOCs) emitted from ecosystems, and they have been
of the global halogen budget [2]. Marine environment
shown to affect a wide range of climate and atmospheric
have been recognized as one of the important natural
chemistries [1, 2]. Of par ticular interest are the
sources of halocarbons. In the ocean, macroalgae and
halocarbons involved in the global halogen budget and
marine phytoplankton produce halocarbons (e.g., [8, 9,
the depletion of tropospheric and stratospheric ozone [3].
10]). Recently, the bacterial production of CH3Cl, CH3Br
Long-lived halocarbons (methyl chloride, CH3Cl and
[11], and CH3I [12] was also reported. However, microbial
methyl bromide, CH3Br) are abundant in the atmosphere
production of halocarbons remains highly uncertain and
and have been studied widely. However, natural sources
requires further study.
of these long-lived halocarbons are insuf ficient to
Several extraction procedures have been available for
compensate for known sinks of these compounds [4, 5].
the measurement of VOCs in natural and biological
As for shor t-lived halocarbons (e.g., methyl iodide,
samples (e.g., [13]). The static headspace (SHS) and
CH 3I; bromoform, CHBr3; and diiodomethane, CH2I 2),
headspace-solid phase microextraction (HS-SPME)
there is few information about short-lived halocarbon
methods have been widely utilized as a means of directly
＊
Graduate School of Integrated Basic Sciences, Nihon University,
3-25Į40, Sakurajosui, Setagaya-ku, Tokyo 156-8550, Japan
Institute for Environmental Sciences, University of Shizuoka,
52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
＊＊＊
Department of Chemistry, College of Humanities & Sciences, Nihon
University, 3-25-40, Sakurajosui, Setagaya-ku, Tokyo 156-8550, Japan
＊＊
─ 231 ─
（ 1 ）
Gen TANIAI, et al.
Exhaust
Inert gas inlet
Inert gas inlet
Helium flow
Helium flow
Absorbent
GC
Sample
MS
Volatiles
a) Concentration
b) Desorption
Fig. 1 Schematic diagram of DHS process.
measuring VOCs in the headspaces of samples [14, 15].
beverages [23]. Our group was the first to report the
However, neither method is sensitive enough to detect
bacterial production of halocarbons using the DHS
−1
VOCs at the environmental concentration level (pmol L
method [11]. However, there are no published data on the
level). To measure trace levels of VOCs, the purge-and-
optimization of an analytical method for halocarbons at
trap (P&T) method is generally prefer red over the
very low concentrations (pico- to nano- mol L− 1 range) in
headspace extraction methods. The P&T method has
aqueous or biological samples that have high cell density.
been used to assess halocarbons in liquid samples such as
In the present study, the optimization of the DHS
fresh water [16], brackish water [17], seawater [18, 19,
extraction conditions combined with GC/MS was carried
20], and phytoplankton cultures [21]. Unfortunately, the
out to develop a sensitive quantitative analysis of 15
P&T method concentrates VOCs on the trap by bubbling
halocarbons, including highly volatile ones such as CH3Cl
an iner t gas (e.g., ultrapure helium) through liquid
and CH3Br. Below, we discuss the development of the
samples, so this method cannot be used to measure VOCs
present analytical method and evaluate the applicability of
in foam-forming samples (e.g., bacterial culture) or in
DHS GC/MS to the analysis of halocarbons in bacterial
solid samples (e.g., sediment).
cultures for the exploration of halocarbon sources.
An alternative method, called the dynamic headspace
(DHS) extraction method, is an extraction technique
2. Materials and methods
concentrating VOCs on the trap tube by passing a
2 – 1. Chemicals
stream of an inert gas into the headspace above the
The mixed standard solution of CH3Cl (2000 Pg mL − 1)
sample (Fig. 1). The DHS extraction method depends on
and CH3Br (2000 Pg mL − 1) was purchased from Supelco
the continuous regeneration of an extracting gas to force
(Bellefonte, Pennsylvania, USA). The standards of C2H5Br
volatiles from a sample into the headspace gas phase by
(99 % pure), CH 3 I (99.5 % pure), C 2 H 5 I (99 % pure),
sweeping the headspace. Compared to the SHS or HS-
(CH3)2CHI (99 % pure), CH2Br2 (99 % pure), CH3(CH2)2I
SPME method, the DHS method may be a more sensitive
(99 % pure), CHBrCl 2 (98 % pure), CH 2ClI (97 % pure),
means of detecting volatile compounds, and it may have
CHBr2Cl (98 % pure), CH2BrI (97 % pure, LOT 09211BJ),
great potential for researching volatile biogenic trace
CHBr3 (99 % pure), CH2I2 (99 %), and (CD3)2CDI (isotopic
gases in a wide variety of samples (e.g., seawater, cultured
purity 98 atom % D) were purchased from Sigma-Aldrich
microorganisms, and sediment). The DHS method has
(Tokyo, Japan). CH 2 BrCl (98 % pure) and pesticide
generally been used for the analysis of volatiles in several
residue-grade solvents were obtained from Wako Pure
kinds of samples, such as fermented foods [22] and
Chemical Industries (Tokyo, Japan). These reagents were
（ 2 ）
─ 232 ─
Simultaneous Determination of 15 Halocarbons at Pico- to Nano - Mol per Liter Levels in Water and Biological Samples Using Dynamic
Headspace Extraction and Gas Chromatography − Mass - Spectrometry
used without further purification.
before each analysis. The dynamic headspace system was
The primary standards were prepared gravimetrically
used to purge the gas phase above each sample (10 mL)
and dissolved in ultrapure methanol. Aqueous working
with ultrapure helium (>99.9999 % pure, Air Liquide Kogyo
standards were prepared by diluting suitable aliquots of
Gas Ltd.; Tokyo, Japan), and halocarbons with boiling
primar y standards with ultrapure water (Milli-Q water;
points between − 24 ℃ (CH3Cl) and 181 ℃ (CH2I2) were
Nihon Millipore, Tokyo, Japan) or culture medium (used
pre-concentrated in the trap column of a glass tube (60
for the microbial experiments). Calibrations using
mm, 4 mm ID, 6 mm OD) containing about 60 mg of
aqueous working standards were carried out approximately
Tenax TA, which was maintained at room temperature.
once per week. A liquid standard of (CD 3) 2CDI was
The halocarbons were released from the trap column
added as an internal standard to each sample at a final
by heating it at 200℃ with a thermal desorption unit (TDU,
−1
to monitor
Gerstel K. K.; Tokyo, Japan), and the desorbed halocarbons
GC/MS sensitivity drift. Instr umental blanks were
were cryofocusing on a liner tube trap of Tenax TA held
measured prior to sample measurements.
at − 150 ℃ with a cooled injection system (CIS 4, Gerstel
concentration of approximately 1 nmol L
K. K.; Tokyo, Japan). After cryofocusing, a liner tube was
2 – 2. Instrumentation
rapidly heated up to 200℃ and halocarbons were introduced
Sample extraction and introduction were fully automated
into the capillar y column (DB-624, length, 20 m; inner
using a Gerstel MPS-2 autosampler configured for the
diameter, 0.18 mm; and film thickness, 1 Pm; Agilent
auto-DHS injection, which was operated with Gerstel
Technologies; Tokyo, Japan). Halocarbons were
Maestro software Ver. 1.4.8.14. The halocarbons in
measured with a gas chromatograph (GC, 6890N, Agilent
aqueous samples contained in glass vials sealed with
Technologies, Inc.; Wilmington, North Carolina, USA)
silicone/PTFE septa (Gerstel K.K.; Tokyo, Japan) were
mass spectrometer (MS, 5975C, Agilent). Analysis of
measured using an automated dynamic headspace
halocarbons was performed by selected ion monitoring to
extraction system (DHS, Gerstel K. K.; Tokyo, Japan). The
attain high sensitivity. The analytical conditions for the
baking of an adsorbent trap (230℃，30 min) was carried out
DHS GC/MS and the target ions are given in Tables 1
Table 1 Experimental conditions for the dynamic headspace extraction system and GC/MS
Dynamic headspace extraction system
Thermostatting
Trap column
Cryo-focus
GC/MS
Capillary column
Carrier gas
Carrier gas flow rate
Oven temperature
Thermostatting time (min)
Agitation speed (rpm)
Trapping material
Trap temperature (℃ )
Purge volume (mL)
Purge flow rate (mL min − 1)
Examined
range
Optimized
condition
1–4
350 – 800
2
500
Tenax TA
20
100
75 – 150
Desorption temperature (℃ )
7.5 – 20
10
200
Trapping material
Trap temperature (℃ )
Desorption temperature (℃ )
Tenax TA
− 150
200
DB-624 (20 m × 0.18 mm × 0.1 Pm)
He
1.0 mL min − 1
Initial 40 ºC for 4 min
programmed to 100 ºC at 10 ºC min − 1
programmed to 200 ºC at 20 ºC min − 1
Ionization mode
Mass mode
EI (electron ionization, 70 eV)
SIM
─ 233 ─
（ 3 ）
Gen TANIAI, et al.
Table 2 Retention time ( RT ), target ions, linearity range, and correlation coefficient ( R 2 ) for the target
compounds
RT a
(min)
Compound
Target ions b
(m / z)
Linearity range
( pmol L − 1 )
Correlation coefficient
( R2 )
52
3,000 – 300,000
0.961
0.989
CH3Cl
2.0
CH3Br
2.3
94
96
100 – 10,000
C2H5Br
3.1
108
110
50 – 5,000
0.968
CH3I
3.1
142
127
70 – 7,000
0.984
50
C2H5I
4.8
156
127
9 – 900
0.995
CH2BrCl
5.1
130
128
30 – 3,000
0.998
(CH3)2CHI
(CD3)2CDI
6.1
6.0
170
177
127
127
7 – 700
7 – 700
0.990
0.994
CH2Br2
CH3 (CH2)2I
7.1
174
93
10 – 1,000
0.999
7.2
170
127
6 – 600
0.993
CHBrCl2
7.3
83
85
8 – 800
0.998
CH2ClI
7.7
176
178
9 – 900
0.999
CHBr2Cl
CH2BrI
9.4
129
127
80 – 800
0.999
9.6
222
220
10 – 1,000
0.999
CHBr3
11.1
173
171
9 – 900
0.999
CH2I2
11.6
141
127
9 – 900
0.998
a
RT: Experimental conditions were as in Table 1.
b
Target ions: Italicized ions represent major ion fragments used for quantitation.
and 2, respectively.
with the composite working standards at least at four
concentrations each that spanned the range of trace gas
2 – 3. Optimization of DHS parameters
concentrations in the bacterial cultures. The detection
Among the possible variables that might exert some
limit was defined as three times the standard deviation of
ef fect on halocarbon recover y, four DHS parameters
the control samples ( 3 V). To evaluate the influence of the
(thermostatting time, agitator speed, purge volume, and
matrix effect, the standard solutions were measured with
purge flow rate) were optimized. To assess the effects of
DHS GC/MS, which enabled us to individually ascertain
sample thermostatting time, agitation speed, purge
calibration cur ves in ultrapure water or in a bacterial
volume, and purge flow rate on the DHS extraction
medium matrix.
efficiency, 10.0 mL of aqueous standard solutions were
used. The concentrations for the 15 halocarbons in the
2 – 5. Application for the analysis of halocarbons in
bacterial culture
aqueous standard solution were as follows: CH3Cl, 1000
−1
−1
−1
; CH3I,
Bacterial culture samples were obtained from batch
50 pmol L − 1 ; C2H5I, 9 pmol L − 1 ; CH2BrCl, 9 pmol L − 1 ;
cultures of Erythrobacter longus JCM6170 T and Alteromo-
(CH3)2CHI, 7 pmol L − 1 ; (CD3)2CDI, 7 pmol L − 1 ; CH2Br2 ,
nas macleodii JCM20772 T(from Riken Bioresource Center,
10 pmol L − 1 ; CH3 (CH2)2I, 6 pmol L − 1 ; CHBrCl2 , 8 pmol
Saitama, Japan; Japan Collection of Microorganisms
pmol L
L
−1
; CH3Br, 40 pmol L
; CH2ClI, 9 pmol L
10 pmol L
−1
−1
; C2H5Br, 20 pmol L
; CHBr2Cl, 8 pmol L
; CHBr3 , 9 pmol L
−1
−1
; CH2BrI,
; and CH2I2 , 9 pmol L
−1
[JCM]). Each strain was preincubated in test tubes. The
.
cells were then collected by centrifugation (12,000 rpm; 3
The examined ranges for thermostatting time, agitation
min), washed three times with fresh medium, inoculated
speed, purge volume, and purge flow rate are listed in
in 10 mL of marine broth 2216 (Difco Laboratories;
Table 1.
Detroit, Michigan, USA) with potassium iodide (KI; final
concentration, 1 Pmol L − 1 for Erythrobacter longus, 1
mmol L − 1 for Alteromonas macleodii) in 20-mL glass
2 – 4. Calibration and assessment of DHS
For the calibration plot of the standard solutions, each
vials sealed with silicone/PTFE septa, and incubated in
10 mL of ultrapure water or culture medium was spiked
the dark at 25 ℃ in the Sanyo MIR-253 incubator (Sanyo,
（ 4 ）
─ 234 ─
Simultaneous Determination of 15 Halocarbons at Pico- to Nano - Mol per Liter Levels in Water and Biological Samples Using Dynamic
Headspace Extraction and Gas Chromatography − Mass - Spectrometry
days, the concentration of halocarbons in the culture
samples of marine bacteria in 20-mL vials were analyzed
using DHS GC/MS. Bacterial growth was monitored by
measuring optical density at 600 nm (OD600) during the
culture period using a WPA CO7500 colorimeter
12,000
CH3Cl
CH3Br
a
4,000
3,000
8,000
2,000
4,000
1,000
0
0
Peak area of CH 3Br
Peak area of CH 3Cl
Tokyo, Japan). After incubation of the cells for several
(Biochrom, Cambridge, UK). After the measurement of
sealed vial was removed, and optical density was measured
with a 10-mm cell. For each measurement of halocarbons
(and optical density) at 0, 1, 2, 3, 4, 8, and 10 d, separate
vials of cultured samples were prepared.
50,000
40,000
CH3I
b
6,000
5,000
10,000
4,000
0
3,000
Peak area
To optimize the conditions for the DHS extraction
procedure, we investigated the effects of four parameters:
7,000
20,000
3,000
extraction
8,000
30,000
3. Results
3 – 1. Optimization of the conditions for DHS
C2H5Br
C2H5I
(CH3)2CHI
(CD3)2CDI
CH3(CH2)2I
CH2BrCl
CH2Br2
CHBrCl 2
CHBr2Cl
CHBr3
CH2ClI
CH2BrI
CH2I2
Peak area of
C2H5I and C 2H5Br
Peak area of CH 3I
halocarbons in cultured samples (10 mL), the cap of the
c
2,000
1,000
0
sample thermostatting time, agitation speed, purge
volume, and purge flow rate. The peak area grew
substantially as the time of thermostatting increased to
9,000
Peak area
around 2 or 3 min for CH3Cl and CH3Br (Fig. 2a). Further
increases in the thermostatting time led to decreases in
the peak areas of CH3Cl and CH3Br. At thermostatting
times of longer than 3 min, CH3I, C2H5Br, and C2H5I were
found to lead to increases in the peak areas (Fig. 2b). As
d
6,000
3,000
0
for the other halocarbons, no significant changes were
observed in the peak area by increasing the thermostatting
2,500
Peak area
time from 1 to 4 min (Fig. 2c–e). To avoid the loss of
CH3Cl and CH3Br, a thermostatting time of 2 min was
chosen as the optimum condition. As regards the
agitation speed during thermostatting and purging, the
2,000
1,500
1,000
500
0
maximum peak area was obser ved at 500 rpm for all
analytes (Fig. 3a–e). Thus, to achieve effective extraction,
e
0
1
2
3
4
5
Thermostatting time (min)
)LJ)
an agitation speed of 500 rpm was selected as the
optimum condition.
Fig. 2
As regards the purge volume (Fig. 4a–e), larger purge
volumes (>100 mL) reduced the peak areas of extremely
volatile halocarbons (e.g., CH3Cl, boiling point: − 24 ℃ ;
CH3Br, boiling point: 3.5 ℃ ) (Fig. 4a), whereas increases
in purge volume tended to enhance the peak areas of other
halocarbons, especially CHBr3 (boiling point: 149 ℃ ) and
CH2I2 (boiling point: 181℃ ) (Fig. 4d–e). Purge volumes
lower than 100 mL appeared to be insufficient to extract
─ 235 ─
Ef fect of thermostatting time on the extraction
efficiency of 15 halocarbons. Agitation speed: 500 rpm,
purge volume: 100 mL, purge flow rate of helium:
10 mL min − 1. Concentrations of spiked halocarbons
were as follows. CH3Cl, 1000 pmol L − 1 ; CH3Br, 40
pmol L − 1 ; C2H5Br, 20 pmol L − 1 ; CH3I, 50 pmol L − 1 ;
C2H5I, 9 pmol L − 1 ; CH2BrCl, 9 pmol L − 1 ; (CH3)2CHI,
7 pmol L −1 ; (CD3)2CDI, 7 pmol L −1 ; CH2Br2 , 10 pmol
L − 1 ; CH3 (CH2)2I, 6 pmol L − 1 ; CHBrCl2 , 8 pmol L − 1 ;
CH2ClI, 9 pmol L − 1; CHBr2Cl, 8 pmol L − 1 ; CH2BrI,
10 pmol L − 1 ; CHBr3 , 9 pmol L − 1 ; and CH2I2 , 9 pmol
L−1
（ 5 ）
10,000
2,000
1,000
5,000
0
40,000
CH3I
C2H5Br
C2H5I
b
6,000
30,000
4,000
20,000
2,000
10,000
0
0
(CH3)2CHI
(CD3)2CDI
CH3I
C2H5Br
C2H5I
b
6,000
4,000
2,000
0
3,000
10,000
8,000
10,000
CH2BrCl
CH2Br2
CHBrCl 2
0
(CH3)2CHI
(CD3)2CDI
CH3(CH2)2I
CH2BrCl
CH2Br2
CHBrCl 2
CHBr2Cl
CHBr3
CH2ClI
CH2BrI
CH2I2
c
2,000
1,000
CHBr2Cl
CHBr3
9,000
d
Peak area
Peak area
6,000
3,000
d
6,000
3,000
0
CH2ClI
CH2BrI
CH2I2
3,000
e
1,500
Peak area
Peak area
0
0
1,000
500
0
200
（ 6 ）
1,000
20,000
0
Fig. 3
2,000
0
0
2,000
3,000
5,000
30,000
5,000
4,000
10,000
c
1,000
CH3Br
a
CH3(CH2)2I
2,000
9,000
CH3Cl
15,000
Peak area
Peak area
3,000
8,000
20,000
Peak area of
C2H5I and C 2H5Br
50,000
0
Peak area of CH 3Cl
a
3,000
Peak area of CH 3I
CH3Br
Peak area of
C2H5I and C 2H5Br
Peak area of CH 3I
CH3Cl
Peak area of CH 3Br
15,000
Peak area of CH 3Br
Peak area of CH 3Cl
Gen TANIAI, et al.
400 600 800 1000
Agitation speed (rpm)
)L
2,000
1,000
0
Effect of agitation speed on the extraction efficiency of 15
halocarbons. Thermostatting time: 2 min, purge volume:
100 mL, purge flow rate of helium: 10 mL min − 1 .
Concentrations of spiked halocarbons were as follows.
CH3Cl, 1000 pmol L − 1 ; CH3Br, 40 pmol L − 1 ; C2H5Br, 20
pmol L − 1 ; CH 3I, 50 pmol L − 1 ; C 2H 5I, 9 pmol L − 1 ;
C H 2 B rC l , 9 p m o l L − 1 ; ( C H 3 ) 2 CH I , 7 p m ol L − 1 ;
(CD 3) 2CDI, 7 pmol L − 1 ; CH 2Br 2, 10 pmol L − 1 ; CH 3
(CH2)2I, 6 pmol L − 1 ; CHBrCl2 , 8 pmol L − 1 ; CH2ClI, 9
pmol L − 1 ; CHBr2Cl, 8 pmol L − 1; CH2BrI, 10 pmol L − 1 ;
CHBr3 , 9 pmol L − 1 ; and CH2I2 , 9 pmol L − 1
Fig. 4
─ 236 ─
e
75
100 125
150
Purge volume (ml)
)L
)LJ
Effect of purge volume on the extraction efficiency of 15
halocarbons. Thermostatting time: 2 min, agitation
speed: 500 rpm, purge flow rate of helium: 10 mL min − 1.
Concentrations of spiked halocarbons were as follows.
CH3Cl, 1000 pmol L − 1 ; CH3Br, 40 pmol L − 1 ; C2H5Br, 20
pmol L − 1 ; CH 3I, 50 pmol L − 1 ; C 2H 5I, 9 pmol L − 1 ;
CH 2 BrCl, 9 pmol L − 1 ; (CH 3 ) 2 CHI, 7 pmol L − 1 ;
(CD 3) 2CDI, 7 pmol L − 1 ; CH 2Br 2, 10 pmol L − 1 ; CH 3
(CH2)2I, 6 pmol L − 1 ; CHBrCl2 , 8 pmol L − 1 ; CH2ClI, 9
pmol L − 1 ; CHBr2Cl, 8 pmol L − 1 ; CH2BrI, 10 pmol L − 1 ;
CHBr3 , 9 pmol L − 1 ; and CH2I2 , 9 pmol L − 1
15,000
CH3Br
a
compounds with high boiling points from the samples,
4,000
3,000
10,000
2,000
5,000
1,000
0
0
CH3I
50,000
C2H5Br
C2H5I
b
40,000
2,000
10,000
0
0
(CH3)2CHI
(CD3)2CDI
Peak area
3,000
the trap column. For the simultaneous analysis of 15
halocarbons, the following experiments were performed
with a purging volume of 100 mL helium. The peak areas
were decreased by increasing the purge flow rates of
8,000
4,000
20,000
to increase the loss of low-boiling-point analytes from
of very volatile halocarbons, such as CH3Cl and CH3Br,
6,000
30,000
whereas purge volumes higher than 100 mL appeared
Peak area of
C2H5I and C 2H5Br
Peak area of CH 3I
CH3Cl
Peak area of CH 3Br
Peak area of CH 3Cl
Simultaneous Determination of 15 Halocarbons at Pico- to Nano - Mol per Liter Levels in Water and Biological Samples Using Dynamic
Headspace Extraction and Gas Chromatography − Mass - Spectrometry
helium from 7.5 to 20 mL min −1 (Fig. 5a–c). On the other
hand, the purge flow rate had no significant effect on the
peak areas of other compounds, such as CHBr3 and CH2I2
(Fig. 5d–e). Since the lowest flow rate (7.5 mL min − 1)
appeared to reduce the precision of the analyses of CH3Cl
and CH3Br, the following experiments were conducted
CH3(CH2)2I
using a purge flow rate of 10 mL min − 1.
c
The optimum conditions for the DHS method are
2,000
summarized in Table 1. The total duration of analysis
1,000
(DHS extraction and GC/MS analysis) was approximately
5 hours for 10 samples.
0
CH2BrCl
CH2Br2
CHBrCl 2
10,000
Peak area
3 – 2. Linearity, detection limits, and precision
Linearity was studied over two orders of magnitude.
The linear range and the correlation coefficient R2 relative
d
8,000
to each compound are shown in Table 2. The calibration
6,000
4,000
curves indicated good correlations with R 2 ≥ 0.990, except
2,000
in the cases of CH 3Cl (0.961), CH 3Br (0.989), C 2H 5Br
0
(0.968), and CH3I (0.984). The limit of detection (LOD)
CH2ClI
CH2BrI
Peak area
2,000
was calculated as 3 V, where V is the standard deviation of
CH2I2
10 consecutive measurements of the standard solutions
e
(Table 3). The relative standard deviation (RSD) of the
1,500
results of the DHS GC/MS measurements were 24.7 % for
1,000
CH3Cl, 16.3 % for CH3Br, 10.5 % for CH3I, and from 1.4 % to
500
0
Fig. 5
CHBr2Cl
CHBr3
8.8 % for the other halocarbons (Table 3).
5
10
15
20
Purge flow rate (ml min -1)
)LJ)
Effect of purge flow rate of helium on the extraction
efficiency of 15 halocarbons. Thermostatting time: 2
min, agitation speed: 500 rpm, purge volume: 100 mL.
Concentrations of spiked halocarbons were as
follows. CH3Cl, 1000 pmol L − 1 ; CH3Br, 40 pmol L − 1 ;
C2H5Br, 20 pmol L − 1 ; CH3I, 50 pmol L − 1 ; C2H5I, 9
pmol L − 1 ; CH2BrCl, 9 pmol L − 1 ; (CH3)2CHI, 7 pmol
L − 1 ; (CD3)2CDI, 7 pmol L − 1 ; CH2Br2, 10 pmol L − 1 ;
CH 3 (CH 2) 2I, 6 pmol L − 1 ; CHBrCl 2, 8 pmol L − 1 ;
CH2ClI, 9 pmol L − 1 ; CHBr2Cl, 8 pmol L − 1 ; CH2BrI,
10 pmol L − 1 ; CHBr3 , 9 pmol L − 1 ; and CH2I2 , 9 pmol
L−1
3 – 3. Effects of bacterial medium matrices
To estimate the influence of the matrix of bacterial
culture medium, standard solutions were separately
prepared in ultrapure water and bacterial medium. The
calibration cur ves showed excellent linearity for
halocarbons at pico- to nano- mol L − 1 level in bacterial
medium (R 2 ≥ 0.987, except in the cases of CH3Cl (0.960),
CH3Br (0.895), C2H5Br (0.906), and CH3I (0.937)) as in
ultrapure water (Table 2). These results indicated that
the DHS analysis using the calibration cur ve of each
matrix is suitable for the analysis of trace levels of
─ 237 ─
（ 7 ）
Gen TANIAI, et al.
Table 3 Relative standard deviation (RSD) and limit of detection (LOD) using DHS GC/MS, LOD
using purge-and-trap (P&T) GC/MS, and LOD using solid-phase microextraction (SPME) GC/MS
DHSa
RSD
(%)
CH3Cl
24.7
CH3Br
16.3
(1000)e
(40)
LOD b
( pmol L − 1 )
LOD
using P&T c
(pmol L − 1 )
LOD
using SPME d
(pmol L − 1 )
762
1.2
̶f
19.1
0.14
̶f
5.6
(20)
4.2
̶
̶f
10.5
6.5
(50)
(9)
17.9
1.1
0.03
̶f
̶f
̶f
CH2BrCl
(CH3)2CHI
4.7
8.5
(9)
(7)
2.4
1.3
0.02
̶f
̶f
̶f
(CD3)2CDI
8.8
(7)
1.4
̶f
̶f
CH2Br2
2.9
(10)
2.0
0.05
̶f
C2H5Br
CH3I
C2H5I
f
CH3 (CH2)2I
8.1
(6)
1.3
̶
̶f
CHBrCl2
CH2ClI
1.4
(8)
1.5
0.02
104
5.1
(9)
1.7
0.02
̶f
CHBr2Cl
CH2BrI
CHBr3
CH2I2
f
2.1
(8)
1.9
0.02
14.4
2.9
(10)
1.2
̶f
̶f
4.1
(9)
2.1
0.03
31.7
2.3
(9)
2.4
0.12
̶f
a
This study
LOD was calculated as 3V, where V is the standard deviation of 10 consecutive measurements of the standard solutions
c
Kurihara et al. 2010
d
Allard et al. 2012
e
−1
Values in brackets are the compound concentrations in pmol L .
f
"̶" indicates no data.
b
halocarbons in aqueous and culture bacterial samples. Since
CHBrCl2, CH2ClI, CHBr2Cl, CH2BrI, CHBr3, or CH2I2) were
the slopes of the calibration curves were different between
detected in the cultures. The OD 600 time courses of the
ultrapure water and bacterial medium, the halocarbons in the
cultured samples indicated that E. longus increased during
bacterial samples were quantified based on the calibration
the incubation period (0.54 at 10 d). These results indicated
curves established by an analysis of halocarbon standards in
that the concentrations of CH3Cl, CH3Br, and CH3I increased
the bacterial medium to correct for the matrix effects. Figure
in the culture as they grew. Alteromonas macreodii incubated
6 shows typical ion chromatograms obtained from the
in culture medium with 1 mmol L − 1 KI, produced CH3Cl,
analysis of the bacterial culture medium spiked with
CH3Br, and CH3I up to 1.58×104±0.25×104, 6.41×103±0.91
halocarbon standards. As shown in Fig. 6, the separation of
× 103, 1.37 × 104 ± 0.33 × 104 nmol L − 1, respectively (n = 3).
every peak was sufficient for quantitative analysis, and there
The DHS GC/MS system can analyze about 1,000 bacterial
were no disturbances due to any other peaks from the
samples within 6 months without being dismantled for
medium.
cleaning.
3 – 4. Application of DHS GC/MS method to analysis of
4. Discussion
4 – 1. DHS GC/MS method
halocarbons in bacterial culture samples
The DHS GC/MS method was used to quantify halocarbons
The DHS GC/MS method was evaluated by comparison
in the cultures of Erythrobacter longus (D - Proteobacteria) and
with established methods: P&T GC/MS and solid-phase
Alteromonas macleodii (J - Proteobacteria). The time courses
microextraction (SPME) GC/MS (Table 3). Table 3 shows
of CH3Cl, CH3Br, and CH3I concentrations in the cultures of
that the DHS method was applicable to the simultaneous
E. longus are shown in Fig. 7. No other halocarbons (i.e.,
analysis of 15 halocarbons at pico- to nano-mol L − 1 levels.
C2H5Br, C2H5I, CH2BrCl, (CH3)2CHI, CH2Br2, CH3 (CH2)2I,
The sensitivity of the P&T method is more sensitive than the
（ 8 ）
─ 238 ─
1200
b) P] 94
CH3Br
800 j) P] 83
CHBrCl2
400
0
Abundance
Abundance
CH2Br2
120
200
800
c) P] 108
C2H5Br
100
120
k) P] 176
CH2ClI
60
d) P] 142
CH3I
0
160 e) P] 156
600 l) P] 129
CHBr2Cl
300
400
0
100 m) P] 222
C2H5I
CH2BrI
80
50
120 f) P] 130
300 n) P] 173
CH2BrCl
CHBr3
200
100
60
80 g) P] 177
Abundance
Abundance
180 i) P] 174
60
0
400
200
Abundance
a) P] 50
CH3Cl
600
Abundance
Abundance
Abundance
Simultaneous Determination of 15 Halocarbons at Pico- to Nano - Mol per Liter Levels in Water and Biological Samples Using Dynamic
Headspace Extraction and Gas Chromatography − Mass - Spectrometry
300 o) P] 141
(CD3)2CDI
60
100
120 h) P] 170
2
CH3(CH2)2I
4
6
8
10
Time (min)
(CH3)2CHI
60
2
4
6
8
10
Fig. 5 Taniai et al
Time (min)
Fig. 6
CH2I2
200
)LJ 7DQLDL HW DO
Ion chromatograms of 15 halocarbon species in a bacterial medium sample using DHS GC/MS
analysis. Single ion chromatogram collected in selected-ion monitoring mode. Concentrations of
spiked halocarbons were as follows. CH3Cl : 7,000 pmol L − 1; CH3Br : 7,000 pmol L − 1 ; C2H5Br : 500
pmol L − 1 ; CH3I, C2H5I, CH2BrCl : 900 pmol L − 1 ; (CD3)2CDI, (CH3)2CHI : 700 pmol L − 1; CH2Br2 :
1,200 pmol L − 1 ; CH3(CH2)2I : 600 pmol L − 1 ; CHBrCl2 : 800 pmol L − 1; CH2ClI : 900 pmol L − 1 ;
CHBr2Cl : 800 pmol L − 1 ; CH2BrI : 1,000 pmol L − 1 ; CHBr3 , CH2I2 : 900 pmol L − 1
DHS method. Unfortunately, however, the P&T method
less sensitive than the DHS method (Table 3) for the
concentrates VOCs on the trap by bubbling an inert gas
halocarbon measurement.
(e.g., ultrapure helium) through liquid samples, so this
It was reported that Tenax TA has low water adsorption
method cannot be used to measure VOCs in foam-
capacity, which is an impor tant proper ty for the
forming samples (e.g., bacterial culture) or in solid
measurement of trace gases in aqueous samples [24].
samples (e.g., sediment). Whereas the SPME method can
Furthermore, Tenax TA exhibits high thermal stability,
be used to measure VOCs in various samples, including
relatively low water retention, and a low bleed rate [25].
bacterial cultures and sediment, the SPME method [15] is
To avoid or at least reduce the effects due to water matrix
─ 239 ─
（ 9 ）
CH3Cl (pmol L -1)
Gen TANIAI, et al.
Alteromonas macreodii ( J - Proteobacteria) produce
8000
halocarbons in the culture. Previous research has
a
6000
indicated that Erythrobacter species produced CH 3Cl,
CH3Br, and CH3I [11], similar to the results of halocarbon
4000
production in the present study. Even though the
2000
detected halocarbon concentrations were as low, i.e., at
sub-nmol L − 1 levels, this DHS GC/MS method confirmed
0
the production of halocarbons in the bacterial samples. In
CH3Br (pmol L -1)
a previous study on bacteria belonging to J - Proteobacteria,
600
Alteromonas macleodii was reported to produce CH3I [12]
b
and Pseudomonas species were repor ted to produce
400
CH3Cl, CH3Br, and CH3I [11] in the culture experiment.
These results suggested that not only Erythrobacter longus
200
(D - Proteobacteria) but also Alteromonas macreodii
( J - Proteobacteria) would produce halocarbons in the
0
marine environment.
CH3I (pmol L -1)
It was repor ted that biogenic marine aggregates,
communities, yield iodocarbons (CH3I, C2H5I, CH3CHICH3,
c
150
CH3CH2CH2I) [26]. During a large aggregation event in the
northern Adriatic, Alteromonadaceae ( J - Proteobacteria)
100
dominated in mucilaginous aggregates [27]. The results
50
0
Fig. 7
which were formed by phytoplankton-bacteria microbial
200
of the present study, taken together with the production
of iodocarbons by marine aggregates, showed that aquatic
0
5
10
Incubation time (d)
15
bacterial communities could be new sources of methyl
halides in marine environments. The present DHS GC/
Time courses of CH3Cl, CH3Br, and CH3I concentrations
in a culture of Erythrobacter longus. The circles and the
er ror bars indicate the mean and the standard
deviation, respectively (n = 3).
MS method would be applicable to the evaluation of
halocarbon production from marine microbial communities
such as marine aggregates.
5. Conclusion
The DHS GC/MS method was optimized for the
and bleeding, Tenax TA was selected and used in the
simultaneous determination of 15 halocarbons. This
present study as a solid sorbent for the stable measurement
method can be used to quantify halocarbons at pico- to
of trace halocarbons.
nano- mol L − 1 levels in aqueous samples. Linear
regression analysis of the standard solution prepared with
4 – 2. Production of halocarbons in bacterial culture
samples
bacterial culture medium indicated that this method was
successfully applied to the analysis of trace levels of
The results shown in Table 3 and Fig. 6 indicate that
halocarbons in bacterial culture. This DHS GC/MS
the DHS method is applicable to the determination of
method was applied to the quantitative determination of
levels of halocarbons in bacterial
sub-nmol L − 1 levels of halocarbons for several days in
medium as an alter native to the P&T method (for
bacterial culture. Besides having sufficient sensitivity, the
example, foam-forming samples when using the P&T
DHS GC/MS system can analyze about 1,000 bacterial
method). The results shown in this study also indicate
samples without being dismantled for cleaning. The
that both Erythrobacter longus (D - Proteobacteria) and
present DHS GC/MS method will be reliable for
pico- to nano- mol L
（ 10 ）
−1
─ 240 ─
Simultaneous Determination of 15 Halocarbons at Pico- to Nano - Mol per Liter Levels in Water and Biological Samples Using Dynamic
Headspace Extraction and Gas Chromatography − Mass - Spectrometry
examinations of halocarbon production or degradation in
Scientific Research (C) (20510013) and a Grant-in-Aid for
marine microbial communities such as marine aggregates,
Scientific Research (B) (23310010) from the Ministry of
sediment, and symbiotic cultures.
Education, Culture, Sports, Science, and Technology of
Japan.
Acknowledgments
This study was supported in part by a Grant-in-Aid for
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