Journal of Chromatography A, 1161 (2007) 89–97

Determination of triazine herbicides in aqueous samples

by dispersive liquid–liquid microextraction with gas
chromatography–ion trap mass spectrometry
Dongari Nagaraju, Shang-Da Huang ∗
Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan
Received 7 February 2007; received in revised form 18 May 2007; accepted 22 May 2007
Available online 29 May 2007

Abstract
A simple and rapid new dispersive liquid–liquid microextraction technique (DLLME) coupled with gas chromatography–ion trap mass spectro-
metric detection (GC–MS) was developed for the extraction and analysis of triazine herbicides from water samples. In this method, a mixture of
12.0 ␮L chlorobenzene (extraction solvent) and 1.00 mL acetone (disperser solvent) is rapidly injected by syringe into the 5.00 mL water sample
containing 4% (w/v) sodium chloride. In this process, triazines in the water sample are extracted into the fine droplets of chlorobenzene. After cen-
trifuging for 5 min at 6000 rpm, the fine droplets of chlorobenzene are sedimented in the bottom of the conical test tube (8.0 ± 0.3 ␮L). The settled
phase (2.0 ␮L) is collected and injected into the GC–MS for separation and determination of triazines. Some important parameters, viz, type of
extraction solvent, identity and volume of disperser solvent, extraction time, and salt effect, which affect on DLLME were studied. Under optimum
conditions the enrichment factors and extraction recoveries were high and ranged between 151–722 and 24.2–115.6%, respectively. The linear
range was wide (0.2–200 ␮g L−1 ) and the limits of detection were between 0.021 and 0.12 ␮g L−1 for most of the analytes. The relative standard
deviations (RSDs) for 5.00 ␮g L−1 of triazines in water were in the range of 1.36–8.67%. The performance of the method was checked by analysis
of river and tap water samples, and the relative recoveries of triazines from river and tap water at a spiking level of 5.0 ␮g L−1 were 85.2–114.5%
and 87.8–119.4%, respectively. This method was also compared with solid-phase microextraction (SPME) and hollow fiber protected liquid-phase
microextraction (HFP-LPME) methods. DLLME is a very simple and rapid method, requiring less than 3 min. It also has high enrichment factors
and recoveries for the extraction of triazines from water.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Dispersive liquid–liquid microextraction (DLLME); Sample preparation; Water samples analysis; Gas chromatography–ion trap mass spectrometry
(GC–MS); Triazine herbicides

1. Introduction herbicide residue level in drinking water in the range of parts

Triazines are important herbicides used in weed control. They
are ubiquitous environmental pollutants in soil and water. Their
use has caused great concern because of their mobility and sol-
ubility in water and they strongly sorb onto soil [1]. Triazines
and their degradation products are toxic and persistent in water,
soil and organisms. Moreover, atrazine is a member of the tri-
azine family and has been classified as human carcinogen [2].
The United States Environmental Protection Agency (EPA) and
European Union (EU) legislation have established a maximum
∗ Corresponding author. Tel.: +886 3 5721194; fax: +886 3 5736979.
E-mail address: sdhuang@mx.nthu.edu.tw (S.-D. Huang).
per billion. In the EU, the maximum allowed limit for each indi-
vidual herbicide has been set at 0.1 ␮g L−1 [3], but the EPA has
set the maximum allowable level of atrazine at 3 ␮g L−1 [4].
Because of these restrictions, analytical methods are required
for monitoring the widespread distribution of trace levels of tri-
azines; it is highly desirable that these be environmental friendly
“green” analytical methods.
Generally, liquid–liquid extraction (LLE), solid-phase
extraction (SPE), and supercritical fluid extraction (SFE) are
widely used to extract triazines from environmental samples
[5–9]. However, LLE is time-consuming and requires large
amounts of organic solvents that are potentially toxic. SPE uses
much less solvent than LLE but can be relatively expensive.
Additionally, evaporation of the final organic extract into a small

0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2007.05.065
90 D. Nagaraju, S.-D. Huang / J. Chromatogr. A 1161 (2007) 89–97
volume is necessary to achieve high enrichment of the analytes.
Batch-to-batch reproducibility continues to be the major concern
for analysts in selecting SPE devices. Elution of sorbed solute
must be performed after sample loading. Solvent evaporation
and redissolution are often required. SFE requires an expensive,
high-pressure delivery system and high-purity carbon dioxide.
Recent research has been directed toward developing
efficient, economical, and miniaturized sample preparation
methods. As a result, solid-phase microextraction (SPME)
[10–12] and solvent microextraction (SME) [13,14] have been
developed, among others. Compared with LLE, SPME devel-
oped by Arthur and Pawliszyn [15], is a solvent-free process
that includes simultaneous extraction and preconcentration of
analytes from aqueous samples or the headspace of the samples.
There are many SPME procedures reported in the literature for
the extraction and determination of triazine herbicides in various
environmental samples such as soil, waste water, and sewage
sludge samples [1,16–21]. However, SPME is expensive, the
fiber used is fragile and has a limited lifetime, and sample carry-
over can be a problem [22]. Jeannot and Cantwell developed a
liquid–liquid microextraction (LLME) system by which extrac-
tion into a single drop was achieved [23]. He and coworkers
reported liquid-phase microextraction (LPME) and single drop
microextraction (SDME) in 1997 [24,25]; these were developed
as solvent-minimized sample pretreatment procedures which are
inexpensive, and since very little solvent is used, there is min-
imal exposure to toxic organic solvents [26,27]. Shen and Lee
also used the hollow-fiber-protected LPME for the extraction of
triazine herbicides from water for quantitation by GC–MS [28].
However, there are some disadvantages in these methods: fast
stirring speed tends to break up the organic drop; air bubbles
are formed [2]; extraction is time-consuming; and equilibrium
could not be attained after a long time in most cases [27].
Very recently, Assadi and coworkers have developed a novel
microextraction technique as a high-performance and powerful
preconcentration method, which they have named as disper-
sive liquid–liquid microextraction (DLLME) [29]. It is based
on a ternary component solvent system like homogeneous
liquid–liquid extraction (HLLE) and cloud point extraction
(CPE). The advantages of the DLLME method are simplicity of
operation, rapidity, low cost, high recovery and enrichment fac-
tors. These workers demonstrated the performance of DLLME
with the determination of polycyclic aromatic hydrocarbons
(PAHs) (recovery 60.3–111.3%, enrichment factors 603–113),
organochlorine pesticides (OCPs), organophosphorus pesti-
cides (OPPs), and benzene, toluene, ethylbenzene and xylenes
(BTEX) from water by using gas chromatography–flame ion-
ization detection and gas chromatography–flame photometric
detection (GC–FID and GC–FPD) [30].
In our present investigation we have developed a simple and
rapid DLLME method coupled with gas chromatography–mass
spectrometry for the extraction of triazine herbicides from aque-
ous samples. In this method, acetone was used as the dispersive
solvent, which contained chlorobenzene as the extractant. Anal-
ysis was done by the GC–electron impact ionization (EI)-MS
detection method; the compounds were identified and moni-
tored using selected-ion storage (SIS). The factors which affect
DLLME extraction were investigated and operating conditions
were optimized. Finally the optimal conditions were used with
the method to extract, detect and identify triazines in river water
samples.
2. Experimental
2.1. Materials and reagents
All triazine herbicides used (atrazine, desmetryn, simazine,
prometryn, propazine, sebuthylazine, secbumeton and sime-
tryn) had purity of >98%; these were purchased from Riedel-de
Haën (Seelze-Hannover, Germany). Tetrachloroethylene and
chlorobenzene (spectroscopy grade) were obtained from J.T.
Baker (Phillisburg, NJ, USA); carbon tetrachloride was obtained
from Riedel-de Haën. Acetone, acetonitrile, and methanol as dis-
perser solvents (HPLC grade,) were obtained from J.T. Baker.
Sodium chloride (analytical-reagent grade) was obtained from
Showa (Tokyo, Japan). Deionized water was purified in a Milli-
Q RO Plus 60 filtration system (Millipore, Bedford, MA, USA)
before use for preparing aqueous solutions.
2.2. Instrumentation
Analysis of triazines was performed on a Varian (Walnut
Creek, CA, USA) Saturn 2000 GC/MS system. Ultra pure
helium (99.9995%) was passed through a molecular sieve trap
and oxygen trap before its use as the carrier gas. The GC CP-3800
was fitted with a DB-5 column (30 m, 0.25-mm i.d., 0.25 ␮m)
obtained from J&W Scientific (Folsom, CA, USA). Helium was
used as the carrier gas at a flow rate of 1.5 mL/min corresponding
to a constant linear velocity of 42 cm/min. The oven temperature
program employed for separation of triazine herbicides was as
follows: 100 ◦ C for 2 min; 20 ◦ C/min to 170 ◦ C, held for 2 min;
then 2 ◦ C/min to 180 ◦ C, held for 3 min; 25 ◦ C/min to 280 ◦ C,
held for 5 min. The injector temperature was 280 ◦ C, and all
injections (1079 split/splitless injector) were made in the split-
less mode. The mass detector was used in the electron impact (EI,
70 eV) mode and scanned over the range m/z 100–500 to confirm
the retention times of the analytes. For determination of triazines,
the selected-ion storage (SIS) mode was used. For identity, con-
firmation of triazines were made by selecting the most abundant
characteristic ions of each triazine, and two characteristic frag-
ment ions were monitored in addition to the molecular ion (see
Table 1). The trap and transfer line temperatures were 150 ◦ C
and 250 ◦ C, respectively. External standard and standard addi-
tion methods were used in the quantitative analysis of spiked
water samples and river water samples, respectively. A Sorvall
5C plus centrifuge (Global Medical, Ramsey, MN, USA) was
used for centrifuging.
2.3. Analytical procedures
0.0100 g of each triazine was dissolved in 10 mL of ace-
tone to obtain a standard stock solution with a concentration
of 1000 ␮g mL−1 . A fresh 10 ␮g mL−1 standard solution con-
taining eight triazines was prepared in acetone every week and
 D. Nagaraju, S.-D. Huang / J. Chromatogr. A 1161 (2007) 89–97
Table 1
List of compounds studied, molecular structure, and m/z selected for SIS mass detection
Herbicide molecular structure (Abbreviation) m/z (R.A., %)c
201a ,186b ,173 (100, 85, 42)
215, 200, 173 (35, 100, 30)
229, 214, 172 (30, 100, 58)
225, 210, 196 (25, 43, 100)
a Underline indicates spectrum base peak.
b Boldface indicates molecular mass peak.
c R.A.: relative abundance.
stored at 4 ◦ C. The working solutions were prepared daily by
using standard solutions with suitable dilutions.
The river water samples used for the evaluation of the
method were collected from different field areas of Khayaashi
River (Hsinchu city, Taiwan) and analyzed in triplicate using
the recommended DLLME procedure. Prior to the analysis,
water samples were filtered through a 0.45 ␮m membrane filter
obtained from Millipore and were stored at 4 ◦ C.
2.3.1. Dispersive liquid–liquid microextraction procedure
(DLLME)
The experimental procedure for DLLME is illustrated in
Fig. 1. A 5.00 mL portion of deionized water was placed in a
10 mL screw cap glass test tube with conical bottom and spiked
with each triazine at concentrations of 2.0 ␮g L−1 , and 4% (w/v)
sodium chloride was added to the solution. One milliliter of ace-
tone (as disperser solvent) containing 12.0 ␮L chlorobenzene as
extraction solvent was rapidly injected into the sample solution
91
Herbicide molecular structure (Abbreviation) m/z (R.A., %)
229, 214, 200 (7.5, 15, 100)
213, 198, 171 (100, 84, 33)
213,198, 170 (100, 17, 35)
241, 226, 184 (74, 65, 100)
with a 1.00 mL syringe (Hamilton, Reno, NV, USA) and then the
mixture was gently shaken. A cloudy solution (water, acetone,
and chlorobenzene) was formed in the test tube; the cloudy state
was stable for a long time. The mixture was then centrifuged
for 5 min at 6000 rpm, causing the dispersed fine droplets of
the extraction phase to settle to the bottom of the conical test
tube. The 2.0 ␮L of settled extraction phase was collected using
a 2.0 ␮L micro syringe (zero dead volume, Hamilton, Reno,
USA) and injected into the GC–MS system. The volume of the
settled phase was determined using a 10 ␮L micro syringe; this
was about 8.0 ± 0.3 ␮L.
2.4. Calculation of enrichment factor, extraction recovery
and relative recovery
The enrichment factor (EF) was defined as the ratio between
the analyte concentration in the settled phase (Cset ) and the initial
92 D. Nagaraju, S.-D. Huang / J. Chromatogr. A 1161 (2007) 89–97

Fig. 1. Dispersive liquid–liquid microextraction procedure. (A) Injection of 1.0 mL of dispersive solvent (acetone) containing extractant (12.0 ␮L chlorobenzene)
into the 5.0 mL aqueous sample solution, (B) dispersion of dispersive solvent and extractant, (C) After 5 min centrifugation at 6000 rpm, and (D) the settled phase
was collected with a 2.0 ␮L syringe and injected into GC–MS.

concentration of analyte (Co ) in the aqueous sample [29,30]: tion process. Some of these parameters are selection of suitable
extraction solvent, disperser solvent, volume of extraction sol-
Cset
EF = (1) vent, volume of disperser solvent, ionic strength of aqueous
Co phase, and extraction time. It is very important to optimize
The Cset was obtained from direct injection of analyte stan- them in order to obtain good recovery. We selected eight triazine
dard solution in the extract solvent. herbicides and studied their behavior under various extraction
The extraction recovery (ER) was defined as the percentage conditions.
of the total analyte (n0 ) which was extracted into the settled
phase (nset ). 3.1. Selection of extraction solvent
nset Cset × Vset
ER = × 100 = × 100 (2) In DLLME, the selection of suitable organic solvents is based
n0 Co × Vaq on the requirement of a higher density than that of water, the sol-
Vset vent’s extraction capability for selected compounds, and good
ER = EF × × 100 (3) gas chromatographic behavior. The selection of an appropri-
Vaq
ate solvent is of high importance for the DLLME process.
where Vset and Vaq are the volumes of the settled phase and Carbon tetrachloride (CCl4 ), tetrachloroethylene (C2 Cl4 ), and
the sample solution, respectively. The relative recovery (RR) is chlorobenzene (C6 H5 Cl) were compared for the extraction
defined by the following equation: of triazines. The density values of the selected organic sol-
Cfound − Creal vents are 1.62 g mL−1 (C2 Cl4 ), 1.59 (CCl4 ), and 1.11 g mL−1
RR = × 100 (4) (C6 H5 Cl). A series of sample solutions were studied by using
Cadded
1.00 mL of acetone containing different volumes of extrac-
Here Cfound , Creal , and Cadded are the concentration of analyte tion solvent to obtain 5.0 ␮L volume of settled phase. This
after addition of a known amount of standard in the real sample, required 8.0, 13.0, and 12.0 ␮L volumes of C2 Cl4 , CCl4 , and
the concentration of analyte in the real sample and the concen- C6 H5 Cl, respectively. The average recovery (n = 3) and standard
tration of known amount of standard which was spiked to the deviation (SD) are shown in Table 2 for the different extrac-
real sample, respectively. tion solvents. It can be seen from Table 2 that chlorobenzene
has the highest extraction efficiency (12.6–95.8%) when com-
3. Results and discussion pared with C2 Cl4 (9.3–74.3%) and CCl4 (5.7–45.1%). This
is probably due to the interaction between the benzene ring
In order to select the optimum DLLME conditions for the of chlorobenzene and the 1,3,5 triazine ring present in all the
determination of triazine herbicides, a procedure was required to selected triazines. ATZ and SMZ are not extracted in C2 Cl4 and
optimize the different parameters that affect the DLLME extrac- CCl4 .
 D. Nagaraju, S.-D. Huang / J. Chromatogr. A 1161 (2007) 89–97 93

Table 2
Extraction efficiencies of different extraction solvents evaluated for extraction
of triazines by DLLMEa
Compound Recovery (%) ± SD (n = 3)

Chlorobenzene Carbon tetrachloride Tetrachloroethylene

SMZ 12.6 ± 0.9 nd nd

ATZ 26.3 ± 2.9 nd nd
PPZ 38.9 ± 3.1 18.2 ± 2.9 28.3 ± 2.6
SBN 21.2 ± 2.0 5.7 ± 3.1 83.9 ± 4.2
SBZ 32.3 ± 2.9 16.5 ± 2.1 31.4 ± 1.8
DMN 27.1 ± 2.2 6.8 ± 1.9 9.3 ± 2.8
SMN 17.7 ± 1.6 6.9 ± 1.3 20.1 ± 3.6
PMN 95.8 ± 4.5 45.1 ± 3.6 74.3 ± 2.2
a Extraction conditions: 5.0 mL water sample was spiked with 2.0 ␮g L−1
of each triazine, dispersive solvent acetone volume 1.0 mL; extraction
Fig. 2. Effect of the volume of chlorobenzene on the recovery of triazines
solvent volume, chlorobenzene (12.0 ␮L), carbon tetrachloride (10.0 ␮L), tetra-
obtained by DLLME. Extraction conditions: aqueous sample volume 5.0 mL;
chloroethylene (8.0 ␮L), settled phase volume, 5.0 ± 0.3 ␮L; room temperature.
disperser solvent volume, 1.0 mL; concentration of each triazine, 2.0 ␮g L−1 ;
room temperature.
3.2. Selection of disperser solvent
were fixed and included the use of 1.00 mL acetone containing
The miscibility of the disperser solvent in the extraction different volumes (i.e., 7.0, 12.0, 17.0, 22.0, and 27.0 ␮L) of
solvent (i.e., organic phase) and aqueous phase is the most C6 H5 Cl. The results are presented in Figs. 2 and 3 which show
important factor affecting the selection of disperser solvent in plots recovery and enrichment factor versus volume of extraction
DLLME. Acetone, acetonitrile, and methanol have this property solvent (C6 H5 Cl), respectively. It was observed that increasing
and were selected for this purpose. A series of sample solu- the volume of C6 H5 Cl from 12.0 to 27.0 ␮L increases the volume
tions was studied by using 1.00 mL of each disperser solvent of the settled phase from 5.0 to 20.0 ␮L. Fig. 2 also indicates
containing 12.0 ␮L chlorobenzene as extraction solvent. The that increasing the volume of C6 H5 Cl results in slight increases
results are represented in Table 3. The ranges of recovery using in extraction recovery for most of the analytes, which remains
acetone, acetonitrile, and methanol as disperser solvents were constant for SMZ and ATZ. However, as the volume of the settled
12.3–92.1%, 8.8–72.6%, and 7.7–33.9, respectively. According phase increases, the enrichment factor decreases with increasing
to Table 3, acetone yields the highest recoveries. It was also used volume of C6 H5 Cl as shown in Fig. 3. At a fairly low volume of
as a disperser solvent with a similar type of observation reported extraction solvent, high enrichment factors and good recoveries
in the literature [29–31]. Therefore, it is selected; in addition, are obtained. A useful gain in sensitivity was achieved by using
acetone has low toxicity and is inexpensive. 12.0 ␮L of C6 H5 Cl.

3.3. Effect of extraction solvent volume

3.4. Effect of disperser solvent volume
To study the effect of extraction solvent volume, solutions
The effect of disperser solvent acetone volume was studied
containing different volumes of chlorobenzene were subjected
over the range of 0.5–2.0 mL, but the variation of the volume of
to the same DLLME procedure. The experimental conditions
acetone (as disperser solvent) causes changes in the volume of
Table 3
Extraction efficiency of disperser solventa

Compound Extraction recovery (%) ± SD (n = 3)b

Acetonitrile Methanol Acetone

SMZ 8.8 ± 0.6 7.7 ± 0.2 12.3 ± 0.6

ATZ 20.8 ± 3.1 23.4 ± 0.4 27.0 ± 3.9
PPZ 27.3 ± 1.1 12.1 ± 0.4 34.6 ± 3.3
SBN 10.3 ± 2.9 10.3 ± 3.9 17.6 ± 2.3
SBZ 19.3 ± 2.1 9.2 ± 3.9 28.4 ± 3.5
DMN 12.5 ± 1.3 7.8 ± 3.2 20.5 ± 2.2
SMN 16.2 ± 0.3 8.8 ± 2.4 21.6 ± 1.4
PMN 72.6 ± 6.6 33.9 ± 5.5 92.1 ± 5.6
a Extraction conditions: 5.0 mL water sample was spiked with 2.0 ␮g L−1

of each triazine, dispersive solvent (each acetonitrile, acetone and methanol)

volume 1.0 mL; chlorobenzene as extraction solvent volume, 12.0 ␮L, settled
phase volume, 5.0 ± 0.3 ␮L; room temperature. Fig. 3. Effect of the volume of chlorobenzene on the enrichment factors of
b Average of three determination (n = 3).
triazines extracted by DLLME. Extraction conditions as in Fig. 2.
94 D. Nagaraju, S.-D. Huang / J. Chromatogr. A 1161 (2007) 89–97
Fig. 4. Effect of the volume of acetone on the recovery of triazines extracted
by DLLME. Extraction conditions: aqueous sample volume, 5.0 mL; settled
phase volume 5.0 ± 0.3 ␮L; concentration of each triazine 2.0 ␮g L−1 ; room
temperature.
the settled phase. Hence, it is impossible to consider indepen-
dently the influence of the volume of acetone on the extraction
efficiency in DLLME. To avoid this problem and in order to
attain a constant volume of settled phase, the volume of acetone
and C6 H5 Cl were changed simultaneously. The experimental
conditions were fixed and included the use of different volumes
of acetone: 0.50, 1.00, 1.50, and 2.00 mL containing 7.3, 8.0,
10.3, and 12.4 ␮L of C6 H5 Cl, respectively. The volume of the
settled phase was constant (5.0 ± 0.3 ␮L) under these condi-
tions. The results are shown in Fig. 4; initially the extraction
efficiency increases and then decreases as the volume of acetone
is increased. At lower volumes of acetone, the cloudy suspen-
sion of droplets is not formed well, resulting in a decrease in
extraction recovery. At higher volumes of acetone, the solubil-
ity of triazines in water increases and the extraction efficiency
decreases. One microliter of acetone was selected as the opti-
mum volume of the disperser solvent.
3.5. Effect of extraction time
Mass transfer is a time dependent process and one of the
salient factors in most of the extraction procedures, especially in
microextraction methods such as SPME and LPME. In DLLME
extraction, time is defined as the time interval between inject-
ing the mixture of disperser solvent (acetone) and extraction
solvent (chlorobenzene) and starting to centrifuge. The effect
of extraction time was studied over the range of 0–60 min,
Table 4
Comparison of proposed DLLME with hollow fiber (HF) protected LPME and SPME for determination of triazines in water samples
Methods Linearity (␮g L−1 ) LOD (␮g L−1 )
SPME–GC–MS naa 0.02–0.15
HF-LPME–GC–MS 2–20 0.007–0.021
SPME–GC–MS 0.002–80 0.002–0.017
SPME–HPLC 5–1000 2.8–3.41
DLLME–GC–MS 0.2–200 0.021–0.12
a na: not assigned.
Fig. 5. Effect of extraction time on the recovery of triazines obtained from
DLLME. Extraction conditions: aqueous sample volume, 5.0 mL; acetone
1.0 mL; 12.0 ␮L chlorobenzene; settled phase volume 5.0 ± 0.3 ␮L; concen-
tration of each triazine 2.0 ␮g L−1 ; room temperature.
with other experimental conditions remaining constant. Fig. 5
shows the extraction recoveries of triazines versus extraction
time. It is seen that as the time increases the extraction recov-
ery decreases significantly for ATZ, but for the other analytes
it is constant. There is no influence of time on extraction effi-
ciency. Evidently, the surface area between the extraction solvent
and the aqueous phase is extremely large, as demonstrated by
Assadi and coworkers using optical microscopic photography
[29]. Transfer of analytes from the aqueous phase (sample) to
the extraction phase is therefore fast. Equilibrium is achieved
quickly, consequently extraction times can be very short. This
is one of the most important advantages of the DLLME tech-
nique. A comparison of the extraction time of triazines from
water samples using DLLME, hollow fiber protected LPME,
and SPME is summarized in Table 4. The comparison indicates
that this novel method requires a much shorter extraction time
(less than 5 min) than the other techniques. Since SPME is a pro-
cess that achieves equilibrium slowly, it requires a longer time
for equilibrium to be established. The time to reach absorption
equilibrium determines the maximum amount of analyte that can
be extracted by the fiber and, therefore, controls the sensitivity of
the method, but the equilibration time for SPME is large. LPME
is a non-equilibrium extraction method in most cases [27,32].
However, DLLME reaches the equilibrium state immediately.
In this method, the time-consuming step is the centrifuging of
the sample solution during the extraction procedure, which takes
about 5 min.
RSD (%) Extraction time (min) Sample vol. (mL) Reference
3.90–16.1 30 3.0 [28]
3.50–4.99 20 3.0 [28]
5.00–8.1 45 1.2 [17]
2.40–7.2 20 3.0 [19]
1.36–8.67 3 5.0 Proposed method
 D. Nagaraju, S.-D. Huang / J. Chromatogr. A 1161 (2007) 89–97
Fig. 6. Effect of salt addition on the recovery of triazines obtained by DLLME.
Extraction conditions: aqueous sample volume, 5.0 mL; acetone 1.0 mL; 12.0 ␮L
chlorobenzene; concentration of each triazine 2.0 ␮g L−1 ; at room temperature.
3.6. Salt addition
The salting-out effect has been used universally in SPME and
LLE. Generally, addition of salt decreases the solubility of ana-
lytes in the aqueous sample and enhances their partitioning into
the adsorbent (for SPME) or organic phase (LLE). In DLLME
the effect of ionic strength of the water sample was evaluated by
adding sodium chloride (NaCl; 0–5%, w/v) into the water sample
spiked with triazines at levels of 2.00 ␮g L−1 . DLLME experi-
mental conditions were the same as those described before. The
recovery and enrichment factor are plotted versus ionic strength
in Figs. 6 and 7. It could be inferred from the results that increas-
ing the NaCl from 0 to 5% results in an increase in the volume
of the settled phase from 5.0 to 9.50 ␮L, due to the decrease
in aqueous solubility of the extraction solvent in the presence
of salt. The triazines showed similar types of behavior in the
presence of sodium chloride (see Fig. 6) for ATZ, PPZ, SBN,
SMN, SBZ, and PMN, the extraction recoveries decrease at 1.0%
NaCl, then increase for most of the analytes as the sodium chlo-
ride concentration is increased from 1 to 4%. At 5% the PPZ
extraction efficiency was decreased. However, salt has an effect
on extraction efficiency as well as on enrichment factors, but the
increase in volume of settled phase did not effect much on the
Fig. 7. Effect of salt addition on the enrichment factors of triazines obtained by
DLLME. Extraction conditions: same as Fig. 6.
95
Fig. 8. GC–MS chromatogram of water sample spiked with all eight triazine
herbicides at 2.0 ␮g L−1 obtained by the DLLME technique.
enrichment factors of most of the analytes (Fig. 7). On the basis
of the above observations, 4% sodium chloride was selected
since this quantity provides good results for all the analytes.
Fig. 8 shows a typical gas chromatography–ion trap mass spec-
trometry (GC/MS-SIS mode) chromatogram of eight triazines
extracted with DLLME from an aqueous sample spiked with
each triazine herbicide at 2.0 ␮g L−1 .
3.7. Quantitative analysis
The optimum DLLME parameters finally selected are as
follows: 5.0 mL sample solution, 12.0 ␮L chlorobenzene as
extraction solvent, 1.0 mL acetone as disperser solvent, 4% (w/v)
NaCl, and 5.0 min centrifugation at 6000 rpm at room temper-
ature. To investigate the linearity of dispersive liquid–liquid
microextraction, 0.2–200 ␮g L−1 solutions of triazines were
prepared in deionized water. All the triazines exhibited good
linearity with correlation coefficients (r2 ) > 0.9995 (Table 5).
The limits of detection (LODs) of triazines which were studied
in the aqueous sample, calculated as three times the standard
deviation obtained from seven replicate runs of aqueous sample
spiked with 1.0 ␮g L−1 of each triazine under MS-SIS condi-
tion, were in the range of 0.021–0.12 ␮g L−1 . The repeatability
of the DLLME was performed by extracting aqueous sample
spiked at 5.0 ␮g L−1 of each triazine (five replicates). The rel-
96 D. Nagaraju, S.-D. Huang / J. Chromatogr. A 1161 (2007) 89–97

Table 5
Quantitative analysis of DLLME and GC–MS of triazines from water samples

Compound %RSDsa EF ER (%) Linearity range (␮g L−1 ) r2 LODb (␮g L−1 )

SMZ 4.31 151 24.2 0.2–200 0.9952 0.120

ATZ 4.33 151 24.2 0.2–200 0.9979 0.060
PPZ 7.38 501 81.4 0.2–200 0.978 0.021
SBN 4.73 145 23.3 0.2–200 0.9996 0.092
SBZ 5.83 495 79.3 0.2–200 0.9998 0.058
DMN 8.67 296 47.5 0.2–200 0.9995 0.085
SMN 1.36 251 40.2 0.2–200 0.9985 0.068
PMN 4.86 722 115.6 0.2–200 0.9985 0.046
a Average of five determination (n = 5) of each triazine spiked to deionized water at 5.00 ␮g L−1 level.
b LOD values are calculated as three times the standard deviation of seven replicated runs of aqueous sample spiked with 1.0 ␮g L−1 of each triazine.

ative standard deviations (RSDs) were calculated for peak area
counts and were found to be 1.36–8.62. The extraction recov-
eries and enrichment factors of this method were 24.2–115.8%
and 151–722, respectively (Table 5)
3.8. Application to real water analysis
River water from Khayaashi River (Hsinchu city, Taiwan) and
tap water from our laboratory were collected, and were extracted
using the optimized DLLME method; and the extracts were ana-
lyzed by GC–MS. The results for river and tap water showed that
they were free of triazines (i.e., triazines were undetectable).
River and tap water were spiked with each triazine standard at
the concentration of 5.0 ␮g L−1 to assess matrix effects. Fig. 9
shows the chromatogram obtained for river water by spiking
triazines. Results of relative recovery of river and tap water
are given in Table 6. The data revealed that for all triazines
the relative recoveries of river and tap water were in the range
of 85.2–114.5% and 87.8–119.4%, respectively. These results
demonstrate that the river and tap water matrices in our present
context had little effect on DLLME.
The extraction capacity of DLLME was studied in more
complex samples with an emulsifying capacity. The effect of
sodium dodecyl sulfate (SDS) concentration (i.e., 0, 2, 10,
and 50 ␮g mL−1 ) was studied on the extraction of triazines in
water. The results inferred that, as the concentration of SDS
increases from 0 –50 ␮g mL−1 , the volume of settled phase
(i.e., extraction solvent volume) decreases insignificantly and
the extraction recoveries of triazines also decreases. This is
because the solubility of analytes increases in aqueous phase as
the SDS concentration increases, so that the extraction efficiency
of analytes into the settled phase decreases (Fig. 10). However,
at higher concentration of SDS, the settled phase may decrease

Fig. 9. Shows GC–MS chromatogram of (A) river water sample and (B) spiked
with eight triazine herbicides at 5.0 ␮g L−1 obtained by DLLME; DLLME
conditions: river water sample volume, 5.0 mL; acetone 1.0 mL; 12.0 ␮L
chlorobenzene; NaCl 4%(w/v); settled phase volume 8.0 ± 0.3 ␮L; concentra-
tion of each triazine 5.0 ␮g L−1 ; room temperature. For peak identification see
Table 1.
Fig. 10. Effect of sodium dodecyl sulfate (SDS) concentration on the recovery of
triazines obtained by DLLME Extraction conditions: aqueous sample volume,
5.0 mL; acetone 1.0 mL; 12.0 ␮L chlorobenzene; 4% (w/v) NaCl, concentration
of each triazine 2.0 ␮g L−1 ; room temperature.
 D. Nagaraju, S.-D. Huang / J. Chromatogr. A 1161 (2007) 89–97 97

Table 6
Analysis of river and tap waters, and relative recovery of triazines spiked river and tap water samples
Compound River water 5 ␮g L−1 spiked river water Tap water 5 ␮g L−1 spiked tap water

Mean ± SD Founded ± SDa RRa (%) Mean ± SD Founded ± SDa RRa (%)

SMZ ndb 5.5 ± 0.1 109.7 nd 5.8 ± 0.31 115.9

ATZ nd 4.3 ± 0.15 85.2 nd 4.5 ± 0.14 90.2
PPZ nd 4.5 ± 0.13 90.4 nd 5.6 ± 0.21 111.8
SBN nd 5.7 ± 0.13 113.3 nd 5.2 ± 0.15 103.1
SBZ nd 5.7 ± 0.2 114.5 nd 5.7 ± 0.41 113.8
DMN nd 5.6 ± 0.08 111.4 nd 5.9 ± 0.46 119.4
SMN nd 5.1 ± 0.15 101.5 nd 4.4 ± 0.31 87.8
PMN nd 4.9 ± 0.15 98.9 nd 5.2 ± 0.2 104.6
a Average of three determination (n = 3); RR: relative recovery.
b nd: not detected.

and finally disappears. This is due to the emulsification of the
extraction solvent with SDS so that the DLLME technique fails
to analyze more complex samples with an emulsifying capacity.
4. Conclusions
A simple, rapid, and inexpensive DLLME recovery and con-
centration technique has been coupled to a GC–MS method
for the determination of triazine herbicides in water samples.
The technique provides good repeatability, high enrichment fac-
tors and good recovery within a short time compared to other
techniques. The method is also applied for the extraction of
triazines from river and tap water with good results. Compar-
ison of this new method with other extraction methods such
as LPME and SPME shows that DLLME is simple, rapid, and
inexpensive.
Acknowledgment
The authors wish to thank the National Science Council of
Taiwan (NSC 95-2113-M-007-035) for financial support.
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