The document summarizes research on analyzing the concentration of the herbicide atrazine in water samples using two microextraction techniques: dispersed liquid-liquid microextraction (DLLME) and headspace liquid phase microextraction (HS-LPME). DLLME was able to recover atrazine from water samples as shown through GC/MS analysis, with the optimal amounts of dispersive solvent (methanol) and extraction solvent (chlorobenzene) determined to be 100 μL and 45 μL, respectively. In contrast, HS-LPME was unsuccessful at extracting atrazine. Further work is needed to validate the DLLME method through calibration and analyzing atrazine levels in collected surface water samples.

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Figure 2. Dispersed Liquid-Liquid Microextraction
Analysis of Atrazine Using Dispersed Liquid-Liquid Microextraction
and Headspace Liquid Phase Microextraction
Eleanor K. Skelton
Owens Research Group
CHEM 4980, Merck Research Methods
Department of Chemistry and Biochemistry, University of Colorado at Colorado Springs
May 7, 2012
1. Introduction
The triazine compound atrazine (Figure 1) is an
herbicide used in both agriculture and urban areas and is
suspected to be a carcinogen, having been implicated in both
ovarian and stomach cancer (1, 2). In 2003, the EPA
announced that it would begin “intensive, targeted monitoring of
raw water entering certain community water systems in areas of
atrazine use” (3). Yet a screening of United States drinking
water in 2008 revealed that atrazine is one of “11 most frequently detected compounds” in
the water supply of over 28 million people and also detected the presence of atrazine in
surface water farther from agricultural areas than expected (4).
The objective of the work presented here was to isolate atrazine from water samples
using green analytical techniques and analyze its concentration levels in the El Paso County
water supply and surface
water. Two extraction
methods were evaluated—
dispersed liquid-liquid
microextraction (DLLME)
and headspace liquid
phase microextraction (HS-LPME). DLLME
involved utilizing a water miscible dispersive solvent and a water immiscible extraction
Figure 1. Structure of Atrazine

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Figure 3. Headspace Liquid Phase Microextraction
solvent with a density (p) greater than 1 (Figure 2). The procedure for the HS-LPME
method was to first partially submerge a headspace vial, which contained a mixture of the
analysis sample and DI H2O, into a room temperature water bath while both solutions were
stirred. Then the syringe was filled with extraction solvent, injected through septum of
headspace vial, and the plunger of the syringe was suppressed until one droplet appeared
(Figure 3).
Both the DLLME and the HS-LPME methods are being
employed more heavily with various compounds as these
methods require considerably smaller amounts of chemicals
for extraction instead of needing higher concentrations to
obtain accurate data. Yet concentrations of atrazine in the
environment have not been evaluated using these methods to
date.
Last July, Chandrasekaran et. al. (ref) published an
article in the Analytical Methods Journal of the Royal Society
of Chemistry about their efforts to analyze concentrations of
uranium (iv) in water using DLLME (5). Similarly, in May
2011, scientists in the Chinese Academy of Sciences found
DLLME to be successful for several analytes such as trace preservatives—methylparaben,
ethylparaben, propylparaben, and butylparaben—in pancakes, “a common food in western
China [….] purchased from roadside kiosks,” using “0.1 mL of 1-octyl-3-methylimidazolium
hexafluorophosphate ([C8MIM][PF6]) as an extraction solvent, 0.1 mL of acetonitrile as a
disperser solvent, 5 min extraction time, and sample ionic strength of 30% sodium chloride
in water sample at pH 6.0” (6).
For HS-LPME, older articles seem to be more readily available. The overall idea—
using one droplet of extraction solvent and a microsyringe—was explored as early as 1997
by professors in Singapore (7). Dr. Alexander Nazarenko, the assistant professor at State
University of New York, wrote about his experimentation with this technique for both

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hydrophilic and hydrophobic hydrocarbons in August 2004. His work involved extracting
ethanol, propanol, and butanols with a water droplet and toluene, ethylbenzene, and
xylenes from carbon disulfide (8). Yet recent articles are also accessible—other Chinese
scientists in Quingdao and Yantai published research about their use of the headspace
method to extract geosmin and 2-methylisoborneol from water into the droplet of the
headspace syringe and used gas chromatography for analysis (9). Overall, worldwide data
shows that chemists have been working with these procedures by determining the best
extraction and dispersive solvents for their target compound—often preservatives,
pesticides, or other contaminants—and deducing their results using a form of mass
spectrometry (10, 11).
2. Experimental
For both the DLLME and the HS-LPME methods, a stock solution of 1,480 µg/mL was
prepared from 37 mg of atrazine, HPLC grade from Fluka Analytical (St. Louis, MO), diluted
in 25 mL methanol. Samples were analyzed by gas chromatography mass spectrometry
(GC/MS) using a Hewlett Packard 5890 Series II Gas Chromatograph with 5971A mass
selective detector. The column employed was a CP-SIL 5CB-MS column (25 m, 0.25 mm
i.d., 0.4 µm thickness; Agilent Technologies, Santa Clara, CA). Helium was used as the
carrier gas at 1.2 cm/s (column flow rate: 15 cm3
/min). The initial column temperature was
70ºC (hold for 2.00 min) with a ramp of 10.0°C/min to a final temperature of 250ºC (hold
for 3.00 min), for a total run time of 18.00 min. A sample volume of 1.0 μL was injected at
250°C in split/splitless mode. The MS detector was maintained at 280 °C and was utilized
in the SIM mode. Ions m/z 200 and 215 were monitored for atrazine. A GC/MS analysis
yielded data to be used as a standard for DLLME and HS-LPME extractions in natural water
sources.
First, the general format for the DLLME method was to pipet 800 μL 18 MΩ DI water,
54 μL of the atrazine stock solution (1480 µg/mL), 200 μL of the dispersive solvent
(methanol), and 30 μL of the extraction solvent (chlorobenzene) into 1.5 mL

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microcentrifuge tubes, sonicate for five minutes (FS2OD Sonicator, Fisher Scientific,
Fairlawn, NJ), centrifuge for three minutes at 10,500xg (TDX centrifuge, Abbott
Laboratories), and extract the bottom layer from GC/MS analysis. A number of dispersive
solvents (n=2 for each solvent) were examined using this procedure, including HPLC grades
(Fischer Scientific, Fairlawn, NJ) methanol, acetonitrile, acetone, ethanol, tetrahydrofuran
(THF), and dimethyl sulfoxide (DMSO).
In the second set of experiments, an evaluation was conducted of the most effective
amount of methanol to use as the dispersive solvent. Again, two tubes each were prepared
with the following amounts of methanol: 50 μL, 100 μL, 150 μL, 200 μL, 300 μL, and 500
μL. Amounts of the extraction solvent, chlorobenzene, were tested in the following
experiment: 15 μL, 30 μL, 45 μL, and 60 μL with n=2 for each.
For the HS-LPME method, the procedure was to first fill a 250 mL beaker with 18 MΩ
DI H2O and partially submerge a headspace vial, also containing 18 MΩ DI H2O, with the
stir bar set at 700 rpm (an appropriately-sized stir bar was placed in both the beaker and
the vial). The GC gas-tight syringe (SGE, Australia) was filled with 1 μL 1-octanol, injected
through septum of headspace vial, and the plunger of the syringe was suppressed until one
droplet appeared. Because the droplet had a tendency to drop off the needle into solution
after five minutes, the system was timed for three minutes, and then the droplet was drawn
back up into syringe for GC-MS analysis.
3. Results and Discussion
In the GC/MS data analyzed (Supplemental Material), not all of the compounds
tested displayed peaks that could be integrated. From the initial results for DLLME,
methanol appeared to be the most effective dispersive solvent to isolate atrazine. The
percent recoveries for varying amounts of methanol, the dispersive solvent, obtained from
GC-MS integration are listed below (Table 1). The results of this experiment to determine
the optimum amount of extraction solvent, chlorobenzene, are also described (Table 2).
Once again, a similar pattern appears—the percent recovery decreases as the amount of

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extraction solvent increases. Unfortunately, using HS-LPME, none of the six headspace
samples displayed peaks upon examination.
4. Conclusion
Ultimately, the HS-LPME method was not successful, but DLLME did recover atrazine
from water, as evidenced by GC/MS analysis. Further experimentation indicated which
dispersive and extraction solvents, as well as what amounts of each, were most effective.
Using the EPA standard of 120% for percent recovery, 45 μL of chlorobenzene and 100 μL
of methanol would be the most effective combination of amounts of dispersive and
extraction solvents for the analysis of atrazine. The 200 μL samples of methanol as the
dispersive solvent will be reprocessed in an attempt to attain more accurate data. Now that
these amounts have been determined, the next steps are to prepare a calibration curve,
possibly using an internal standard, to validate the method by determining the percent
recovery, and to use this developed DLLME protocol to analyze atrazine in collected
samples. This summer, we will collect samples from various surface waters to determine if
detrimental concentrations of atrazine are present in and around El Paso County.
Amount of Methanol Percent Recovery
50 μl 229.3%
100 μl 135.4%
150 μl 50.6%
200 μl 24.2%
300 μl 47.0%
500 μl 40.8%
Amount of Chlorobenzene Percent Recovery
15 μl 310.56%
30 μl 182.71%
45 μl 89.94%
60 μl 40.02%
Table 2. Evaluation of Amount of Extraction SolventTable 1. Evaluation of Amount of Dispersive Solvent

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Figure 4. Gas Chromatogram for 1,480 µg/mL stock solution of atrazine
Supplemental Material

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Figure 5. Gas Chromatogram and Mass Spectrum for 1,480 µg/mL stock solution of atrazine

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