MINDANAO UNIVERSITY OF SCIENCE AND TECHNOLOGY

C.M. Recto Ave., Lapasan, Cagayan de Oro City

ANALYSIS OF ORGANOCHLORINES IN WATER

Experiment No. 6

Submittted by:
John Mark C. Saburao
BS Chem- 3H1

March 12, 2016

OBJECTIVES
At the end of this experiment, the students should be able to:
1. Study the instrumentation and be familiarized with the parts of a gas chromatograph
2. Extract and purify an organochlorine compound present in a water sample by solid phase
extraction
3. Analyze a stagnant water sample for presence of organochlorine compounds by gas
chromatography
4. Interpret and analyze the chromatogram of the eluate generated by the gas chromatograph
5. Determine the concentration of the organochlorine compound present in the original water
sample based on external standard calibration

THEORETICAL BACKGROUND
Organochlorines are a class of compounds with a similar chemical structure that persist in
the environment. A commonly used organochlorine compound are pesticides which include
substances that kill weeds (herbicides), insects (insecticides), and fungus (fungicides) among
others. The persistence of organochlorine compounds, such as pesticides, in the environment
leads to incorporation into ecosystems and food chains which remain for years. Thus,
organochlorine compounds become persistent organic pollutants (POPs) which are resistant to
environmental degradation. The most common application of organochlorine compounds is for
agricultural uses. Exposure to organochlorine compounds not only affect the environment but
also human health which can cause illness, cancer and in a worst case scenario, death. For this
reason, various analytical methods are employed to strictly control and monitor the presence of
these compounds. One of the methods that had been developed to extract, purify, and analyze
such compounds in water samples (e.g. agricultural wastewaters) is through solid phase
extraction (SPE) and gas chromatography. SPE extraction procedure is carried out to streamline
the sample preparation process for determining the organochlorine pesticides.

For gas

chromatography, the instrument employed is a gas chromatograph with electron capture detector
(ECD). The presence of chlorine atoms in organochlorines makes ECD an ideal detector for the
analysis.

MATERIALS AND CHEMICALS

wash bottle

filter paper

deionized water

solid phase extraction set-up

beaker

vacuum filtration set-up

graduated cylinder

water sample

Petri dish

hexane-acetone mixture (50:50)

micropipette

hexane

forceps

methanol

PROCEDURE
A. Solid Phase Extraction Conditioning
The Thermo Scientific HyperSep C18 extraction column was added with 10 mL hexaneacetone mixture (50:50). The solvent was allowed to drain into solvent waste without allowing
the cartridge to dry out. Next, the column was conditioned with 10 mL methanol and again, the
solvent was allowed to drain into solvent waste without allowing cartridge to dry out. Lastly, the
column was added with 10 mL of deionized water and drained as usual.
B. Solid Phase Extration
In a Thermo Scientific HyperSep C18 extraction column, a 250 mL aliquot of the water
sample was loaded at a rate of 8-10 mL per minute. Then, the column was washed with 20 mL
deionized water. After which, it was dried using maximum vacuum pressure for 15 to 30
minutes. After the column was dried, elution was carried out by adding 4 mL of hexane-acetone
mixture (50:50) twice. Then it was followed by the addition of 4 mL pure hexane. When elution
was done, a keeper solvent was added to the column. The keeper solvent added was methanol of
volume 500 L and then the whole mixture was evaporated to 5 mL.
C. Sample Analysis
The instrument used was Shimadzu GC- 2014 gas chromatograph equipped with
Shimadzu ECD- 2014 detector and fitted with a capillary column. The gas chromatograph (GC)
set-up was undergone to test runs. After which, operating conditions for sample analysis were

carried out. First, an aliquot of the blank solution was injected into the GC column. Then it was
followed by injection of an aliquot of 1 L of the water sample. The GC was then run for 15
minutes. The retention time of the peak obtained was compared with the previously obtained
from the injection of the individual standard solutions. The peak response of the peak was then
measured. Afterwards, the concentration of the organochlorine compound present in the eluate
was determined based on the external standard calibration curve. Lastly, the concentration of the
compound present in the original sample was calculated by multiplying the result for the eluate
by the dilution factor of 5/250.

DATA
Table 1. Peak Response and Retention Time of Pentachloronitrobenzene
Compound Name

Retention time

Pentachloronitrobenzene

9.222

Area

Height

10394.6 4671.5

Figure 1. External standard calibration curve of pentachloronitrobenzene.

Figure 2. Gas chromatogram of the water sample (5 mL eluate).

CALCULATIONS
Concentration of Pentachloronitrobenzene (Original sample):
From the external standard, pentachloronitrobenzene has an area of 292578 and a
concentration of 39.347 ppb. By multiplying the area obtained from the sample eluate to the
external standard, the concentration of pentachloronitrobenzene in the eluate can be obtained. To
calculate the concentration of pentachloronitrobenzene in the original sample, the concentration
of the compound in the eluate is multiplied by a dilution factor of 5/250.

DISCUSSION
Solid phase extraction methods and gas chromatography were employed to analyze the
presence of organochlorines in a water sample obtained along the Limketkai Road. The
instuments used were Thermo Scientific HyperSep C18 extraction column and Shimadzu GC2014 gas chromatograph equipped with Shimadzu ECD-2014 detector.
The water sample was subjected to solid phase extraction procedures. It was employed to
concentrate the quantity of the compound present in the water sample for it to be measured
accurately. This ensures the largest response in the detection system and minimizes errors in

precision caused by background noise. A possible chromatogram is depicted below if the original
sample would directly be used in the analysis:

SPE was also done to remove the interfering compounds that would mask the analyte during the
analysis thereby giving clearly identifiable signals from the extracted components in the sample.
Lastly, employing SPE converts the sample into a form that is compatible with the gas
chromatograph by removal of sample matrix. Prior to solid phase extraction, the extraction
columns were first conditioned. Conditioning was done to make the sorbent compatible with
sample solution for close contact in small channels. In addition, conditioning activates the C-18.
The solvent used for washing is a mixture of hexane-acetone (50:50). Washing removes the
interferences coadsorbed from the SPE column. Also the use of hexane-acetone mixture as
washing solvent improves the percent recovery of the compound.
The extracted sample from SPE was then analyzed by gas chromatography. An aliquot of
1L of the eluate was injected into the gas chromatograph column. The sample enters the gas
stream which is transported by the carrier gas into a separation tube called the column. The
carrier gas in the experiment was helium. The column used in the GC instrument is a packed
column which is made up of copper which is 3 meters long and has a diameter of 15 cm that is
formed as coils. The packed column is used to have efficient separation and precise reading.
Inside the column, the various components in the sample was separated. The detector measures
the quantity of the components that exit the column. The GC instrument used in the experiment
was equipped with an electron capture detector (ECD) which selectively responds to halogencontaining compounds, such as pesticides. In the detector, the sample eluate from the column is
passed over the radioactive emitter, nickel-63. The electron from the emitter causes ionization
of the helium gas and produces a burst of electrons. In the presence of organochlorines which has
an electronegative functional group that tend to capture electrons, the current decreases markedly

and thus detected with high sensitivity. The detections are then displayed as peaks in the
computer in which the retention time and the area of the organochlorine compound is shown.
The results of the analysis showed that there is a significant peak with area 10394.6 and
height 4671.5 which was determined to be pentachloronitrobenzene. The concentration of the
organochlorine compound was calculated by converting the area reading into concentration
based on the external standard method. The concentration of pentachloronitrobenzene in the
original sample was calculated to 0.0280 ppb. This is not a typical result of chromatography
because the concentration level is not within the range of approximately 0.1 ppb to
approximately 5 ppb.

CONCLUSION
Solid phase extraction is a useful sample preparation technique that utilizes extraction
selectivity enrichment of trace concentration of samples with very low concentration levels. Gas
chromatography is an analytical method that can be used to analyze an organochlorine
compound in water sample. The water sample was analyzed to contain the organochlorine,
pentachloronitrobenzene which is present in concentrations of 0.0280 ppb. The concentration
level of the compound in the water sample is not significant since typical results should be within
the range of approximately 0.1 ppb to approximately 5 ppb.

REFERENCES
Simpson, N. J. (2000). Solid-Phase Extraction: Principles, Techniques, and Applications. New
York, Basel: Marce Dekker.
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