Experiment 7

Tittle
Analysis of Chloropyrifos in Water by Solid Phase Extraction (SPE) and Gas
Chromatography-Electron Capture Detector (GC-ECD).
Abstract
A pesticide multiresidue method for determining dichlorvos, naled. lindane, diazinon.
chlorpyri-fos-methyl, dichlofluanid, chlorpyrifos, folpet, and -endosulfan, endosulfansulphate, fen-propathrin and acrinathrin in water samples at the levels required by the EEC
Drinking Waters Directive has been developed. The pesticides were selected among the most
used during die last 20 years in Almeria (Spain), where there is a high agricultural activity.
Solid Phase Extraction (SPE) was selected as extraction method after being compared with
Liquid-Liquid Extraction (LLE).
Solid Phase Extraction (SPE) is becoming popular to extract pesticides from may
sample matrices. This method is more economical and few operational steps are required for
the extraction process when compared to liquid-liquid extraction. In the study, C-18 bonded
silica was utilized as a solid phase to extract pesticides form water. Water samples (500 ml)
were passed through the C-18 cartridges (500 mg) at a constant flow rate (12 ml/min) using
vacuum manifold. Adsorbed analytes were eluted with ethyl acetate and analysed by
GC/ECD, NPD. Acceptable recoveries (range: 80% -120%) were obtained for alachlor,
captan, chlorpyrifos, diazinon, profenophos and oxyfluorfen.
Several liquid-liquid extraction methods have been developed to determine
organohelide and organophosphate pesticides and these are time consuming methods.
Dichloromethane is the most commonly used solvent for the extraction processes. Discharge
of dichloromethane to the environment is a serious air pollution issue.
The aim of this work was to apply the SPME technique for determining two important
pesticides (chlorpyrifos and chlorpyrifos-methyl) in waste water and to show the effective
application of SPME methods for determining these residues.

Reagents and Solvents

a. Analytical grade methanol.
b. Analytical grade hexane.
c. Standard chlorpyrifos (100 ppm).
Apparatus
a. C18 solid phase extraction catridges (500 mg).
b. Glass fibre filter paper.
Sample
a. Waste water (50 mL) was adjusted to pH 2 using HCl and 10% methanol was added.
Instrument
Gas Chromatograph (Agilent Technologies 6890N) equipped with an electron capture detector
(ECD) and 30 m x 250 m x 0.25 m HP5 MS capillary column.
Analytical Procedure
a. Water sample was filtered through a glass fibre filter paper by a laboratory assistant.
b. Solid phase extraction procedure.
i.
C18 SPE catridge was conditioned by passing 10 mL of methanol.
ii.
Then, the catridge was rinsed by passing 6 mL of deionized water without
iii.

applying vacuum.
The filtered water sample (50 mL) was passed through the preconditioned
column using a vacuum manifold at ~ 6 mL per min (48 drops per min).

iv.
v.

The column was not allowed to dry during this sample enrichment step.
The column was dried by vacuum for 15 minutes.
After that, the interference was removed by eluting the column with 10 mL

vi.

of deionized water. Then, again the catridge was dried for 10 minutes.
Lastly, the pesticide was eluted using 5 mL of hexanee. By blowing down
using gentle nitrogen, the sample was concentrated to 1 mL. The sample
was ready for GC analysis.

c. Instrument set up.

i.
Injector temperature
ii.
Detector temperature
iii.
Carrier gas flow rate

: 280 C.
: 300 C.
: 20.0 mL min-1 (nitrogen).

iv.

Column temperature

: Initial temperature 165 C for 3 min, increased to

260 C at 3 C min-1 with a final time of 2 min.

d. Quantitative analysis of chlorpyrifos.
i.
1 L of sample was injected onto the column. A reproducible peak area was
ii.

got because the sample injection was repeated.

1 L of standard chlorpyrifos was injected. A reproducible peak area was got

iii.

because the sample injection was repeated.

By using the data from standard solution, the concentration ot chlorpyrifos in
the sample was calculated.

Results
a. Comparison in retention time of standard and samples
Retention time of standard

Sample.

(ppm).

Retention time of

Average retention

sample (min).

time of sample
(min).

1
2
3

6.722

Trial 1
6.723
6.724
6.724

Trial 2
6.724
6.723
6.724

6.724
6.724
6.724

b. Calculation of responce factor (RF) of standard compund.

Responce factor (RF) = 11 202 ppm/Hz*s
(Calculation can see at appendix)

c. Amount of chlorpyrifos in samples.

Sample

Area (Hz*s)

Average Area (Hz*s)

Amount of
chlorpyrifos (ppm)

1
2
3

Trial 1
485893
507099
624548

Trial 2
519338
506456
667543

502615
506777
646045

Average amount of chlorpyrifos in sample = 49.26 ppm

44.87
45.24
57.67

d. Percentage recovery of samples.

Sample

Amount of chlorpyrifos in sample

Percentage recovery (%)

1
2
3

(ppm)
44.87
45.24
57.67

149.57
150.80
192.23

Average percentage recovery of samples = 164.2%

Discussion
The determination of chlorpyrifos at low levels in wastewater requires highly selective and
sensitive analytical procedures. The removal of matrix components during sample preparation
results in significant benefits towards reducing the matrix effects during GC analysis.
Therefore this experiment describes a simple method to enrich and clean up chlorpyrifos from
waste water usin solid phase extraction (SPE). In our experiment, the first step start with
conditioning C18 catridge by using methanol. This conditioning method will ensure that there
is no other interference include in the catridge and will ensure that the column will perform
effeciently. The column also was rinsed with deionized water before eluting the pesticide to
remove interference.

Final analysis was performed by gas chromatography. Gas chromatography with

electron capture detector is used because the analyte to be analysed is halogenated compound.
ECD only can detect analytes which contain electronegative functional groups that can
capture electrons such as halogens, peroxides, quinones and nitro groups. The result of
chromatogram also was analysed to evaluate each of the value. In this experiment, using and
appropriate formula, we had calculated the response factor (R f) and the value is 11 202
ppm/Hz*s. The calculation can be seen in the appendix. After that, we calculated the amount
of chlorpyrifos in the sample by dividing the average area (Hz*s) with the responcse factor,
(Rf) and the average value is 49.26 ppm . Recovery for chlorpyrifos ranged between 150 % to
190 %. Based on our research, the amount of the chlorpyrifos should be below than the
standard value which is 30 ppm. But, for our experiment, the value is 49.26 ppm.

So, we had do some further learning to find out what is the error for our experiment.
Data may be wrong because the chromatographer uses his equipment only 8 hours per day,
switches off energy and gases and restarts his equipment without the early morning test.
The latter can be just a well selected quantitative test mixture to be injected together with a
non sorbed but detectable inert gas, may be methan. The quantitative test values must
correlate with with late evening value which means it is a good idea to check the whole
working period per day by inclusion into two test run values. Each column has a limited
working range for the nature of substances : their molecular weight, their polarity, their
temperature stability. As the majority of all quantitative GC analyses is done by temperature
programming we have working range limits for the low starting temperature, the upper end of
the temperature program and the heating rate. For isocratic GC the selection of the column
temperature is limited by working ranges. The higher quantity of chlorphyrifos that our group
got because of the waste water that had been collected at the area which is freshly spray with
insectices. We assumed that, the insecticide was absorbed by the soil and water was flow in
them.
Conclusion
As the conclusion, the average amount of chlorpyrifos in sample is 49.26 ppm and the
percentage recovery is 164.2 %.

References
1.
2.
3.
4.

http://npic.orst.edu/factsheets/chlorpgen.pdf
https://www.epa.gov/ingredients-used-pesticide-products/chlorpyrifos
http://www.interchromforum.com/html/qnt_err_gc.html
Analytical Separation Methods laboratory Guide, 2nd Edition, n.d, Norashikin Saim,
Ruziyati Tajudin and Mardiana Saaid.

Appendix
1. Calculation of responce factor (RF) of standard compund.
Rf =

areaof standard peak

amount of standard
3.3606e5
30 ppm

= 11 202 ppm/Hz*s
2. Calculation of amount of chlorpyrifos in sample.
Sample amount (1) =

Peak area
R f standard

502615
11202

= 44.87 ppm
Sample amount (2) =

506777
11202

= 45.24 ppm
Sample amount (3) =

646045
11202

= 57.67 ppm
3. Average amount of chlorpyrifos.
=

44.87+ 45.24+57.67
3

= 49.26 ppm
4. Percentage recovery of samples.
Sample (1) =

amount of samples
100
amount of standard
44.87 ppm
100
30 ppm

= 149.57%
Sample (2) =

45.24 ppm
100
30 ppm

= 150.80%
Sample (3) =

57.67 ppm
100
30 ppm

= 192.23%
5. Average percentage recovery of samples.

149.57+ 150.80+ 192.23

3

= 164.20%