EXPERIMENT 1

Gas Chromatography (GC): Method

Development

NAME: NIK ADRIANA BINTI ROSLI

MATRIC NO: 2020996935

CLASS: RAS2453B

DATE OF SUBMISSION: 19/1/2021

LECTURER’S NAME: DR SHARIZAL HASAN

OBJECTIVE
To determine effects of separation using Gas Chromatography (GC) by changing their
volatility of compound, the effect of length of the column, the effects of column temperature
and flow rate of carrier gas.

INTRODUCTION
Method development is a process used to prove whether the analytical method that has been
done is acceptable or not. The efficiency of compounds’ separation in Gas Chromatography
(GC) are depends on the compounds travelling through the column at different rates. There
are factors that affect GC separation such as volatility of compound, column temperature,
flow rate of gas through the column, length of the column, column polarity and polarity of the
compounds. In this experiment, the factors that has been focuses the most only four factors
which are volatility of compound, column temperature and flow rate of the gas through the
column. All factors affecting the separation based on their properties. For volatility of
compound, when the boiling point is low which is the volatility of compound is high because
they evaporate more so the compound will travel faster through the column than the
compound with high boiling point. Next, for the column temperature when the column
temperature increase, it will speed up the elution process of the compound. Then, for the flow
rate of column. When the flow rate of column increase, the gas will flow through the column
faster so it elutes first. Compounds that have shorter elution time will produce a sharp and
narrow peak with has a better separation. The last one is for the length of the column. If the
column is longer, it will take some time for the compound to elute. So, the compound will
elute later than other analyte. As for this experiment, the resolution is calculated to determine
whether the peak has better separation or not. If the resolution is 1.5, the peak has better
separation. If the resolution is above 1.5, the peak does have better resolution, but the
retention time is longer. The resolution is calculated as below:
Rs= [2(Tr2-Tr1)/W1+W2]
Rs = resolution
Tr1 and Tr2 = retention times of two peaks
W1 and W2 = baseline width of the peaks

REAGENTS AND SOLUTIONS

a. Individual methyl esters compounds: methyl laurate, methyl myristate, methyl
palmitate, methyl stearate, methyl oleate, methyl linoleate.
b. Standard mixture: methyl laurate (0.20 mg m L−1 ¿, methyl myristate (0.20 mg m L−1 ¿,
methyl palmitate (1.0 mg m L−1 ¿, methyl stearate (0.70 mg m L−1 ¿, and methyl
linoleate (0.35 mg m L−1 ¿
INSTRUMENT
Gas chromatography (Agilent Technologies 6890N) equipped with flame ionisation detector
(FID) and 30m × 0.25µm HP5-MS capillary column.

ANALYTICAL PROCEDURE

1. The instrument set up as below:

Injection port: split (40:1)
Injection port temperature: 250℃
Column temperature: 210℃
Carrier gas flow rate: 30 cm sec-1
Detector temperature: 250℃

2. Effect of carrier gas flow rate on the isothermal GC separation of methyl esters.
0.40μL standard mixture isothermally was injected at 210°C at gas flow rate 30 cm
sec-1. The flow rate then increased to 50 cm sec -1. The system was allowed for a few
minutes for it to equilibrate and before standard mixture was injected again. The same
procedure was repeated at flow rate 70 cm sec -1. Based on the result, the optimum was
determined to be 70 cm sec-1.
3. Effect of column temperature on isothermal GC separation of methyl esters.
0.4µL standard mixture was injected isothermally at 170℃, followed by 190℃ at
optimum carrier gas flow rate. The effect of column temperature on separation,
resolution and analysis time were evaluated.
4. At optimum flow rate, standard mixture was injected using temperature range from
100℃ to 290℃. Alter the temperature programming to improve resolution of
compounds. The methyl ester was injected individually to identified various
components in standard mixture using optimized GC conditions.

RESULTS

Effect on resolution based on same temperature but different flow rate.

Condition Injection Retention Peak width Resolution Average

time (min) (min) Resolution
Temperature: 2 Peak 2: Peak 2: 34.7692 35.0642
210°C 4.570 0.0467
Peak 3: Peak 3:
Flow rate:
6.491 0.0638
30 m/s
3 Peak 2: Peak 2: 35.3591
 4.568 0.0450

Peak 3: Peak 3:
6.488 0.0636
Temperature: 1 Peak 2: Peak 2: 30.1039 31.8834
210°C 2.740 0.0351
Peak 3: Peak 3:
Flow rate:
3.899 0.0419
50 m/s
2 Peak 2: Peak 2: 33.6628
2.738 0.0304
Peak 3: Peak 3:
3.896 0.0384
Temperature: 2 Peak 2: Peak 2: 25.0904 25.3061
210°C 1.956 0.0288
Peak 3: Peak 3:
Flow rate:
2.789 0.0376
70 m/s
4 Peak 2: Peak 2: 25.5218
1.959 0.0314
Peak 3: Peak 3:
2.791 0.0338
Table 1 shows effect on resolution based on constant temperature but different flow rate.

Condition Injection Retention Peak width Resolution Average

time (min) (min) Resolution
Column 1 Peak 2: Peak 2: 61.8101 60.8707
temperature: 3.644 0.0390
Peak 3: Peak 3:
170°C
7.127 0.0737
Flow rate:
2 Peak 2: Peak 2: 59.9312
70 m/s
3.631 0.0445
Peak 3: Peak 3:
7.116 0.0718
Temperature: 2 Peak 2: Peak 2: 36.9381 36.1247
190°C 2.578 0.0333
Peak 3: Peak 3:
Flow rate:
4.279 0.0588
70 m/s
3 Peak 2: Peak 2: 35.3112
2.579 0.0356
 Peak 3: Peak 3:
4.281 0.0608
Temperature: 2 Peak 2: Peak 2: 25.0904 25.3061
210°C 1.956 0.0288
Peak 3: Peak 3:
Flow rate:
2.789 0.0376
70 m/s
4 Peak 2: Peak 2: 25.5218
1.959 0.0314
Peak 3: Peak 3:
2.791 0.0338
Table 2 shows effect on resolution based on constant flow rate different temperature

Standard mixture Retention time (min)

Methyl linoleate 4.002
Methyl myristate 1.938
Methyl laurate 1.559
Methyl palmitate 2.728
Methyl stearate 2.553
Table 3 shows retention of standard mixture of methyl ester.

DISCUSSION

Gas chromatography is a separation technique of molecule from its sample mixture. Detector
used for this experiment which is flame ionization detector (FID) will detect the components
whether it present or not and if it present, it will show by chromatogram in the form of
different peaks. This experiment conducted to know effect on peak separation when changing
with different flow rate and column temperature and to know optimum flow rate and
temperature for better separation. When changing the flow rate, based on the result high flow
rate give lower retention time. But it causes the peak to broad due to mass transfer which is
the C-term in Van Deemter Plat because the solute does not fully interact with stationary
phase. The peak has better separation when the peak is narrow and sharp and when the peak
is not farther apart from each other. Optimum gas flow rate must be use in this experiment to
reduce retention time and to create better separation. In this experiment the optimum gas flow
rate is 70 m/s with average resolution 25.3061 which is the nearer to ideal resolution, 1.5
compared to other flow rate.

Changing temperature also affect retention time and resolution of the peak. Retention time
is inversely proportional to the column temperature. When increase the temperature, time for
the analyte to flow through the column will decrease but some peaks might be overlapped to
each other. When lower the temperature, the peak does give better separation, but it has
higher retention time. So, the analyte might take some time to pass through the column. In
order to reduce retention time and to make the compounds separate sufficiently, optimum
temperature must be used. Result shows the optimum temperature is at 210°C. The
experiment concludes, to separate methyl, the optimum gas flow rate, 70 m/s and column
temperature 210°C will give the best resolution and better peak of separation. From the last
data, the retention time used to know the boiling point of standard mixture. When the boiling
point is low, time for sample from injection to detection is faster. It is because the sample is
more volatile when it has lower boiling point, so it increases the speed of sample to pass
through the column. The result shows the lowest boiling point is methyl laurate while the
highest boiling point is methyl linoleate.

CONCLUSION

Gas chromatography is type of chromatography used in analytical chemistry for separating

sample from its mixture. Mobile phase for gas chromatography is gas carrier while the
stationary phase is a chemical that can selectively attract components in a sample mixture.
From the experiment, it concludes that the best resolution and more efficient peak can be
achieve without worsen the quality of the peak by optimizes the gas flow rate and column
temperature. The optimum gas flow rate is 70 m/s while the optimum temperature is 210°C.
The first peak that out after the solvent peak is correspond to methyl laurate followed with
methyl myristate and end with methyl linoleate.

REFERENCES
Analytical Laboratories Applications GC with Electron Capture Detector (GC-ECD). (n.d.). Retrieved
from AIR PRODUCTS : http://www.airproducts.com.my/industries/Analytical-
Laboratories/analytical-lab-applications/product-list/gc-with-electron-capture-detector-gc-
ecd-analytical-laboratories.aspx?
itemId=2ED69212C574443C9354860ABEFCFE2B#:~:text=Gas%20Chromatography
%20%E2%80%9

Solid Phase Extraction (SPE). (2020, June 9). Retrieved from Chem.LibreTexts:
https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Supplemental_Modules_(An
alytical_Chemistry)/Analytical_Sciences_Digital_Library/Active_Learning/Contextual_Modul
es/Sample_Preparation/03_Solid-Phase_Extraction

Solid-Phase Extraction. (n.d.). Retrieved from ScienceDirect:

https://www.sciencedirect.com/topics/chemistry/solid-phase-extraction#:~:text=The
%20basic%20principle%20of%20SPE,greater%20affinity%20for%20the%20analytes.

What is Solid-Phase Extraction (SPE)? (n.d.). Retrieved from Waters:

https://www.waters.com/waters/en_US/Goals-and-Benefits-of-SPE/nav.htm?
cid=10083495&locale=en_US

APPENDIX

FIGURE 1 STANDARD MIXTURE OF METHYL ESTER

TEMPERATURE, °C FLOW RATE, m/s INJECTION
210 30 2
 FIGURE 2 STANDARD MIXTURE OF METHYL ESTER
TEMPERATURE, °C FLOW RATE, m/s INJECTION
210 30 3
 FIGURE 3 STANDARD MIXTURE OF METHYL ESTER
TEMPERATURE, °C FLOW RATE, m/s INJECTION
210 50 1
 FIGURE 4 STANDARD MIXTURE OF METHYL ESTER
TEMPERATURE, °C FLOW RATE, m/s INJECTION
210 50 2
 FIGURE 5 STANDARD MIXTURE OF METHYL ESTER
TEMPERATURE, °C FLOW RATE, m/s INJECTION
210 70 2
 FIGURE 6 STANDARD MIXTURE OF METHYL ESTER
TEMPERATURE, °C FLOW RATE, m/s INJECTION
210 70 4
 FIGURE 7 STANDARD MIXTURE OF METHYL ESTER
TEMPERATURE, °C FLOW RATE, m/s INJECTION
170 70 1
 FIGURE 8 STANDARD MIXTURE OF METHYL ESTER
TEMPERATURE, °C FLOW RATE, m/s INJECTION
170 70 2
 FIGURE 9 STANDARD MIXTURE OF METHYL ESTER
TEMPERATURE, °C FLOW RATE, m/s INJECTION
190 70 2
 FIGURE 10 STANDARD MIXTURE OF METHYL ESTER
TEMPERATURE, °C FLOW RATE, m/s INJECTION
190 70 3
 FIGURE 11 STANDARD MIXTURE OF METHYL ESTER WITH
METHYL LINOLEATE
TEMPERATURE, °C FLOW RATE, m/s INJECTION
210 70 1
 FIGURE 12 STANDARD MIXTURE OF METHYL ESTER WITH
METHYL MYRISTATE
TEMPERATURE, °C FLOW RATE, m/s INJECTION
210 70 1
 FIGURE 13 STANDARD MIXTURE OF METHYL ESTER WITH
METHYL LAURATE
TEMPERATURE, °C FLOW RATE, m/s INJECTION
210 70 1
 FIGURE 14 STANDARD MIXTURE OF METHYL ESTER WITH
METHYL PALMITATE
TEMPERATURE, °C FLOW RATE, m/s INJECTION
210 70 1
 FIGURE 15 STANDARD MIXTURE OF METHYL ESTER WITH
METHYL STEARATE
TEMPERATURE, °C FLOW RATE, m/s INJECTION
210 70 1
 EXPERIMENT 2
High Performance Liquid
Chromatography (HPLC): Method
Development

NAME: NIK ADRIANA BINTI ROSLI

MATRIC NO: 2020996935

CLASS: RAS2453B

DATE OF SUBMISSION: 19/1/2021

LECTURER’S NAME: DR SHARIZAL HASAN

OBJECTIVE
To separate mixture of 5 compounds using High Performance Liquid Chromatography
(HPLC) by differed their mobile phase composition.

INTRODUCTION
High Performance Liquid Chromatography (HPLC) is one of the analytical techniques used
to separate and determine each of the component in a mixture. Mobile phase of this
chromatography is liquid while the stationary phase is also liquid. The separation is based on
differences in polarity of analyte. The analyte that elute first is analyte that less interact to the
stationary phase and analyte that elute late is the one that interact the most with the stationary
phase. Reversed phase chromatography used in this experiment. Reversed phase is where the
mobile phase is polar while the stationary phase is nonpolar. Changing the polarity of the
mobile phase change will affect the interaction between the analyte and stationary phase. The
efficiency of separation of the compound will also been affected. The composition of the
mobile phase can be differed by two method. First, by isocratic elution where the
composition of mobile phase remains the same throughout the analysis. Second, by using
gradient elution. The composition of the mobile phase is different during separation either
continuously or in step to separate analyte with different range of polarity.

REAGENTS AND SOLUTIONS

HPLC grade acetonitrile
Deionised water
Standard mixture of caffeine, acetone, ethyl benzoate, phenatole and phenanthrene (around
100 ppm)

INSTRUMENT
Liquid chromatograph (Agilent G1314A HPLC) equipped with diode array detector (DAD),
RPC 18 column and 2μL sample loop.

ANALYTICAL PROCEDURE
1. The instrument set up as below:
Detector wavelength: 254 nm
Flow rate: 1.5 mL/min
Mobile phase: acetonitrile:water
 2. Effect of mobile phase on HPLC separation.
The mobile phase using polar solvent which acetonitrile and deionized water with the
ratio 50:50. The standard mixture was injected. Then, the composition of mobile
phase was changed to ratio 70:30.
3. Identification of components in the mixture
To investigate the component in the mixture using the selected high performance
liquid chromatography or HPLC specification, each compound was injected
individually in automatic injection.
4. Separation using gradient elution
Based on the separation, gradient elution separation was performed to improve the
efficiency of the column by enhance the ratio of the column.

RESULT
1. The consequences of the change composition of mobile phase on resolution by
isocratic elution:

2(t R 2−t R 1)
Rs = W 2 +W 2

Composition Peak no. Retention Base width of Resolution Average

of mobile time (min) peak (min) resolution
phase (ACN:
H 2O)
50:50 2-1 1.356, 1.135 0.1319, 0.1674 1.4768 12.8583
3-2 3.964, 1.356 0.2201, 0.1319 14.8182
4-3 6.887, 3.964 0.2767, 0.2210 11.7673
5-4 26.141, 6.887 1.3710, 0.2767 23.3708
70:30 2-1 1.234, 1.169 0.1069, 0.1435 0.5192 6.1078
3-2 2.089, 1.234 0.1247, 0.1069 7.3834
4-3 2.800, 2.089 0.1303, 0.1247 5.5765
5-4 6.377, 2.800 0.5229, 0.1303 10.9522

Average resolution of isocratic elution

12.8583+ 6.1078
= 2
= 9.4831
 2. The impact of the composition of the mobile phase by gradient solution.

Composition Peak no. Retention Base width of Resolution Average

of mobile time (min) peak (min) resolution
phase (ACN:
H 2O)
50:50 (0 min 2-1 1.278, 1.138 0.0991, 0.1540 1.1063 6.0542
– 1.8 min)
3-2 2,582, 1,278 0.1934, 0.0991 8.9162
70:30 (after
1.8 min – 8.0 4-3 3.487, 2.582 0.1521, 0.1934 11.7673
min)
5-4 5.403, 3.487 0.2758, 0.1934 23.3708
50:50 (0 min 2-1 1.278, 1.136 0.1013, 0.1598 1.0877 5.7411
– 1.8 min)
3-2 2.582, 1.278 0.2222, 0.1013 8.0618
70:30 (after
1.8 min – 3.0 4-3 3.489, 2.582 0.1507, 0.2222 4.8646
min)
5-4 5.403, 3.489 0.2770, 0.1507 8.9502
85:15 (3.0
min until 8.0
min)

Average resolution of gradient elution

6.0542+ 5.7411
=
2
= 5.8977

3. The retention time of the components that modify HPLC mode (70:30) ration of
(ACN: H 2 O )

Standard mixture of the Retention time on

components individual standard (min)
Caffeine 1.121
Acetone 1.332
Methyl benzoate 2.095
Phenatole 2.812
Phenanthrene 6.353
DISCUSSION
High performance liquid chromatography (HPLC) is process of chromatography used to
separate sample mixture into small compounds with mobile phase and stationary phase are in
the form of liquid. Method development of HPLC is a process to prove the stability of the
sample for future used by changing the column temperature, injection volume, flow rate of
the sample and others. In this experiment, the polarity of the analyte is the one that being
changed. Analyte that has low interaction between the stationary phase will elute first and
analyte that stay the most at stationary phase will elute later. Polarity of analyte is directly
proportional to the retention time. When the polarity of analyte increase, the retention time
will also increase. Lower retention time will give sharp and narrow peak. The polarity of
analyte measured by composition of the mobile phase. Based on experiment conducted,
reversed phase is used. Reversed phase chromatography is liquid chromatography technique
where the stationary phase is non polar and mobile phase is polar. Reversed phase
chromatography used to separate molecules based on hydrophobic interactions between the
solute molecules in the mobile phase and the ligands attached to the stationary phase.
Gradient elution mode used in this experiment to separate mixture with wide polarity and
change the polarity of the composition of mobile phase continuously or in step throughout the
analysis. By changing the composition of mobile phase, the eluent strength will increase
during the separation and the analysis time also will decrease. It will produce the separation
more efficient and has good resolution. Strong eluent strength is where the amount of the of
organic solvent used in the ratio is higher than amount of water. For the experiment, the
composition that have strong eluent is 70:30 (ACN: H 2 O ) compared to other composition
50:50 (ACN: H 2 O ). In composition 70:30, the analytes come out faster than the one with
composition 50:50. The peak has better separation when it has ideal resolution 1.5. The peak
that has resolution more than 1.5 will has better separation but the space between the peaks
much longer. Resolution less than 1.5 produce inadequate separation between peak or the
peak has already overlapped to each other. Both average resolutions of isocratic elution and
gradient elution is more than 1.5 which are 9.4831 and 5.8977. So, it concludes that the peaks
have better separation but have longer retention time for the analyte to be eluted. But the
average resolution of for gradient elution is lower than the isocratic. Supposedly the
resolution of the gradient elution is much higher than isocratic elution. The qualitative
analysis was done to identify the components in the mixture by comparing the peaks in the
mixture with the peaks of the standard compound. The one that elute first with shorter
analysis time is the first peak. So, there is caffeine as the first peak followed by other
compounds such as acetone, methyl benzoate, phenatole and phenanthrene.

CONCLUSION
High performance liquid chromatography (HPLC) is chromatography technique that used
pressure instead of gravity to separate mixture into small components but it has to develop
method to prove the stability of mixture for further used. In this experiment, polarity of
analyte has been changed. Polarity indicates to composition of mixture. Based on the result,
the most suitable composition of mixture is 70:30 (ACN: H 2 O ). When the polarity of organic
solvent increases the strength eluent also increase and the retention time will decrease. The
first peak is corresponding to caffeine followed by other compound which are acetone,
methyl benzoate, phenatole and phenanthrene peak.

REFERENCES
Analytical Laboratories Applications GC with Electron Capture Detector (GC-ECD). (n.d.). Retrieved
from AIR PRODUCTS : http://www.airproducts.com.my/industries/Analytical-
Laboratories/analytical-lab-applications/product-list/gc-with-electron-capture-detector-gc-
ecd-analytical-laboratories.aspx?
itemId=2ED69212C574443C9354860ABEFCFE2B#:~:text=Gas%20Chromatography
%20%E2%80%9

Solid Phase Extraction (SPE). (2020, June 9). Retrieved from Chem.LibreTexts:
https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Supplemental_Modules_(An
alytical_Chemistry)/Analytical_Sciences_Digital_Library/Active_Learning/Contextual_Modul
es/Sample_Preparation/03_Solid-Phase_Extraction

Solid-Phase Extraction. (n.d.). Retrieved from ScienceDirect:

https://www.sciencedirect.com/topics/chemistry/solid-phase-extraction#:~:text=The
%20basic%20principle%20of%20SPE,greater%20affinity%20for%20the%20analytes.

What is Solid-Phase Extraction (SPE)? (n.d.). Retrieved from Waters:

https://www.waters.com/waters/en_US/Goals-and-Benefits-of-SPE/nav.htm?
cid=10083495&locale=en_US
APPENDIX
Figure 1 STANDARD MIXTURE gradient elution (injection 1)
Figure 2 STANDARD MIXTURE gradient elution (injection 2)
Gradient Program Time (min) %ACN:%Water

0 – 1.8 50:50

1.8 – 8.0 70:30

Figure 3 STANDARD MIXTURE ISOCRATIC ELUTION 70%: 30% ACN: Water
Figure 4 STANDARD ELUTION Individual standard CAFFEINE at 70%: 30% ACN: Water
Figure 5 STANDARD ELUTION Individual standard ACETONE at 70%: 30% ACN: Water
70% ACN: 30% Water

Figure 7 STANDARD ELUTION Individual standard PHENATOLE at 

70% ACN: 30% Water
Figure 8 STANDARD ELUTION Individual standard PHENANTHRENE at 
70% ACN: 30% Water
Figure 9 STANDARD MIXTURE ISOCRATIC ELUTION, 50% ACN: 50% Water
Pea Retation time Width
k

4 6.887 0.2767

5 26.141 1.3710

EXPERIMENT 3
Analysis of Chlorpyrifos in Water using
Solid Phase Extraction (SPE) and Gas
Chromatography Electron Capture
Detector (GC-ECD)

NAME: NIK ADRIANA BINTI ROSLI

MATRIC NO: 2020996935

CLASS: RAS2453B

DATE OF SUBMISSION: 19/1/2021

LECTURER’S NAME: DR SHARIZAL HASAN

OBJECTIVE
1) To determine the analysis of chlorpyrifos in water by using Solid-phase Extraction
(SPE) and Gas Chromatography-Electron Capture Detector (GC-ECD) methods.
2) To calculate amount of chlorpyrifos in each sample and their percentage of recovery
in average.

INTRODUCTION
Chlorpyrifos is white crystal-like solid which also an insecticide. It is not soluble in water.
This compound widely used for homes and farms. At homes, this insecticide used to control
cockroaches, fleas, and termites while on the farm, it is used to control ticks on cattle and as a
spray to control crop pests. Solid phase extraction (SPE) is one of the extraction methods
used to isolate analyte from the solution by using solid and liquid phase. Separation of the
compound is based on their physical and chemical properties. Aim of using this extraction
method is to simplify complex sample matrices into less complex compound, to purify and
reduce the ion suppression in mass spectrometry applications. Advantages of using SPE are
lower solvent and reagent consumption, the techniques of separation only include fewer steps
and for the safety, it is safe because it is less expose to the toxic and dangerous agents. Steps
in performing SPE are preparation of the sample which includes dilution and adjustment of
pH, condition of cartridge, load the sample then lastly elution of sample.

REAGENTS AND SOLVENTS

Analytical grade methanol
Analytical grade hexane
Standard chlorpyrifos (30 ppm)

APPARATUS
C 18 solid phase extraction cartridges (500 mg)
Glass fibre filter paper
SAMPLE
Wastewater (50 mL). pH was adjusted to 2 using HCl and add 10% methanol.

INSTRUMENT
Gas Chromatograph (Agilent Technologies 6890 N) equipped with an electron capture
detector (ECD) and 30 m x 250 μm x 0.25 μm HP5-MS capillary column.

ANALYTICAL PROCEDURE
1. The water sample was filtered through a glass filter paper.
2. Solid phase extraction procedure
i) Condition C 18 SPE cartridge by passing 10 mL of methanol.
ii) The cartridge was rinsed by passing 6 mL of deionized water without applying
vacuum.
iii) 50 mL of filtered water sample was passed through preconditioned column at
6 mL per min (48 drops/min). The column did not allow to dry during this
sample enrichment step.
iv) The column wad dried by vacuum for 15 minutes.
v) The interference was removed by eluting the column with 10 mL of deionized
water and again the cartridge was vacuumed dry for 30 minutes.
3. Instrument was set-up as below:
Injector temperature: 250 °C
Detector temperature: 300 °C
Carrier gas flow rate: 50 cm sec-r (nitrogen)
Column temperature: Initial temperature 165 °C for three minutes, increase to 260 °C
at 3 °C min−1with a final time of two minutes.
4. Quantitative analysis of chlorpyrifos
i) 1 μL of sample was injected onto the column. The injection was repeated to
get reproducible peak areas.
ii) 1 μL of standard chlorpyrifos was injected. The injection was repeated to get
reproducible peak areas.
iii) By using data from the standard solution, the concentration of chlorpyrifos in
the sample was calculated.

RESULTS
Injection Concentration Peak Area (Hz*s) Response Factor
(ppm) [ppm/(Hz*s)]

1 30 2.05954e^6 1.4566e^(-5)

2 30 2.05954e^6 1.4566e^(-5)
 Average 1.4566e^(-5)

Table 1 shows the Response Factor of Standard Chlorpyrifos

Injection Response factor Peak area Concentration

[ppm/(Hz*s) (Hz*s) (ppm)
6
Sample 1 1 1.4566e^(-5) 1.19924e 17.47
6
2 1.4566e^(-5) 1.18178e 17.21
Average 17.34
6
Sample 2 1 1.4566e^(-5) 1.38315e 20.15
6
2 1.4566e^(-5) 1.36710e 19.91
Average 20.03
Sample 3 1 1.4566e^(-5) 2.51821e 6 36.68
6
2 1.4566e^(-5) 2.53506e 36.93
Average 36.81
Table 2 shows the concentration of Chlorpyrifos for each sample

Chlorpyrifos Average concentration of TOTAL average

each sample (ppm) concentration (ppm)
Sample 1 17.34
Sample 2 20.03 24.73
Sample 3 36.81
Table 3 shows the average concentration of Chlorpyrifos

CALCULATION
Percent recovery of Chlorpyrifos
= (concentration in sample/ concentration of standard) x 100%
= (24.73 ppm / 30 ppm) x 100%
= 82.42%

DISCUSSION
Chlorpyrifos is a compound that does not mix well with water but it can mix with oily
liquids. Low concentration of chlorpyrifos make it difficult to determine the sample in the
compound, so solid phase extraction (SPE) is used to separate compound that dissolved in
liquid mixture from other compound depends on their physical and chemical properties. SPE
have rapid separation compared to other method and the purification prior to the
chromatographic analysis. In SPE, solid phase must have greater affinity compared to the
sample matrix. The compounds that retained on solid phase can be removed by eluting
solvent with a greater affinity for the analytes. Gas chromatography in Electron Capture
Detector (GC-ECD) also used in this experiment because this technique is used to analyse
halogenated compounds and the compounds used in this experiment is halogenated
compounds. ECD only can detect analytes that contain electronegative functional groups
which can capture electrons such as halogens, peroxides, quinones and nitro groups. The
weaknesses of using ECD is it involve radioactive components so it can be easily affecting
the peak of analysis. Response factor is used to calculate amount of chlorpyrifos in the
sample which based on standard compound. The average amount of chlorpyrifos getting from
the result is 1.4566e−5 ppm/( Hz∗s). Data of percent recovery calculated is 82.42% in
average. In order to get higher amount of chlorpyrifos or percentage recovery, the SPE must
be carried out carefully so it will extract more chlorpyrifos efficiently.

In this analysis, GC with Electron Capture Detector (ECD) 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 disadvantages of ECD is it involve radioactive component.
The amount of chlorpyrifos in samples is calculated by using response factor calculation that
based on the standard compound. The average amount of chlorpyrifos in the sample is 1.4566
e−5 ppm/( Hz∗s). The percentage recovery calculated is 82.42% in average. In order to get
higher amount of chlorpyrifos or percentage recovery, the SPE must be carried out carefully
so it will extract more chlorpyrifos efficiently.

CONCLUSION
Solid phase extraction (SPE) and Gas chromatography in Electron Capture Detector (GC-
ECD) is used in this experiment to analyse chlorpyrifos in water. From the result and
calculation, average amount of chlorpyrifos in sample is 1.4566e−5 ppm/(Hz∗s) and the
percent recovery calculated based on result is 82.42% in average.
REFERENCES
Analytical Laboratories Applications GC with Electron Capture Detector (GC-ECD). (n.d.). Retrieved
from AIR PRODUCTS : http://www.airproducts.com.my/industries/Analytical-
Laboratories/analytical-lab-applications/product-list/gc-with-electron-capture-detector-gc-
ecd-analytical-laboratories.aspx?
itemId=2ED69212C574443C9354860ABEFCFE2B#:~:text=Gas%20Chromatography
%20%E2%80%9

Solid Phase Extraction (SPE). (2020, June 9). Retrieved from Chem.LibreTexts:
https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Supplemental_Modules_(An
alytical_Chemistry)/Analytical_Sciences_Digital_Library/Active_Learning/Contextual_Modul
es/Sample_Preparation/03_Solid-Phase_Extraction

Solid-Phase Extraction. (n.d.). Retrieved from ScienceDirect:

https://www.sciencedirect.com/topics/chemistry/solid-phase-extraction#:~:text=The
%20basic%20principle%20of%20SPE,greater%20affinity%20for%20the%20analytes.

What is Solid-Phase Extraction (SPE)? (n.d.). Retrieved from Waters:

https://www.waters.com/waters/en_US/Goals-and-Benefits-of-SPE/nav.htm?
cid=10083495&locale=en_US
APPENDIX

1) Standard Chlorpyrifos (Example)

2) Chlorpyrifos in Sample (Example)