Determination Of Polycyclic Aromatic Hydrocarbon In Waste Oil Biology Essay

Published:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

Polycyclic aromatic hydrocarbons (PAHs) are also known as polynuclear aromatic hydrocarbons or poly-aromatic hydrocarbons. They consist of fused aromatic rings and do not contain heteroatoms or substituents. The pure PAHs are usually in colourless, white or pale yellowish green colour solids. These compounds have their major sources in fossil or synthetic fuels or are produced as by products from the incomplete combustion or high-temperature reactions of coal, oil and gas, automobile exhaust as well as organic materials. Furthermore, incomplete combustion of fuel in an internal combustion engine not only causes the formation of PAHs, other gaseous and particulates will also be emitted. A few PAHs can be used in medicines and some can be used to make plastics, pesticides, dyes and so on.

Nowadays, PAHs is one of the largest groups of environmental carcinogens which are present in air, food and drinking water. As the byproducts of degradation and combustion of organic materials, PAHs has become widespread in the environment. They are pollutants which are carcinogenic, teratogenic and mutagenic. The United State Environment Protection Agency (EPA) has selected thirty-two compounds of PAHs to be main pollutants. Among these 32 PAHs compounds, the EPA has categorised seven PAH compounds to be the possible carcinogens which can affect human health which are benzo(a)anthracene, chrysene, benzo(a)pyrene, benzo(b)fluoranthene, benzo(k)fluoranthene, dibenz(a,h)anthracene and indeno(1,2,3-cd)pyrene. Therefore, to avoid these 7 PAHs compounds exist excessively in the environment and threaten human's health, the presence of PAHs has to be monitored regularly in the most environmental department and industrial concern. The principle of this project is to determine the concentration of these 7 PAH compounds present in motor oil samples which are from the car factory and then identify whether they are within the limit set by the Department of Environment (DOE). If the concentration of the 7 PAHs in the waste oil exceeds the limit, the waste oil has to be treated before being disposed. Besides that, any punishment may also be given to the owner. Used motor oils which contain PAHs are mutagenic; especially from the motor vehicles which using leaded petrol whereas used oils from vehicles which using unleaded petrol or diesel fuel are less mutagenic. Since the method used is considered as a new method which is imported from other country. Thus, I was given a chance to partially validate the method whether the recoveries of sample analytes can be obtained through this method.

PAHs are lipophilic, so they are easier to mix with oil rather than water. Therefore, PAHs are mostly present in soil and oily substances in the environment when they are in larger sizes, but sometimes, they also exist in particulate matters which suspended in the air. PAHs in the soil can flow into underground water and then contaminate the water. Furthermore, PAHs can also enter into water through discharges from treatment plants of industrial and sewage water. However, most of the PAHs are not soluble in water, so they prefer combining with solid particles and then settle to the bottom of rivers and exist in sediment. Sediments, soils, water and other substances will be contaminated when PAHs is present. After a period of weeks or months, PAHs can be broken down by microorganisms, so they will not exist forever.

Toxicity of PAHs depends on the structures but not on their sizes. With isomers, they can be from non toxic to extremely toxic. Although some effects of PAHs have not been seen in human so far, animal experiments have proved that PAHs are very carcinogenic. After exposing to the PAHs no matter for a short or long period of time, they can cause harmful health effects such as on the skin, body fluids, and also the ability to fight diseases. For instance, laboratory animals were infected by skin cancer when their skin were exposed to the PAHs for a long time, lung cancer when they breathed air which containing PAHs as well as stomach cancer when they consumed food containing PAHs. Furthermore, in the animal studies on mice, the mice which were injected high level of benzo(a)pyrene during pregnancy would find difficulties when reproducing. Besides that, their offspring also had higher probability of birth defects, decreased body weights and IQ if they highly exposed to the benzo(a)pyrene before being given birth. These effects may also occur in human. Luckily, most of the PAHs which enter the body will leave through faeces and urine within a few days.

The following seven structures are the seven PAHs which are the possible carcinogens of human beings and they are the target analytes of this project:

Properties of Polycyclic Aromatic Hydrocarbon

IUPAC Name

Molecular Structure

Molecular Formula

Appearance

Carcinogenicity

Sources

Benzo(a) anthracene

C18H12

Solid

Carcinogenic

Mineral oil

Benzo(b) fluorenthene

C20H12

Off-white to tan powder

Strongly carcinogenic

Oysters

Benzo(k) fluorenthene

C20H12

Pale yellow solid (needles)

Not carcinogenic

Oysters

Roasted Coffee

Benzo(a) pyrene

C20H12

Pale yellow solid

Strongly carcinogenic

Smoked foods

Oysters

Cigarettes

Oils

BBQ beef & pork

Wax

Charcoal-broiled steaks

Roasted coffee

Polluted water

Fruits and vegetables

Chrysene

C18H12

Colourless with blue or red-blue fluorescence orthorhombic bipyramidal plates

Carcinogenic

Oysters

Wax

Smoked foods

Roasted coffee

Dibenz(a,h) anthracene

C22H14

Colourless solid

Strongly carcinogenic

Charcoal-broiled steaks

Indeno(1,2,3-cd)pyrene

C22H12

Yellow plates

Carcinogenic

Own work

3.3.2 Surrogate

Surrogate is a pure analyte which is not possible to be found in any samples, but its characteristic is the same as the sample analytes, so that there is no overlapping of retention time between the surrogate and any sample analytes. Surrogate solution with known concentration is added into the sample prior to extraction and any other processes. After that, it is determined by using the same procedures which are used to measure the sample analytes, and then its recovery is measured. The function of the surrogate is to monitor the method performance of with each sample. Individual surrogate recoveries are used to correct the concentrations of specific analyte according to the retention time. The surrogate used in this project is terphenyl-d14.

3.3.3 Internal Standard

An internal standard is a pure analyte with known concentration and different from sample analyte. It is added into a sample, extract or standard solution. The function of internal standard is to measure the relative response or concentration of other method analytes and surrogates which are components of the same solution with it by comparing the signal from analyte with the signal from the internal standard.

Internal standard is very useful for analysing a sample in which the sample's quantity or the response of instrument will slightly alter from run to run. The right concentration of analyte can be derived, only if the concentration of internal standard is known. Besides that, internal standard is commonly used in the chromatography because the little amount of sample solution which is injected into the chromatograph is not very reproducible in some tests. In this project, perylene-d12 is used as internal standard to calculate the concentration of other unknown analytes.

3.4 Principle of Method:

Samples are made up in solution, spiked with surrogate (terphenyl-d14), and then transferred into a silica gel clean-up column. By elution with hexane and dichloromethane/hexane 1:1, samples are fractionated into saturate fractions (F1) and aromatic fractions (F2). Then, both fractions are concentrated by evaporation under dry nitrogen "blow-down" apparatus. The seven compounds of PAHs are in the aromatic fractions (F2) whereas saturate fractions (F1) contains saturated hydrocarbon. For the purpose of determination of PAHs, aromatic fraction (F2) is spiked with internal standard (perylene-d12), accurately top up to the pre-injection volume and then analyzed by GC/MS in the scan mode.

3.5 Apparatus and Reagent:

3.5.1 Apparatus:

Analytical balance - must be able to accurately weigh 0.0001g

Volumetric flasks - 1mL, 5mL and 10mL

Volumetric flasks must be rinsed with three portions each of DCM/hexane 1:1 and hexane successively before use.

Graduated cylinders - 5mL, 10mL and 20mL

Micropipettes - 10µL-1000µL

Sample clean-up column - 300mm long x 10.5mm internal diameter plugged with glass wool at the bottom and a Teflon stopcock. Fritted glasses discs are not recommended because they will be very difficult to be cleaned after high concentration of extracts have been passed through.

15mL Centrifuge tubes - calibrated, graduated and with ground-glass stopper

Centrifuge tube and its stopper must be rinsed with three portions each of DCM/hexane 1:1 and hexane successively before use.

Dry-nitrogen "blow-down" device

Vials - 1mL, with Teflon-lined screw cap septa

Funnel

Gas chromatograph with mass spectrometer (GC/MS)

GC column used for analysis is 30m long x 0.25mm internal diameter, 60oC-325oC temperature limit, 0.25µm film thickness capillary DB-5MS (Durabond-5 Mass Spectrum).

Note: All glassware has to be rinsed with solvent (DCM and hexane) before being used.

3.5.2 Reagents:

Acetone

Dichloromethane

Dichloromethane(DCM)/hexane 1:1

Hexane

Glass wool

Before use, glass wool is placed into a large pre-cleaned column. Then, it is eluted with 2 volume of column of DCM followed by 2 volume of column of hexane. DCM and hexane are then discarded. A beaker is pre-cleaned with DCM and hexane. The clean cotton wool is poured into the beaker, covered loosely with solvent-rinsed aluminum foil. It is then dried overnight in fume hood. After that, the cotton wool is stored in an oven at temperature ranging from 160-200oC until ready for use.

Silica gel

- Before use, it must be serially rinsed with acetone, DCM and hexane, then completely dried at 50oC followed by activation for at least 20 hours at 160-180oC in a shallow glass tray, loosely covered with foil.

Anhydrous sodium sulphate

Before use, sodium sulphate must be cleaned by using solvent. The sodium sulphate is poured into a pre-cleaned column. Then, it is eluted with 2 volume of column of DCM followed by 2 volume of column of hexane. DCM and hexane are then discarded. A beaker is pre-cleaned with DCM and hexane. The clean sodium sulphate is poured into the beaker, covered loosely with solvent-rinsed aluminum foil. It is then dried overnight in fume hood. After that, the sodium sulphate is dried in an oven at approximately 180oC overnight. Sodium sulphate is stored in an oven at temperature ranging from 180±20oC until ready for use.

PAH stock mix standard solution - 1000ppm

Internal standards - Perylene-d12 - 1000ppm

Surrogate - Terphenyl-d14 - 1000ppm

3.6 Procedures:

3.6.1 Preparation of Internal Standard (Perylene-d12) and Surrogate (Terphenyl-d14)

The stock internal standard and surrogate solutions which are 1000ppm were diluted with hexane into 100ppm respectively.

Then, the 100ppm of internal standard and surrogate solutions was further diluted into the other concentration that was required in the following steps.

3.6.2 Calibration

Calibration standards were prepared from the 1000ppm of PAH stock mix standard solution.

1000ppm of the PAH stock mix standard solution was diluted with hexane to 5ppm, 10ppm, 20ppm, 50ppm and 100ppm in 1mL volumetric flask.

The five volumetric flasks with different concentration of mix standard were spiked with surrogate with the concentrations which are same as their respective mix standard.

Then, each of the volumetric flasks was spiked with 0.2mL of 100ppm of internal standard to make the concentration of internal standard to be 20ppm.

Hexane was added to the calibration mark and then the 5 solutions were transferred into 5 different of 1mL vials.

After that, the 5 calibration standards were analyzed by GC/MS in the scan mode. The condition of GC/MS will be indicated at Section3.6.3 (C).

The calibration graphs of 7 PAHs compounds and surrogate were printed out and the linearity of graphs were determined.

3.6.3 Determination of PAHs in waste oil samples

(A) Waste Oil Sample preparation

Sample with surrogate but without mix standard ( Mixture of 7 compounds PAHs)

0.5000g of waste oil sample was weighed and pipette into a 5mL volumetric flask.

Surrogate was spiked into the volumetric flask.

Then, hexane was added to the calibration mark.

The volumetric flask was shaked vigorously.

Sample with surrogate and mix standard ( Mixture of 7 compounds PAHs)

0.5000g of waste oil sample was weighed and pipette into a 5mL volumetric flask.

Surrogate followed by mix standard were spiked into the volumetric flask.

Then, hexane was added to the calibration mark.

The volumetric flask was shaked vigorously.

(B) Sample Cleanup

A 0.5cm layer of cotton wool was placed into a 300mm long, 10.5mm internal diameter of chromatographic column.

Approximately 3g of activated silica gel was then placed into the column.

The column was tapped gently to settle the silica gel.

A 0.5cm layer of anhydrous sodium sulphate was added on the top of silica gel.

The column was preconditioned with 20mL of hexane, the eluent was discarded.

When hexane has drained to the top of the column bed, 500µL of concentrated sample and 3mL of hexane were transferred into the column. 3mL of eluent was then discarded.

Before sodium sulphate exposed to the air, 12mL of hexane was added into the column. This eluent was collected in a calibrated and graduated centrifuge tube and this fraction was labeled as "F1".

Before sodium sulphate exposed to the air, 15mL of DCM/hexane 1:1 was added into the column. The eluent was then collected in another calibrated and graduated centrifuge tube and this fraction was labeled as "F2".

After that, both fractions were concentrated gently to about volumes of 0.6-0.8mL by evaporation under dry nitrogen in the "blow-down" apparatus.

The concentrated fractions were spiked with 2mL of 100ppm of internal standard to make the internal standard to be 20ppm.

Both fractions were topped up to the pre-injection volume of 1 ± 0.1mL and then transferred into 1mL of vials with properly labeled Teflon-lined screw cap septa. Vials should be immediately closed to avoid evaporation of solvent. The fractions have to be stored refrigerated, in the dark, for later analysis.

(C) Determination of PAHs by GC/MS

The seven compounds of PAHs were analyzed by GC/MS in the scan mode.

The selected characteristic ions used for analysis of PAHs are listed in the Appendix.

The operating conditions of GC/MS are the following:

Mass range : 35-500amu

Scan time : 1sec/scan

Initial temperature : 40oC, hold for 4 minutes

Temperature program : 40-270oC at 10oC/min

Final temperature : 300oC, hold about 15 minutes until all compounds elute

Injector temperature : 250-300oC

Transfer line temperature: 250-300oC

Injector : splitless (normally for low concentration of analyte)

Injection volume : 1µL

Carrier gas : helium at 30cm/sec

After that, the results were printed out and analyzed.

3.6.4 Quality Control (QC) Protocol

Mid level concentration of calibration standards which is 20ppm of PAH stock mix standard solution, spiked with 20ppm of internal standard was analyzed by GC/MS in the scan mode. This is used to check the performance of instrument. QC has to be analyzed after each 10 samples are run to ensure that samples are running within the instrument performance.

3.7 Results and Calculation:

Calculation for the volumes of 1000ppm of stock mix standard solution used to prepare 5ppm, 10ppm, 20ppm, 50ppm and 100ppm of mix standard solution:

By using formula,

M1V1=M2V2

For 5ppm, M1V1=M2V2

1000ppm x V1 = 5ppm x 1mL

V1 = 0.005mL

For 10ppm, 1000ppm x V2 = 10ppm x 1mL

V2 = 0.01mL

For 20ppm, 1000ppm x V3 = 20ppm x 1mL

V3 = 0.02mL

For 50ppm, 1000ppm x V4 = 50ppm x 1mL

V4 = 0.05mL

For 100ppm, 1000ppm x V5 = 100ppm x 1mL

V2 = 0.1mL

Calculation for the volume of surrogate used in the sample preparation

For example, 20ppm of surrogate was spiked into the sample,

To avoid overloading, no more than 50mg of oil can be placed on the column.

Therefore, we decided to place 50mg of oil on the column. This is because we can save the amount of surrogate's stock solution used.

Concentration of oil = mass of sample used / volume of solution

=

= 100000 mg/L

1 L : 100000mg where x be the volume of concentrated sample which is x L : 50mg spiked into the column

x =

= 5 x 10-4 L

= 500µL

So, to avoid overloading, the maximum volume of concentrated sample can be spiked into the column is 500µL.

M1V1=M2V2

M1 x 500µL = 20ppm x 1mL

M1 = 40ppm

M3V3=M4V4

100 x V3 = 40ppm x 5mL

V3 = 2mL

Where M1 = M4 = Concentration of sample

M2 = Concentration of surrogate obtained after sample cleanup

M3 = Concentration surrogate's stock solution (100ppm)

V1 = Volume of concentrated sample spiked into column

V2 = Pre-injection volume

V3 = Volume of surrogate's stock solution used

V4 = Volume of sample solution prepared

Therefore, 2mL of surrogate was used in the sample preparation to make a concentration of 20ppm.

Note: To calculate other concentration of surrogate and mix standard spiked into the sample, the same calculation steps are used.

3.7.1 Calibration

The calibration graphs are attached behind.

3.7.1.1 Calculation of Relative Response Factor (RRF) for each analyte in the calibration mixtures at each of the concentration levels

Relative Response Factors, RRF =

Where:

As = Area for the target analyte to be measured

AIS = Area for the internal standard (IS)

CIS = Concentration of the internal standard (20ppm)

Cs = Concentration of the target analyte

The values of As, AIS and Cs can be referred to the quantitation report attached behind.

Table 3.1: Relative Response Factor for 5ppm mix standard

Name of compound

As (x106)

AIS

(x106)

CIS

Cs

RRF

Terphenyl-d14

7.1

30.9

20

4.41

1.042057

Benz(a)anthracene

7.3

30.9

20

3.71

1.273563

Chrysene

6.5

30.9

20

3.58

1.175173

Benzo(b)fluorenthene

5.3

30.9

20

2.47

1.388834

Benzo(k)fluorenthene

10.4

30.9

20

4.5

1.495865

Benzo(a)pyrene

4.5

30.9

20

2.16

1.348436

Indeno(1,2,3-cd)pyrene

6.1

30.9

20

4.9

0.805759

Dibenzo(a.h)anthracene

4.9

30.9

20

3.5

0.906149

Table 3.2: Relative Response Factor for 10ppm mix standard

Name of compound

As

(x106)

AIS

(x106)

CIS

Cs

RRF

Terphenyl-d14

18.5

42.1

20

11.42

0.76958

Benz(a)anthracene

19.3

42.1

20

9.75

0.940374

Chrysene

19.9

42.1

20

10.85

0.871307

Benzo(b)fluorenthene

18.2

42.1

20

8.41

1.028071

Benzo(k)fluorenthene

27.2

42.1

20

11.73

1.101587

Benzo(a)pyrene

17.1

42.1

20

8.1

1.002903

Indeno(1,2,3-cd)pyrene

17.1

42.1

20

10.06

0.807507

Dibenzo(a.h)anthracene

14.5

42.1

20

7.56

0.911159

Table 3.3: Relative Response Factor for 20ppm mix standard

Name of compound

As

(x106)

AIS

(x106)

CIS

Cs

RRF

Terphenyl-d14

30.5

41.4

20

18.81

0.783323

Benz(a)anthracene

39.2

41.4

20

19.81

0.955941

Chrysene

39

41.4

20

21.25

0.886616

Benzo(b)fluorenthene

40.2

41.4

20

18.53

1.048046

Benzo(k)fluorenthene

49.1

41.4

20

21.14

1.122034

Benzo(a)pyrene

37.1

41.4

20

17.56

1.020655

Indeno(1,2,3-cd)pyrene

20.3

41.4

20

12.17

0.805815

Dibenzo(a.h)anthracene

30.3

41.4

20

16.01

0.914284

Table 3.4: Relative Response Factor for 50ppm mix standard

Name of compound

As

(x106)

AIS

(x106)

CIS

Cs

RRF

Terphenyl-d14

92.3

44

20

56.89

0.737468

Benz(a)anthracene

91.4

44

20

46.14

0.900422

Chrysene

91.4

44

20

49.79

0.834414

Benzo(b)fluorenthene

106.5

44

20

49.08

0.98633

Benzo(k)fluorenthene

142.1

44

20

61.13

1.056616

Benzo(a)pyrene

93.7

44

20

44.3

0.96142

Indeno(1,2,3-cd)pyrene

83.4

44

20

46.94

0.807607

Dibenzo(a.h)anthracene

105.7

44

20

52.54

0.914455

Table 3.5: Relative Response Factor for 100ppm mix standard

Name of compound

As

(x106)

AIS

(x106)

CIS

Cs

RRF

Terphenyl-d14

156.9

43.4

20

96.68

0.747871

Benz(a)anthracene

202.2

43.4

20

102.06

0.91299

Chrysene

183.4

43.4

20

99.84

0.846516

Benzo(b)fluorenthene

219.3

43.4

20

101.04

1.000197

Benzo(k)fluorenthene

218.7

43.4

20

94.06

1.07148

Benzo(a)pyrene

219.3

43.4

20

103.67

0.974823

Indeno(1,2,3-cd)pyrene

180.9

43.4

20

103.1

0.808575

Dibenzo(a.h)anthracene

198.3

43.4

20

99.85

0.915198

3.7.1.2 Percent relative standard deviation (%RSD) for the five RRFs for each analyte

% RSD

=

Standard deviation of the RRFs

Average of the RRFs

X 100%

Standard deviation =

Where xi = each RRF value used to calculate the mean RRF

= the mean of n values

n = total number of values = 5

All data is referred to the quantitation report attached behind.

Table 3.6: Percent relative standard deviation (%RSD) for the 5 RRFs for each analyte

Name of compound

Relative Response Factor

Average

Standard Deviation

%RSD

5 ppm

10 ppm

20 ppm

50 ppm

100 ppm

Terphenyl-d14

1.042057

0.769580

0.783323

0.737468

0.747871

0.816060

0.12760581

15.64

Benz(a)anthracene

1.273563

0.940374

0.955941

0.900422

0.912990

0.996658

0.156336095

15.69

Chrysene

1.175173

0.871307

0.886616

0.834414

0.846516

0.922805

0.142552036

15.45

Benzo(b)

fluorenthene

1.388834

1.028071

1.048046

0.986330

1.000197

1.090296

0.168603741

15.46

Benzo(k)

fluorenthene

1.495865

1.101587

1.122034

1.056616

1.07148

1.169516

0.184207691

15.75

Benzo(a)pyrene

1.348436

1.002903

1.020655

0.96142

0.974823

1.061647

0.161989879

15.26

Indeno(1,2,3-cd)

pyrene

0.805759

0.807507

0.805815

0.807607

0.808575

0.807053

0.001228422

0.15

Dibenzo(a.h)

anthracene

0.906149

0.911159

0.914284

0.914455

0.915198

0.912249

0.00374513

0.41

3.7.2 Verification of Calculation for PAHs

Concentration of the analyte of interest in the sample (µg/g) =

Where:

As = Area for the analyte in the sample

AIS = Area for the internal standard (IS)

WIS = Amount of internal standard added to the sample = 20µg

D = Dilution factor (dimensionless) = 5

Ws = Weight of sample (g)

Table 3.7: Calculation of Concentration for 7 PAH in sample waste W

(no surrogate spiked and without sample clean-up) 26 Jun 2010

Name of compound

Area for the target analyte

(x106)

Area of IS

(x106)

Conc. of IS (ppm)

Conc. of target analyte (ppm)

RRF

Conc.(µg/g)

DOE limit (ug/g)

Terphenyl-d14

0.04

12

20

0.03

2.222222222

0.3

-

Benz(a)anthracene

3.9

12

20

1.99

3.266331658

19.9

100

Chrysene

4

12

20

2.22

3.003003003

22.2

100

Benzo(b)fluorenthene

1.4

12

20

0.68

3.431372549

6.8

100

Benzo(k)fluorenthene

1.4

12

20

0.61

3.825136612

6.1

100

Benzo(a)pyrene

1.4

12

20

0.71

3.286384977

7.1

10

Indeno(1,2,3-cd)pyrene

1.3

12

20

2.69

0.805452292

26.9

100

Dibenzo(a.h)anthracene

0.1

12

20

0.21

0.793650794

2.1

10

Table 3.8: Calculation of Concentration for 7 PAHs in sample waste A

(no surrogate spiked and without sample clean-up) 25 Jun 2010

Name of compound

Area for the target analyte

(x106)

Area of IS

(x106)

Conc. of IS (ppm)

Conc. of target analyte (ppm)

RRF

Conc.(µg/g)

DOE limit (ug/g)

Terphenyl-d14

0.07

37.7

20

0.05

0.74270557

0.5

-

Benz(a)anthracene

0.2

37.7

20

0.11

0.964552689

1.1

100

Chrysene

0.1

37.7

20

0.1

0.530503979

1

100

Benzo(b)fluorenthene

0.08

37.7

20

0.04

1.061007958

0.4

100

Benzo(k)fluorenthene

0.09

37.7

20

0.04

1.193633952

0.4

100

Benzo(a)pyrene

0.1

37.7

20

0.05

1.061007958

0.5

10

Indeno(1,2,3-cd)pyrene

0.01

37.7

20

0.01

0.530503979

0.1

100

Dibenzo(a.h)anthracene

0.05

37.7

20

0.03

0.884173298

0.3

10

Table 3.9: Calculation of Concentration for 7 PAHs in waste W1

(20ppm surrogate spiked and with sample clean-up) 28 Jun 2010

Name of compound

Area for the target analyte

(x106)

Area of IS

(x106)

Conc. of IS (ppm)

Conc. of target analytev (ppm)

RRF

Conc.(µg/g)

DOE limit (ug/g)

Terphenyl-d14

74.3

44

20

45.81

0.737234824

458.1

-

Benz(a)anthracene

3.9

44

20

2

0.886363636

20

100

Chrysene

2.4

44

20

1.32

0.826446281

13.2

100

Benzo(b)fluorenthene

2.9

44

20

1.35

0.976430976

13.5

100

Benzo(k)fluorenthene

2.8

44

20

1.25

1.018181818

12.5

100

Benzo(a)pyrene

2.9

44

20

1.4

0.941558442

14

10

Indeno(1,2,3-cd)pyrene

1.2

44

20

0.7

0.779220779

7

100

Dibenzo(a.h)anthracene

0.1

44

20

0.07

0.649350649

0.7

10

Table 3.10: Calculation of Concentration for 7 PAHs in waste W2

(20ppm surrogate spiked and with sample clean-up) 30 Jun 2010

Name of compound

Area for the target analyte

(x106)

Area of IS

(x106)

Conc. of IS (ppm)

Conc. of target analyte (ppm)

RRF

Conc.(µg/g)

DOE limit (ug/g)

Terphenyl-d14

14.3

13.2

20

8.92

2.428998505

89.2

-

Benz(a)anthracene

0.8

13.2

20

0.43

2.81888654

4.3

100

Chrysene

0.4

13.2

20

0.26

2.331002331

2.6

100

Benzo(b)fluorenthene

0.3

13.2

20

0.16

2.840909091

1.6

100

Benzo(k)fluorenthene

0.5

13.2

20

0.24

3.156565657

2.4

100

Benzo(a)pyrene

0.4

13.2

20

0.23

2.635046113

2.3

10

Indeno(1,2,3-cd)pyrene

0.2

13.2

20

0.51

0.594177065

5.1

100

Dibenzo(a.h)anthracene

0

13.2

20

0

0

0

10

Table 3.11: Calculation of Concentration for 7 PAHs in waste A1

(20ppm surrogate spiked and with sample clean-up) 28 Jun 2010

Name of compound

Area for the target analyte

(x106)

Area of IS

(x106)

Conc. of IS (ppm)

Conc. of target analyte (ppm)

RRF

Conc.(µg/g)

DOE limit (ug/g)

Terphenyl-d14

46.3

39

20

28.53

0.832232378

285.3

-

Benz(a)anthracene

0.05

39

20

0.03

0.854700855

0.3

100

Chrysene

0.06

39

20

0.04

0.769230769

0.4

100

Benzo(b)fluorenthene

0.02

39

20

0.01

1.025641026

0.1

100

Benzo(k)fluorenthene

-0.002

39

20

0

0

0

100

Benzo(a)pyrene

0.1

39

20

0.06

0.854700855

0.6

10

Indeno(1,2,3-cd)pyrene

0

39

20

0

0

0

100

Dibenzo(a.h)anthracene

0

39

20

0

0

0

10

Table 3.12: Calculation of Concentration for 7 PAHs in waste A2

(20ppm surrogate spiked and with sample clean-up) 30 Jun 2010

Name of compound

Area for the target analyte

(x105)

Area of IS

(x105)

Conc. of IS (ppm)

Conc. of target analyte (ppm)

RRF

Conc.(µg/g)

DOE limit (ug/g)

Terphenyl-d14

423.2

360.1

20

26.38

0.891000078

263.8

-

Benz(a)anthracene

0.3

360.1

20

0.02

0.833101916

0.2

100

Chrysene

0.3

360.1

20

0.02

0.833101916

0.2

100

Benzo(b)fluorenthene

0

360.1

20

0

0

0

100

Benzo(k)fluorenthene

0.1

360.1

20

0.01

0.555401277

0.1

100

Benzo(a)pyrene

1

360.1

20

0.05

1.110802555

0.5

10

Indeno(1,2,3-cd)pyrene

0

360.1

20

0

0

0

100

Dibenzo(a.h)anthracene

0

360.1

20

0

0

0

10

Note: The concentrations of analytes which are not detectable are assumed as zero.

3.7.3 Determination of the Sample Recovery

Concentration of target analyte in the mix standard

= Concentration of target analyte - Concentration of target analyte in the blank

Recovery percentage of mix standard =

Concentration of target analyte in the mix standard

Concentration of mix standard spiked

x 100%

Table 3.13: Calculation of recovery percentage of mix standard for waste W1+ 10ppm mix standard (20ppm surrogate through column) 30 Jun 2010

Name of compound

Concentration of target analyte (ppm)

Concentration of target analyte in the blank (ppm)

Concentration of target analyte in the mix standard (ppm)

Recovery percentage of mix standard (%)

Terphenyl-d14

26.24

0.03

26.21

131.05

Benz(a)anthracene

13.34

1.99

11.35

113.50

Chrysene

11.53

2.22

9.31

93.10

Benzo(b)fluorenthene

10.36

0.68

9.68

96.80

Benzo(k)fluorenthene

11.78

0.61

11.17

111.70

Benzo(a)pyrene

10.11

0.71

9.4

94.00

Indeno(1,2,3-cd)pyrene

4.34

2.69

1.65

16.50

Dibenzo(a.h)anthracene

4.47

0.21

4.26

42.60

Table 3.14: Calculation of recovery percentage of mix standard for waste W2 + 10ppm mix standard (20ppm surrogate through column) 1 July 2010

Name of compound

Concentration of target analyte (ppm)

Concentration of target analyte in the blank (ppm)

Concentration of target analyte in the mix standard (ppm)

Recovery percentage of mix standard (%)

Terphenyl-d14

43.16

0.03

43.13

215.65

Benz(a)anthracene

12.84

1.99

10.85

108.50

Chrysene

12.24

2.22

10.02

100.20

Benzo(b)fluorenthene

10.81

0.68

10.13

101.30

Benzo(k)fluorenthene

11.66

0.61

11.05

110.50

Benzo(a)pyrene

10.42

0.71

9.71

97.10

Indeno(1,2,3-cd)pyrene

6.96

2.69

4.27

42.70

Dibenzo(a.h)anthracene

6.72

0.21

6.51

65.10

Note: The concentration of target analyte in the blank of waste W is taken from the concentration of target analyte in the table 3.1.

Table 3.15: Calculation of recovery percentage of mix standard for waste A1 + 20ppm mix standard (40ppm surrogate through column) 1 July 2010

Name of compound

Concentration of target analyte (ppm)

Concentration of target analyte in the blank (ppm)

Concentration of target analyte in the mix standard (ppm)

Recovery percentage of mix standard (%)

Terphenyl-d14

84.77

0.05

84.72

211.80

Benz(a)anthracene

18.77

0.11

18.66

93.30

Chrysene

20.26

0.1

20.16

100.80

Benzo(b)fluorenthene

18.39

0.04

18.35

91.75

Benzo(k)fluorenthene

20.33

0.04

20.29

101.45

Benzo(a)pyrene

17.64

0.05

17.59

87.95

Indeno(1,2,3-cd)pyrene

10.91

0.01

10.9

54.50

Dibenzo(a.h)anthracene

11.26

0.03

11.23

56.15

Table 3.16: Calculation of recovery percentage of mix standard for waste A2 + 20ppm mix standard (40ppm surrogate through column) 1 July 2010

Name of compound

Concentration of target analyte (ppm)

Concentration of target analyte in the blank (ppm)

Concentration of target analyte in the mix standard (ppm)

Recovery percentage of mix standard (%)

Terphenyl-d14

88.11

0.05

88.06

220.15

Benz(a)anthracene

21.5

0.11

21.39

106.95

Chrysene

21.04

0.1

20.94

104.70

Benzo(b)fluorenthene

19.54

0.04

19.5

97.50

Benzo(k)fluorenthene

21.23

0.04

21.19

105.95

Benzo(a)pyrene

18.88

0.05

18.83

94.15

Indeno(1,2,3-cd)pyrene

14.67

0.01

14.66

73.30

Dibenzo(a.h)anthracene

15.22

0.03

15.19

75.95

Note: The concentration of target analyte in the blank of waste A is taken from the concentration of target analyte in the table 3.2.

Table 3.17: Summary of Recovery percentage of mix standard

Name of compound

Recovery percentage of mix standard (%)

Waste W1

Waste W2

Waste A1

Waste A2

Terphenyl-d14

131.05

215.65

211.8

220.15

Benz(a)anthracene

113.50

108.50

93.30

106.95

Chrysene

93.10

100.20

100.80

104.70

Benzo(b)fluorenthene

96.80

101.30

91.75

97.50

Benzo(k)fluorenthene

111.70

110.50

101.45

105.95

Benzo(a)pyrene

94.00

97.10

87.95

94.15

Indeno(1,2,3-cd)pyrene

16.50

42.70

54.50

73.30

Dibenzo(a.h)anthracene

42.60

65.10

56.15

75.95

3.7.4 Quality Control

Table 3.18: Calculation of recovery percentage of mix standard for QC 20ppm

(no surrogate spiked) 29 Jun 2010

Name of compound

Concentration of target analyte (ppm)

Recovery percentage of mix standard (%)

Benz(a)anthracene

21.62

108.1

Chrysene

22.67

113.35

Benzo(b)fluorenthene

22.16

110.8

Benzo(k)fluorenthene

22.16

110.8

Benzo(a)pyrene

20.18

100.9

Indeno(1,2,3-cd)pyrene

15.3

76.5

Dibenzo(a.h)anthracene

17.34

86.7

3.8 Observations:

3.8.1 The eluent in the F2 which contains PAHs was in yellow colour whereas the eluent in F1 which contains saturated aliphatic hydrocarbon was colourless.

Fraction 1: colourless Fraction 2: yellow colour

3.8.2 When the fractions were exposed to the ultraviolet light, there was fluorescence occurred in F2 but not in F1. This is the preliminary identifying for the presence of PAHs in the waste oil sample. This can be shown by the following pictures:

Fraction 1: fluorescence did not occur Fraction 2: fluorescence occurred

3.8.3 The intensity of yellow colour of F2 in waste W is higher than in waste A.

3.9 Discussions:

3.9.1 Discussion on chromatograph

Refer to the chromatograph of 20ppm of PAHs mix standards attached behind, there are 9 peaks represent to the 7 PAHs, internal standard and surrogate. It shows the retention time of the 7 target PAHs as well as internal standard (perylene-d12) and surrogate (terphenyl-d14). The retention time of the 7 PAHs, internal standard and surrogate are:

Terphenyl-d14 : 24.46 min

Benz(a)anthracene : 26.82 min

Chrysene : 26.91 min

Benzo(b)fluorenthene : 29.25 min

Benzo(k)fluorenthene : 29.30 min

Benzo(a)pyrene : 29.90 min

Perylene-d12 : 30.02 min

Indeno(1,2,3-cd)pyrene : 32.48 min

Dibenzo(a,h)anthracene : 32.56 min

From the chromatograph, we can observe that the splitting of the 9 peaks is moderate but they are still acceptable. There are slightly overlapping of some peaks such as benzo(b)fluorenthene with benzo(k)fluorenthene and indeno(1,2,3-cd)pyrene with dibenzo(a,h)anthracene because their retention time is quite close. If the peaks are overlapping too much, response of the respective analyte will be very difficult to be calculated and errors will also occur. This is because response is calculated from the area occupied by a peak, so if 2 peaks are overlapping, it is very difficult to determine the actual area of each peak and the response obtained will just be an approximation. This will indirectly influence our results. To improve the splitting, temperature programming can be increased.

3.9.2 Linearity of Calibration

The calibration check of the five different concentration of PAHs mix standard can be used to establish the instrument's linear dynamic range. From the results above, we found that the relative response factors of each analyte for the points are constant because the percent RSD is less than 20 over the working range. Besides that, based on the calibration graphs obtained, the sample coefficient of determination (r2) of all the analytes except benzo(k)fluorenthene which is 0.976 are greater than 0.99, so they are considered as the best least-squares fit. Although r2 of benzo(k)fluorenthene is not greater than 0.99, its value (0.976) is only slightly different from 0.99, so it can still be accepted as a good least-squares fit. Due to these two reasons, the instrument can be considered to have linear response over the range of minimum to maximum concentration. Calibration should be checked at the beginning of every project.

3.9.3 Verification of Calculation for PAHs

From the table 5.7 to table 5.12, I can verify that the concentration of each analyte (unit of µg/g) in every sample which I calculated by using the formula given above matches with the results obtained from the GC/MS. As a results, I can guarantee that the concentration of analyte calculated by the GC/MS itself does not have error and it is reliable. Moreover, I found that the concentrations of each analyte in the unit of µg/g which are calculated from the formula are ten times of that concentration given by the GC/MS which is in the unit of µg/mL. To convert the unit of µg/mL to µg/g, the value obtained from the GC/MS has to be multiplied with the volume of sample solution prepared which is 5mL and divided by the mass of waste oil sample used which is 0.5g. Since 5mL divided by 0.5g will obtain 10mL/g, the concentration in the unit of µg/g will be 10 times of the concentration in the unit of µg/mL. Generally, the results obtained from the GC/MS must be converted into the unit of µg/g because the limits of concentration of PAHs present in the waste oil which are given by the Department of Environment (DOE) are all in the unit of µg/g. Hence, it will be more convenient for us to compare the results with the DOE limits.

Based on the table 5.7 and table 5.8, we can preliminarily know the concentrations of the seven PAHs compounds in waste W and waste A samples and we also found that they are within the DOE limits. This is because the waste oil samples were directly run with GC/MS and they were not gone through the column, so we assumed that there is no loss or gain of any amount of compounds when being analyzing in the GC/MS. The results can be further confirmed by doing sample recovery which will be discussed in the Section 5.9.4. On the other hand, the results from the table 5.9 to table 5.12 cannot be used to determine the concentration of PAHs in the two samples because the samples did go through the sample clean-up column, however, the recoveries of surrogate obtained are not good as the recoveries are already out of the range (80-120%). Thus, the results obtained are not reliable because we do not know whether the recoveries of sample analytes are good or not by referring the surrogate recovery. Based on the recovery of surrogate, we can predict that there might be loss or gain of some amount of PAHs during the sample clean-up process.

Furthermore, compared the concentration of each analyte in waste W to waste A in the table 5.7 and table 5.8 respectively, we found that the concentration of each PAH compound in waste W is higher than in waste A. This explains why the colour of eluent in waste W is more intense than in waste A. The higher the concentration of PAHs in the oil sample, the more intense the colour is.

3.9.4 Sample Recovery

Since the recoveries of surrogates from the table 5.9 to table 5.12 are not good, we cannot ensure that whether the recoveries of samples are good or not. Therefore, we used another way to determine the recoveries of samples. We spiked the known concentration of mix standard into the waste oil sample and then followed by the sample clean-up process. After that, the results obtained minus with the concentration of each analyte in the waste oil samples in order to get the recoveries of mix standard. The concentration of each analyte in the waste W and waste A can be obtained from the table 5.7 and table 5.8 respectively. From determining the recoveries of mix standard, we can also know the recoveries of oil samples.

According to the results above (table 5.17), we found that the recovery percentage of the first five analytes (Benz(a)anthracene, Chrysene, Benzo(b)fluorenthene, Benzo(k)fluorenthene and Benzo(a)pyrene) in the mix standard are considered good which is 87.95% - 113.50%, so we can conclude that the recovery of these analytes in the samples is also good, and we assumed that there is no or a little loss or gain of the amount of samples throughout the process. Nevertheless, the recoveries of indeno(1,2,3-cd)pyrene and dibenzo(a,h)anthracene in the mix standard are not good, there recovery percentages are less than 80% . This might be because these two analytes are not suitable for being extracted at the concentration of mix standard that we spiked. For waste W sample in which the concentration of mix standard spiked is 10ppm, the recovery of these two analytes is lower than waste A sample in which the concentration of mix standard spiked is 20ppm. This means that the recovery of these two compounds might be increased if we increase the concentration of mix standard spiked into the samples.

In addition, the recovery of surrogate for the samples is also not good but the recovery percentage is quite consistent. The bad recovery of surrogate might be caused by the glassware such as column that we used during the experiment. The column might not be cleaned properly with dichloromethane and hexane after previously used. Hence, there might be some contaminants or surrogate of the previous experiments left on the column. This will affect our results and cause us cannot obtain a good recovery of surrogate. Besides that, the concentration of surrogate itself (100ppm) before being spiked into the sample might be altered. This is because the 100ppm of surrogate was diluted from the 1000ppm of surrogate stock solution with hexane, so there might be some hexane evaporated when exposed to the air before being spiked. If hexane evaporates, the concentration of surrogate will be increased, so the recovery percentage of surrogate will be very high.

3.9.5 Quality Control

From the table 5.18, we observed that the recovery of the 20pppm of mix standard is good enough because it is within the range from 80% to120%. Therefore, we can ensure that the performance of instrument is good and there is no any leakage of gas in the gas chromatography. If the recovery is out of the range, problems have to be discovered and solved before samples are run.

3.9.6 Others

Both fraction 1 and 2 cannot be over concentrated by evaporation under dry nitrogen in the "blow-down" apparatus until being totally dry because this may lead to loss of amount of PAHs and then our results will be affected.

Hexane is used to extract saturated aliphatic hydrocarbons which is the eluent collected in the Fraction 1 (F1). This is because hexane is a non-polar solvent and saturated aliphatic hydrocarbons are also non-polar. Therefore, when the column is eluted with hexane, non-polar compounds such as aliphatic hydrocarbons will come out together with the hexane. On the other hand, 1:1 DCM/hexane is used to extract aromatic hydrocarbon compounds which are collected in F2. This is because 1:1 DCM/hexane has an intermediate polarity which is similar to the polarity of PAHs so they are eluted with 1:1 DCM/hexane.

In the part of reagents, I mentioned that prior to use, glass wool, silica gel and sodium sulphate have to be rinsed with solvent. This is because impurities can be removed and they can pre-adapt the environment after rinsing with the solvent. Besides that, during the sample clean-up, we should ensure that the silica column is packed. If not, the silica column will be cracked when solvent and samples pass through the column. Our results will be affected if column is cracked. In addition, both fractions 2 which contain PAHs were fluorescent when exposed to ultraviolet (UV) light. This is because when the molecules of PAHs absorb UV light, they will be excited, and then emitting characteristic wavelengths of light.

Furthermore, internal standard has to be added after sample clean-up and nitrogen blowing but not added when the sample preparation like surrogate and mix standard. This is because the addition of internal standard is used to calculate the concentration of other unknown analytes. If it is added into the sample during the sample preparation and then go through sample clean-up, amount of internal standard might be lost during the process. Hence, the concentration of analytes which is calculated depending on the concentration internal standard will be affected. To avoid this problem occur, internal standard is added after sample clean-up.

3.10 Conclusion

The recoveries of benz(a)anthracene, chrysene, benzo(b)fluorenthene, benzo(k)fluorenthene and benzo(a)pyrene in waste oil samples are evenly good which is from 87.95% - 113.50%. This percentage range is within the acceptance criterion (80%-120%). Therefore, we can ensure that these five analytes can be determined by using this method without any loss of amount. However, the recoveries of indeno(1,2,3-cd)pyrene and dibenzo(a,h)anthracene are poor. Thus, modification has to be applied to increase the recovery such as increasing the concentration of mix standard spiked into the samples.

Writing Services

Essay Writing
Service

Find out how the very best essay writing service can help you accomplish more and achieve higher marks today.

Assignment Writing Service

From complicated assignments to tricky tasks, our experts can tackle virtually any question thrown at them.

Dissertation Writing Service

A dissertation (also known as a thesis or research project) is probably the most important piece of work for any student! From full dissertations to individual chapters, we’re on hand to support you.

Coursework Writing Service

Our expert qualified writers can help you get your coursework right first time, every time.

Dissertation Proposal Service

The first step to completing a dissertation is to create a proposal that talks about what you wish to do. Our experts can design suitable methodologies - perfect to help you get started with a dissertation.

Report Writing
Service

Reports for any audience. Perfectly structured, professionally written, and tailored to suit your exact requirements.

Essay Skeleton Answer Service

If you’re just looking for some help to get started on an essay, our outline service provides you with a perfect essay plan.

Marking & Proofreading Service

Not sure if your work is hitting the mark? Struggling to get feedback from your lecturer? Our premium marking service was created just for you - get the feedback you deserve now.

Exam Revision
Service

Exams can be one of the most stressful experiences you’ll ever have! Revision is key, and we’re here to help. With custom created revision notes and exam answers, you’ll never feel underprepared again.