The location of deposit samples station is at Pulau Ketam, Selangor ( N03o 00 ‘ 20 ” N 03o 05 ‘ E101o 09 ‘ E101o 18 ‘ ) . The samples are taken on 5th August 2010. The survey country is shown in the Figure 3.1. Three deposit samples were taken at the same location.

Passs of Malacca

Figure 3.1: Pulau Ketam, Selangor is the trying location.

The circle shows the location of survey country.


The deposits sample was obtained from Pulau Ketam, Selangor. The deposit samples were wrapped by utilizing aluminium foil and so the deposit samples kept in nothing log bag. After that, the deposit samples were conveyance to research lab and stored in deep-freeze under temperature -18 oC until farther analysis. The following are the deposit samples that used for this survey:

Table 3.1: Types and parts of fish that will analyze

Sample Number

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Longitude / Latitute

Dry weight of the sample ( g )


N03o00 ‘ 20 ”

E101o15 ‘ 55 ”



N03o00 ‘ 20 ”

E101o15 ‘ 55 ”



N03o00 ‘ 20 ”

E101o15 ‘ 55 ”


Experimental Apparatus and glasswork cleansing process

Before we start the experiment in research lab, we must clean the setup and glasswork that will utilize in experiment. The intent is to do certain that the glasswork is non contaminanted. This may do interfere to the consequence while utilizing GC-MS. All setup and glasswork were washed with phosphate free detergent until frothing and followed by rinsing exhaustively with abundant of tap H2O. Next, all setup and glasswork were so rinsed with distilled H2O for six times followed by six times of methanol dissolver, propanone dissolver and n-hexane dissolver consequently. All setup and glasswork were so wrapped with aluminium foil after rinsing with dissolvers antecedently and so dried in the oven for minimal two hours at temperature of 60EsC. For the measuring setup such as mensurating cylinder can non dried in the oven. This is because glass will spread out when in high temperature. Therefore, this may act upon the measuring readings. The dried setup and glasswork were so allow to chill at room temperature and kept decently in the closet before usage. Figure 2 show the analytical process to analyse the deposit samples.

Figure 3.2: Analytic Procedure for analysis of Sediment Samples

( edited from: Zakaria et Al )

Freeze prohibitionist ( Dry weight finding )

20 g of sediment sample was dried by utilizing lyophilized. This is to do certain that the extra H2O content in sample was removed. After the sediment sample traveling lyophilized, the dry weight of fish samples were determined by utilizing the undermentioned expression ;

— — — — — — — — — — Equation ( I )

— — — — — — — — — — Equation ( two )

Soxhlet Extraction

The dried deposit sample was scoop into the cellulose thimble. Then, 250 milliliter of methylene chloride ( DCM ) was poured into the unit of ammunition underside flask and 2 or 3 glass beads were added into that unit of ammunition bottom flack catcher. The glass beads were added to forestall the edifice of force per unit area in the flask that may do detonation. After that, the cellulose thimble that contains the sample was placed into the Soxhlet extractor. The Soxhlet was attached to the capacitor at the underside and attached to the unit of ammunition underside flask ( contain DCM and glass beads ) at the underside. The capacitor will go around with the ice-bath H2O to do a condensation procedure. Every fond regards portion at the Soxhlet extractor must be sealed with Teflon tape to forestall the escape and avoid taint from environing. The unit of ammunition underside flask and Soxhlet extractor was wrapped with aluminum foil to avoid the solvent exposure to sunlight because it may do the reaction occur in the dissolver. The Soxhlet extraction procedure was carried out at 30oC for eight hours.

Copper Treatment

To fix activated Cu, the inactivated Cu were soaked into 3N of hydrochloric acid ( HCl ) solution and stirred for 3-5 proceedingss. The hydrochloric acerb solution was discarded and the Cu was rinsed with distilled H2O several times. Following, the Cu was rinsed by methyl alcohol, followed by propanone, and n-hexane dissolver severally. This measure is to take the taint at the Cu. After that, the Cu was kept in methylene chloride ( DCM ) dissolver and the flask covered with aluminium foil until it is used. Then, half spatula of activated Cu added to the infusion in the unit of ammunition underside flask and the unit of ammunition underside flask was wrapped with aluminum foil and left overnight.

Rotary Vaporization

The temperature of H2O bath was set at 30oC. The unit of ammunition underside flask that contains extraction was attached to the rotary evaporator. Then, the unit of ammunition underside flask was revolving around 60-70rpm with vacuity of 600-700 millimeter Hg. The intent of this procedure is to cut down the infusion volume to about 5 milliliters. After the rotary vaporization coating, the extraction in the unit of ammunition underside flask was transportation into the 25mL pear form flask by utilizing grazing land pipette. The bottom flask was rinsed with DCM for several times to do certain that all of the extraction was transferred.

First measure silica gel column chromatography

The 9 millimeter internal diameter glass column was rinsed by utilizing hexane for three times. Then, hexane was poured into the glass column and followed by 5 % of deactivated silicon oxide gel. The silicon oxide gel was transferred into the column by utilizing grazing land pipette and so the silicon oxide gel was packed by utilizing electrical vibrator until 9 centimeters tallness. The electrical vibrator is used to pack the silicon oxide gel in the column and to do certain that no bubble in the column. Anhydrous Na sulfate was poured into the glass column until 1 centimeters height to take extra H2O in the infusion while the extract inject into the column subsequently. The hexane in the column reduced by unfastened the pipette stopper. Then, 20 mL dissolver of Hexane: DCM with ratio 3:1 was rinsed passed through the glass column. After that, the infusion was pipette to the column. The eluted sample was collected in the pear form flask. The flask must wrapped with aluminium foil to forestall reaction occur in the elute sample until the following measure.

Second measure silicon oxide gel column chromatography

The 2nd measure column was prepared same as the first measure column readying. But in 2nd measure silicon oxide gel column chromatography, the 4.5 internal diameter glass column and 100 % activated silica gel are used. After the column prepared, the eluted sample that burden from first measure column chromatography was transportation to 2nd measure column chromatography by utilizing grazing land pipette. To guarantee that all elute sample were transferred into the column, the pear form flask was rinsed with the dissolver and utilize the sonicator to roll up the hydrocarbon in that flask. The pear form flask was rinsed in the ageless sequence of 0.3 milliliters, 0.3 milliliter, 0.4 milliliter, 0.5 milliliter, and the staying dissolver loaded into the glass column straight. Then, 4 milliliter of the hexane ( HPLC class ) dissolver was added into the column to obtain first fraction ( n-alkanes ) . The n-alkane fraction collected in a little pear form flask. Then, the pear form flask that contain n-alkane fraction was removed and replaced with another pear form flask to roll up LABs fraction in following elution. After that, 4 milliliter of hexane was load into the column to obtain 2nd fraction ( LABs ) , it will roll up in pear form flask. To obtain 3rd fraction ( PAHs ) , 16 milliliter of Hexane: DCM with ratio of 3:1 have to lade into the column. All pear form flask that contains n-alkane, LABs and PAHs fraction must wrapped with aluminium foil to forestall reaction occur in the elute sample until the following measure.

Nitrogen blowdown

The LABs fraction was concentrated by utilizing rotary evaporator until about 1 milliliters and transferred into vial by utilizing grazing land pipette. The pear form flask was rinsed three times with hexane dissolver with sonication and transferred the rinsed dissolvers into the phial. The concentrated of LABs fraction was evaporated to dryness under a gender steam of N gas. The vial supports tight to forestall loss through vaporization by utilizing movie tape. The phial was stored in the icebox at 4oC before analyzed by utilizing Gas Chromatography-Mass Spectrometry ( GC-MS ) instrument.

Gas Chromatography-Mass Spectrophotometer ( GC-MS )

50 AµL of IIS added into the phials prior before the injection for the GC-MS analysis. ( GCMS-QP5050 A, GC-17A, SHIMADSU Model )


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