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The location of sediment 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 study area is shown in the Figure 3.1. Three sediment samples were taken at the same location.
Straits of Malacca
Figure 3.1: Pulau Ketam, Selangor is the sampling location.
The circle shows the location of study area.
The sediments sample was obtained from Pulau Ketam, Selangor. The sediment samples were wrapped by using aluminium foil and then the sediment samples kept in zip log bag. After that, the sediment samples were transport to laboratory and stored in freezer under temperature -18 oC until further analysis. The following are the sediment samples that used for this study:
Table 3.1: Types and parts of fish that will analyses
Longitude / Latitute
Dry weight of the sample (g)
220.127.116.11 Apparatus and glassware cleaning procedure
Before we start the experiment in laboratory, we must clean the apparatus and glassware that will use in experiment. The purpose is to make sure that the glassware is not contaminanted. This may cause interfere to the result while using GC-MS. All apparatus and glassware were washed with phosphate free detergent until foaming and followed by washing thoroughly with abundant of tap water. Next, all apparatus and glassware were then rinsed with distilled water for six times followed by six times of methanol solvent, acetone solvent and n-hexane solvent accordingly. All apparatus and glassware were then wrapped with aluminum foil after rinsing with solvents previously and then dried in the oven for minimum two hours at temperature of 60ËšC. For the measurement apparatus such as measuring cylinder cannot dried in the oven. This is because glass will expand when in high temperature. Therefore, this may influence the measurement readings. The dried apparatus and glassware were then let to cool at room temperature and kept properly in the wardrobe before use. Figure 2 show the analytical procedure to analyze the sediment samples.
Figure 3.2 : Analytical Procedure for analysis of Sediment Samples
(edited from: Zakaria et al )
Freeze dry (Dry weight determination)
20 g of sediment sample was dried by using freeze-dried. This is to make sure that the excess water content in sample was removed. After the sediment sample going freeze-dried, the dry weight of fish samples were determined by using the following formula;
-------------------- Equation (i)
-------------------- Equation (ii)
The dried sediment sample was scoop into the cellulose thimble. Then, 250 mL of dichloromethane (DCM) was poured into the round bottom flask and 2 or 3 glass beads were added into that round bottom flak. The glass beads were added to prevent the building of pressure in the flask that may cause explosion. After that, the cellulose thimble that contains the sample was placed into the Soxhlet extractor. The Soxhlet was attached to the condenser at the bottom and attached to the round bottom flask (contain DCM and glass beads) at the bottom. The condenser will circulate with the ice-bath water to make a condensation process. Every attachments part at the Soxhlet extractor must be sealed with Teflon tape to prevent the leakage and avoid contamination from surrounding. The round bottom flask and Soxhlet extractor was wrapped with aluminium foil to avoid the solvent exposure to sunlight because it may cause the reaction occur in the solvent. The Soxhlet extraction process was carried out at 30oC for eight hours.
To prepare activated copper, the inactivated copper were soaked into 3N of hydrochloric acid (HCl) solution and stirred for 3-5 minutes. The hydrochloric acid solution was discarded and the copper was rinsed with distilled water several times. Next, the copper was rinsed by methanol, followed by acetone, and n-hexane solvent respectively. This step is to remove the contamination at the copper. After that, the copper was kept in dichloromethane (DCM) solvent and the flask covered with aluminium foil until it is used. Then, half spatula of activated copper added to the extract in the round bottom flask and the round bottom flask was wrapped with aluminium foil and left overnight.
The temperature of water bath was set at 30oC. The round bottom flask that contains extraction was attached to the rotary evaporator. Then, the round bottom flask was rotating around 60-70rpm with vacuum of 600-700 mm Hg. The purpose of this process is to reduce the extract volume to approximately 5 mL. After the rotary evaporation finish, the extraction in the round bottom flask was transfer into the 25mL pear shape flask by using pasture pipette. The bottom flask was rinsed with DCM for several times to make sure that all of the extraction was transferred.
First step silica gel column chromatography
The 9 mm internal diameter glass column was rinsed by using hexane for three times. Then, hexane was poured into the glass column and followed by 5% of deactivated silica gel. The silica gel was transferred into the column by using pasture pipette and then the silica gel was packed by using electrical vibrator until 9 cm height. The electrical vibrator is used to pack the silica gel in the column and to make sure that no bubble in the column. Anhydrous sodium sulphate was poured into the glass column until 1 cm height to remove excess water in the extract while the extract inject into the column later. The hexane in the column reduced by open the pipette stopper. Then, 20 mL solvent of Hexane:DCM with ratio 3:1 was rinsed passed through the glass column. After that, the extract was pipette to the column. The eluted sample was collected in the pear shape flask. The flask must wrapped with aluminium foil to prevent reaction occur in the elute sample until the next step.
Second step silica gel column chromatography
The second step column was prepared same as the first step column preparation. But in second step silica 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 load from first step column chromatography was transfer to second step column chromatography by using pasture pipette. To ensure that all elute sample were transferred into the column, the pear shape flask was rinsed with the solvent and use the sonicator to collect the hydrocarbon in that flask. The pear shape flask was rinsed in the eternal sequence of 0.3 mL, 0.3 mL, 0.4 mL, 0.5 mL, and the remaining solvent loaded into the glass column directly. Then, 4 mL of the hexane (HPLC grade) solvent was added into the column to obtain first fraction (n-alkanes). The n-alkane fraction collected in a small pear shape flask. Then, the pear shape flask that contain n-alkane fraction was removed and replaced with another pear shape flask to collect LABs fraction in next elution. After that, 4 mL of hexane was load into the column to obtain second fraction (LABs), it will collect in pear shape flask. To obtain third fraction (PAHs), 16 mL of Hexane:DCM with ratio of 3:1 have to load into the column. All pear shape flask that contains n-alkane, LABs and PAHs fraction must wrapped with aluminium foil to prevent reaction occur in the elute sample until the next step.
The LABs fraction was concentrated by using rotary evaporator until approximately 1 mL and transferred into vial by using pasture pipette. The pear shape flask was rinsed three times with hexane solvent with sonication and transferred the rinsed solvents into the vial. The concentrated of LABs fraction was evaporated to dryness under a gender steam of nitrogen gas. The vial keeps tight to prevent loss through evaporation by using film tape. The vial was stored in the refrigerator at 4oC before analyzed by using Gas Chromatography-Mass Spectrometry (GC-MS) instrument.
Gas Chromatography-Mass Spectrophotometer (GC-MS)
50 ÂµL of IIS added into the vials prior before the injection for the GC-MS analysis. (GCMS-QP5050 A, GC-17A, SHIMADSU Model)