EXPERIMENTAL

Prior to fractionation, the sample was analysed by whole oil gas chromatography. Whole oil gas chromatography was carried out using a Varian 3400 GC equipped with a flame ionization detector (FID) and on-column injector. The column was a 30m x 0.25mm i.d x 0.25um thickness DB1. The temperature program was 33°C for 6 minutes, ramped to 63°C at a rate of 3°C/min, then to 320°C at a rate of 6°C/min and held for 10 minutes. Gasoline range hydrocarbons (C5-C8) in the original oil sample were analyzed on a HP 5890 Gas Chromatograph with a flame ionization detector (FID) connected to an OI Analytical 4460 Sample Concentrator. The column was a 60 m DB-1 0.32 mm i.d. with a 1µm film thickness. The temperature program was 30 to 40°C at a rate of 1°C/min. The initial and final temperatures were held for 10 and 25 minutes respectively. Total analysis time was 45 minutes.

As only small amount of oil was available, it was not distilled prior to fractionation. Fractionation was carried out using liquid chromatography. A mixture of 28-200 mesh Silica Gel (MCB) and 80-200 mesh alumina (ALCOA) (1:3 by weight respectively) was used as a support for the column. The support is activated by heating at 120°-150°C for 12 hours. A glass wool plug is placed at a bottom of the column and covered with a 1 cm thick layer of sand. The support, weighed as 1 g of support/10 mg of sample, is slowly settled in pentane and any air trapped is released by gentle tapping on the column. The sample, is then added to the column. Saturates are recovered by eluting with pentane (3.5 ml/g support), aromatics with a 50:50 mixture of pentane and dichloromethane (4 ml/g support), resins with methanol (4 ml/g support) and any remaining asphaltenes with chloroform. The solvents are rotary-evaporated, separate fractions transferred to tared 1 dram vials, dried in a slow stream of nitrogen and weighed to constant weight.

Saturated and aromatic hydrocarbon fractions were analyzed using gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Gas Chromatography used a Varian 3800 FID gas chromatograph with a 30 m DB-1 column with a temperature program from 60°C to 300°C at a rate of 6°C/min and then held for 30 minutes at 300°C. GC-MS analysis were performed on a HP 6890 Series GC coupled to a HP 5973 Series Mass Selective Detector operated in selective ion monitoring (SIM) mode. The GC was fitted with a 30 m DB-5ms column. During the saturate fraction analysis the temperature was initially held at 100°C for 2 min and then programmed at 40°C/min to 180°C and at 4°C/min to 320°C, then held for 7 min. The aromatic fraction analysis was initiated at 100°C, programmed at 3°C/min to 300°C, then held for 10 min.The column was an Agilent DB-5MS, 30 m x 0.32 mm i.d. and a film thickness of 0.25 microns. The addidtion of 100 ul of internal standard (D4 cholestane, 0.1 mg/ml) during the saturate fraction analysis allows absolute quantification of detected biomarker compounds.


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