**5.4.5 C29/C30 hopanes ratios**

C29/C30 hopanes ratios are generally high (>1) in oils generated from organic rich carbonates and evaporates (Connan et al., 1986).

#### **5.4.6 Steranes/17α (H)-hopanes ratio**

The regular steranes /17α(H)-hopanes ratio reflects input of eukaryotic (mainly algae and higher plants) versus prokaryotic (bacteria) organisms to the source rock. The sterane/hopane ratio is relatively high in marine organic matter with values generally approaching unity or even higher. In contrast, low steranes and sterane/hopane ratios are more indicative of terrigenous and/or microbially reworked organic matter (Suzuki et al.,1996).

#### **5.4.7 Bisnorhopanes**

It is believed that sediments containing large amounts of bisnorhopane were deposited under anoxic conditions (Mello et al., 1988). Bisnorhopanes are types of pentacyclic triterpanes present in significant concentrations in oil. Bisnorhopanes are observed in Guatemalan evaporites (Connan et al., 1986) and frequency reported in other biogenic siliceous rocks of the circum-Pacific region (Katz and Elrood, 1983).

#### **5.5 Metalloporphyrins**

Porphyrins are the tetrapyrole compounds; the porphyrin nucleus consists of four pyrrole rings joined by four methine bridges giving a cyclic tetrapyrrole structure. The majority of these compounds are thought to originate from various chloropigments produced by phototrophic organisms of the geological past (Yui et al., 2007). Metalloporphyrins has become a valuable tool in the determination of the origin and maturity of the organic matter (Doukkali et al., 2002; Chikaraishi et al., 2005 and Ohkouchi et al., 2006). The porphyrin structure consists of a porphyrin nucleus with various groups of side chains occupying some or all of its peripheral positions.

Metalloporphyrins were extracted from asphaltene and maltene fractions using adsorption column chromatography (Faramawy et al., 2010). Porphyrins occur as etioporphyrin (Etio), Benzo-etio, deoxophylloerythroetioporphyrin (DPEP), Benzo-DPEP and tetrahydrobenzo-DPEP (THBD). The distribution of different types of metalloporphyrins is useful for interpreting transformation of kerogen into bitumen, depositional environments and maturation levels of deposited organic matters.

The ratio of Ts (trisnorneohopane) to Tm (trisnorhopane) more than (0.5) was found to increase as the portion of shale in calcareous facies increases (Hunt, 1996). Van Grass (1990) stated that Ts/Tm ratios begin to decrease quite late during maturation but Waples and Machihara (1991) reported that Ts/Tm ratio does not appear to be appropriate for

C29/C30 hopanes ratios are generally high (>1) in oils generated from organic rich

The regular steranes /17α(H)-hopanes ratio reflects input of eukaryotic (mainly algae and higher plants) versus prokaryotic (bacteria) organisms to the source rock. The sterane/hopane ratio is relatively high in marine organic matter with values generally approaching unity or even higher. In contrast, low steranes and sterane/hopane ratios are more indicative of terrigenous and/or microbially reworked organic matter (Suzuki et

It is believed that sediments containing large amounts of bisnorhopane were deposited under anoxic conditions (Mello et al., 1988). Bisnorhopanes are types of pentacyclic triterpanes present in significant concentrations in oil. Bisnorhopanes are observed in Guatemalan evaporites (Connan et al., 1986) and frequency reported in other biogenic

Porphyrins are the tetrapyrole compounds; the porphyrin nucleus consists of four pyrrole rings joined by four methine bridges giving a cyclic tetrapyrrole structure. The majority of these compounds are thought to originate from various chloropigments produced by phototrophic organisms of the geological past (Yui et al., 2007). Metalloporphyrins has become a valuable tool in the determination of the origin and maturity of the organic matter (Doukkali et al., 2002; Chikaraishi et al., 2005 and Ohkouchi et al., 2006). The porphyrin structure consists of a porphyrin nucleus with various groups of side chains occupying

Metalloporphyrins were extracted from asphaltene and maltene fractions using adsorption column chromatography (Faramawy et al., 2010). Porphyrins occur as etioporphyrin (Etio), Benzo-etio, deoxophylloerythroetioporphyrin (DPEP), Benzo-DPEP and tetrahydrobenzo-DPEP (THBD). The distribution of different types of metalloporphyrins is useful for interpreting transformation of kerogen into bitumen, depositional environments and

siliceous rocks of the circum-Pacific region (Katz and Elrood, 1983).

**5.4.4 Ts/Tm** 

al.,1996).

**5.4.7 Bisnorhopanes** 

**5.5 Metalloporphyrins** 

some or all of its peripheral positions.

maturation levels of deposited organic matters.

quantitative estimation of maturity.

carbonates and evaporates (Connan et al., 1986).

**5.4.6 Steranes/17α (H)-hopanes ratio** 

**5.4.5 C29/C30 hopanes ratios** 

Fig. 6. Structures of different types of metalloporphyrins.

#### **6. Developments in GC-MS instrumentation**

The low biomarker concentrations in oils (often in the range of several parts per million) in the presence of a highly complex petroleum hydrocarbon matrix especially weathered oils, the variety of chemical classes present in oils and the possible co-elutions in conventional chromatographic separations make the identification of biomarkers a more difficult task.

The development of more reliable, highly selective, fast and sensitive separation and identification tools for biomarker analysis purposes can be considered as one of the most important research points in this field for a meaningful biomarker analysis.

Biomarkers 177

Data analysis is an important part of chemical fingerprinting and a broad collection of

After separation and identification of biomarkers, principal component analysis PCA, a mathematical procedure, can be used for analyses of chromatograms using a fast and objective procedure with more comprehensive data usage compared to other fingerprinting methods. The discriminative power of PCA can be enhanced by deselecting the most

For example, preprocessing of GC-MS chromatograms followed by principal component analysis (PCA) of oil spill samples collected from the coastal environment in the weeks after the Baltic Carrier oil spill and from the tank of the Baltic Carrier (source oil) was carried out (Christensen et al., 2005). The preprocessing consists of baseline removal by derivatization, normalization, and alignment using correlation optimized warping. The method was applied to chromatograms of *m*/*z* 217 (tricyclic and tetracyclic steranes) of oil spill samples and source oils. The four principal components were interpreted as follows: boiling point range (PC1), clay content (PC2), carbon number distribution of sterols in the source rock (PC3), and thermal maturity of the oil (PC4). The method allows for analyses of chromatograms using a fast and objective procedure and with more comprehensive data

The Western Desert covers about 700,000 square kilometers (equivalent in size to Texas) and accounts for about two-thirds of Egypt's land area. This immense desert to the west of the Nile spans the area from the Mediterranean Sea south to the Sudanese border. The chemical fingerprinting of oils in this area is a great interesting research area for many proposes as for example identifying the sources of petroleum oil or complex environmental pollutants. The original sources of complex mixtures can often be identified by the relative abundance of some major individual compounds (e.g n-alkanes) forming a chemical pattern by ratios of specific constituents or by identifying source-specific compounds or markers (e.g triterpanes) in the environmental sample being investigated (Peters et al., 2005). These parameters depend mostly on the preburial environments of the living organisms, the depositional environments of the organic matter and the diagenetic processes in the source

In their work, Roushdy et al. (2011) utilize biomarkers characteristics together with bulk geochemical parameters to identify and characterize the crude oils and to assess the respective depositional environments and maturation. Variation of crude oil-gravities in the Western Desert reflects different stages of oil migration and accumulation as well as different oil source rocks in the same and different ages (Zein El Din et al., 1990).The authors attempt to assess the correlation between the crude oil samples and the potential source rocks to confirm the indigenous sources for the petroleum generation of some oilfields of the North Western Desert. This target was made throughout the study in detail of the analytical results for three crude oil samples collected from three oilfields in the North Western Desert oilfields (Meleiha, Misaada and Qarun) as well as three extract samples (Baharia, Kharita and Khtataba) from formations ranging in age from Upper Cretaceous to Middle Jurassic.

**7. Data analysis by computerized techniques** 

usage compared to other fingerprinting methods.

**8. Previous studies in Egypt 8.1 Egyptian Western Desert** 

rocks.

statistical techniques has been used for evaluation of data.

uncertain variables or scaling them according to their uncertainty.

The use of comprehensive two-dimensional gas chromatography (GC × GC) coupled to time-of-flight mass spectrometry (TOFMS) was found to be a powerful tool for overcoming some problems and limitations since it (i) separates substances using two interconnected capillary columns containing different stationary phases and (ii) uses the fast data acquisition of time-of-flight analyzer as a robust registry for GC × GC (Aguiar et al., 2001).

In their work, Aguiar et al. (2001) used this technique to overcome the co-elution between tri- and pentacyclic terpanes separated by extracted ion chromatograms (EIC) for ions of mass-to-charge ratio (m/z) 191. The biomarker analysis by GC × GC–TOFMS was much better than in previous works using one-dimensional GC. Co-elutions between tri- and pentacyclic terpanes were clearly resolved in the second column. Noteworthy separation between the C30 hopane and C30 dimethylated homohopane was achieved and overlap of hopanes with steranes in the m/z 217 was eliminated. Besides hopanes, dimethylated triand tetracyclic terpanes were identified. These findings indicate the superiority of GC × GC–TOFMS as a technique for separation and identification of biomarkers in oils due to its high sensitivity, specificity and capability to elucidate compounds structure with high spectral resolution.

Comprehensive two-dimensional gas chromatography (GC×GC) has also been used to separate and identify alkylated aromatics (naphthalenes, biphenyls, fluorenes, phenanthrenes and chrysenes), sulfur-containing aromatics (dibenzothiophenes, benzonaphthothiophenes), steranes, triterpanes, and triaromatic steranes. These biomarkers were separated into easily recognizable bands in the GC×GC chromatogram. Methods used to identify the bands included peak matching with chemical standards and comparison with GC/MS extracted ion chromatograms (Frysinger and Gaines, 2001). By designing mass spectrometers that can determine m/z values accurately to four decimal places, it is possible to distinguish different formulas having the same nominal mass. Since a given nominal mass may correspond to several molecular formulas, lists of such possibilities are especially useful when evaluating the spectrum of an unknown compound.

GC/MS/MS is an operation based on the covariant scan of electrostatic magnetic fields on the trisector double focusing mass spectrometer providing more accurate data. The quadraupole is a common mass separator gives a sufficient sensitivity and selectivity however, high resolution mass spectrometry (HRMS) is also used due to its ability to provide quantitative data for compounds present in complex mixtures for biomarkers analysis. Triple quadrupole GC/MS offers a viable alternation for the rapid, routine analysis providing excellent precision, sensitivity, selectivity, and dynamic range (Thermoapplication note 10261).

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) benefits from ultra-high mass resolving power (greater than one million), high mass accuracy (less than 1 ppm) and rapid analysis which make it an attractive alternative for the analysis of different and wide range of petroleum products (Klein et al., 2003).

It should be noted, however, that there is no single fingerprinting technique that can fully and readily meet the objectives of biomarkers investigation and quantitatively allocate hydrocarbons to their respective sources, particularly for complex hydrocarbon mixtures or extensively weathered and degraded oil residues. Combined and integrated multiple tools are often necessary under such situations.

The use of comprehensive two-dimensional gas chromatography (GC × GC) coupled to time-of-flight mass spectrometry (TOFMS) was found to be a powerful tool for overcoming some problems and limitations since it (i) separates substances using two interconnected capillary columns containing different stationary phases and (ii) uses the fast data acquisition of time-of-flight analyzer as a robust registry for GC × GC (Aguiar et al., 2001). In their work, Aguiar et al. (2001) used this technique to overcome the co-elution between tri- and pentacyclic terpanes separated by extracted ion chromatograms (EIC) for ions of mass-to-charge ratio (m/z) 191. The biomarker analysis by GC × GC–TOFMS was much better than in previous works using one-dimensional GC. Co-elutions between tri- and pentacyclic terpanes were clearly resolved in the second column. Noteworthy separation between the C30 hopane and C30 dimethylated homohopane was achieved and overlap of hopanes with steranes in the m/z 217 was eliminated. Besides hopanes, dimethylated triand tetracyclic terpanes were identified. These findings indicate the superiority of GC × GC–TOFMS as a technique for separation and identification of biomarkers in oils due to its high sensitivity, specificity and capability to elucidate compounds structure with high

Comprehensive two-dimensional gas chromatography (GC×GC) has also been used to separate and identify alkylated aromatics (naphthalenes, biphenyls, fluorenes, phenanthrenes and chrysenes), sulfur-containing aromatics (dibenzothiophenes, benzonaphthothiophenes), steranes, triterpanes, and triaromatic steranes. These biomarkers were separated into easily recognizable bands in the GC×GC chromatogram. Methods used to identify the bands included peak matching with chemical standards and comparison with GC/MS extracted ion chromatograms (Frysinger and Gaines, 2001). By designing mass spectrometers that can determine m/z values accurately to four decimal places, it is possible to distinguish different formulas having the same nominal mass. Since a given nominal mass may correspond to several molecular formulas, lists of such possibilities are especially

GC/MS/MS is an operation based on the covariant scan of electrostatic magnetic fields on the trisector double focusing mass spectrometer providing more accurate data. The quadraupole is a common mass separator gives a sufficient sensitivity and selectivity however, high resolution mass spectrometry (HRMS) is also used due to its ability to provide quantitative data for compounds present in complex mixtures for biomarkers analysis. Triple quadrupole GC/MS offers a viable alternation for the rapid, routine analysis providing excellent precision, sensitivity, selectivity, and dynamic range (Thermo-

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) benefits from ultra-high mass resolving power (greater than one million), high mass accuracy (less than 1 ppm) and rapid analysis which make it an attractive alternative for the analysis of different

It should be noted, however, that there is no single fingerprinting technique that can fully and readily meet the objectives of biomarkers investigation and quantitatively allocate hydrocarbons to their respective sources, particularly for complex hydrocarbon mixtures or extensively weathered and degraded oil residues. Combined and integrated multiple tools

useful when evaluating the spectrum of an unknown compound.

and wide range of petroleum products (Klein et al., 2003).

are often necessary under such situations.

spectral resolution.

application note 10261).

## **7. Data analysis by computerized techniques**

Data analysis is an important part of chemical fingerprinting and a broad collection of statistical techniques has been used for evaluation of data.

After separation and identification of biomarkers, principal component analysis PCA, a mathematical procedure, can be used for analyses of chromatograms using a fast and objective procedure with more comprehensive data usage compared to other fingerprinting methods. The discriminative power of PCA can be enhanced by deselecting the most uncertain variables or scaling them according to their uncertainty.

For example, preprocessing of GC-MS chromatograms followed by principal component analysis (PCA) of oil spill samples collected from the coastal environment in the weeks after the Baltic Carrier oil spill and from the tank of the Baltic Carrier (source oil) was carried out (Christensen et al., 2005). The preprocessing consists of baseline removal by derivatization, normalization, and alignment using correlation optimized warping. The method was applied to chromatograms of *m*/*z* 217 (tricyclic and tetracyclic steranes) of oil spill samples and source oils. The four principal components were interpreted as follows: boiling point range (PC1), clay content (PC2), carbon number distribution of sterols in the source rock (PC3), and thermal maturity of the oil (PC4). The method allows for analyses of chromatograms using a fast and objective procedure and with more comprehensive data usage compared to other fingerprinting methods.
