Field of the invention
[0001] The present invention relates, generally, to separation of biomarker compounds from petroleum. More particularly, the present invention relates to a method for biomarker compounds separation, identification and enrichment utilizing combination of molecular sieves of different structural variations.
Background of the invention
[0002] Petroleum or crude oil biomarkers are composed essentially of group of complex hydrocarbon compounds found in rock and soil extracts. Biomarkers are separated from petroleum for carrying out various studies as chemical structure of biomarkers remain unchanged during oil generation processes. For example, studies may include, but not limited to, identifying the source rocks of petroleum, biological organisms which generated the crude oil, degree of biodegradation, age (taxon) specific biomarker studies, determining genetic correlation of oils, environmental conditions prevailing at the time of crude oil formation, crude oil formation history, determining oil and rock relations, geochemical analysis etc. Further, examples of petroleum biomarkers may include, but are not limited to, hopane, sterane, bicadinane, pristine, phytane etc. Further, typically, different biomarker compounds are separated or isolated from other biomarker compounds for source rocks identification, determining age of oil and source rock, genetic correlation determination etc. It has been observed that some biomarker compounds may occur in traces with other biomarker compounds in a complex mixture of biomarker compounds. The separation and analysis of such biomarker compounds from the complex mixture is a difficult task.

[0003] It has been observed that conventional techniques utilized for biomarker compounds separation does not efficiently separate and enrich biomarker compounds from petroleum. Further, it has been observed that the age and source related biomarkers, which usually occur in traces, are masked in the gas chromatography (GC), gas chromatography -mass spectrometry - mass spectrometry chromatograms (GC-MS-MS) by peaks from other components of complex mixtures and therefore could not be identified effectively. Further, various fractions of biomarker compounds present in the complex mixture of biomarkers are extracted for analysis, and it has been observed that utilizing conventional methods all the fractions during separation are not retrievable for further geochemical analysis. Furthermore, conventional biomarker separation techniques fail to yield mixtures that can be resolved fully by GC, GC-MS-MS chromatograms. Furthermore, it has been observed that the essential sterane compounds are not identified with reliability using conventional techniques. Yet further, the conventional biomarker separation techniques are not user friendly and are time consuming, thereby increasing the cost for biomarker separation processes.
[0004] In light of the aforementioned drawbacks, there is a need for a method which provides effective and efficient separation, identification and enrichment of biomarker compounds from petroleum. Further, there is a need for a method which provides for effectively retrieving maximum biomarker compounds present in a complex mixture of biomarker compounds. Yet further, there is a need for a method which provides biomarker separation which is user friendly, less time consuming and cost effective.
Summary of the invention
[0005] In various embodiments of the present invention, a method for one or more biomarker compounds separation,

identification and enrichment is provided. The method comprises soxhletting a saturated fraction of petroleum crude oil with 5A° zeolite molecular sieve for extraction of one or more n-alkane hydrocarbons from one or more branched and cyclic alkane hydrocarbons present in the saturated fraction of crude oil. Further, the branched and cyclic alkane hydrocarbons are soxhletted with zeolite 13X molecular sieve for extracting one or more hopane hydrocarbon compounds and one or more sterane hydrocarbon compounds from the branched and cyclic alkane hydrocarbons. The sterane hydrocarbon compounds and the hopane hydrocarbon compounds are separately extracted as a fraction 1 and a fraction 2 respectively. Finally, the fraction 1 and the fraction 2 is separately treated with ultra-stable-Y (US-Y) zeolite molecular sieve.
Brief description of the accompanying drawings
[0006] The present invention is described by way of embodiments illustrated in the accompanying drawings wherein:
[0007] FIG. la illustrates a representation of a Total Ion Chromatogram (TIC) applying m/z 191 with branched and cyclic alkane after separation with activated 5A° molecular sieve, in accordance with an embodiment of the present invention;
[0008] FIG. lb illustrates a representation of a chromatogram of branched and cyclic alkane after separation with activated 5A° molecular sieve applying m/z 183, in accordance with an embodiment of the present invention;
[0009] FIG. 2a illustrates a representation of a chromatogram of gas chromatography-mass spectrometry (GC-MS) m/z 191 showing dominant presence of diahopane and non-hopanoids, in accordance with an embodiment of the present invention;

[0010] FIG. 2b illustrates a representation of a
chromatogram of GC-MS m/z 217 showing better separation of
C21, C?8 and C29 sterane, in accordance with an embodiment of
the present invention;
[0011] FIG. 3a illustrates a representation of a chromatogram of GC-MS m/z 191 showing hopanes and norhopane peaks, in accordance with an embodiment of the present invention;
[0012] FIG. 3b illustrates a representation of a
chromatogram of GC-MS m/z 217 showing absence of sterane
peaks, in accordance with an embodiment of the present
invention;
[0013] FIG. 4a illustrates a representation of a chromatogram of sterane after separation by activated US-Y molecular sieve by applying m/z 217, in accordance with an embodiment of the present invention;
[0014] FIG. 4b illustrates a representation of a chromatogram of bicadinane after separation by activated US-Y molecular sieve by applying m/z 412, in accordance with an embodiment of the present invention; and
[0015] FIG. 4c illustrates a representation of a chromatogram of hopane and enrichment of Oleanane (OL) after separation by activated US-Y molecular sieve by applying m/z 191, in accordance with an embodiment of the present invention.
Detailed description of the invention
[0016] The present invention discloses a method for biomarker compounds separation, identification and enrichment. The invention provides a method for retrieving maximum biomarker compounds present in a complex mixture of biomarker compounds. The invention provides a method for identifying and enriching age (taxon) specific and source

related biomarkers from complex biomarker compounds. Further, the invention provides a method for complete separation and enrichment of trace components of a biomarker for identification and quantification of such components in a reliable manner. Furthermore, the invention provides a method for unmasking of peaks of biomarker compounds which occur in traces in a complex biomarker mixture from other biomarker compounds analyzed utilizing gas chromatography (GC) and gas chromatography-mass spectrometry - mass spectrometry (GC-MS-MS) techniques. Yet further, the invention provides a method for biomarker separation which is user friendly, less time consuming and cost effective.
[0017] The disclosure is provided in order to enable a person having ordinary skill in the art to practice the invention. Exemplary embodiments herein are provided only for illustrative purposes and various modifications will be readily apparent to persons skilled in the art. The general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the invention. The terminology and phraseology used herein is for the purpose of describing exemplary embodiments and should not be considered limiting. Thus, the present invention is to be accorded the widest scope encompassing numerous alternatives, modifications and equivalents consistent with the principles and features disclosed herein. For purposes of clarity, details relating to technical material that is known in the technical fields related to the invention have been briefly described or omitted so as not to unnecessarily obscure the present invention.
[0018] The present invention would now be discussed in context of embodiments as illustrated in the accompanying drawings.
[0019] In various embodiment of the present invention, the biomarker compounds present in a complex mixture of biomarker

compounds are separated from petroleum crude oil utilizing molecular sieves of different types, diameters and sizes. The molecular sieves of one or more types are effective in separating and concentrating selected biomarkers from petroleum. In an embodiment of the present invention, the method for separation and enrichment of biomarker compounds is carried out based on structural variations of combination of molecular sieves. In an exemplary embodiment of the present invention, dehydrated zeolites or molecular sieves may be utilized for separating and concentrating biomarker compounds from petroleum crude oil. The dehydrated zeolites or molecular sieves have sieve-like properties which provide efficient separation of biomarker compounds, for analysis, present in a complex biomarker mixture as trace compounds. For example, zeolites are hydrated crystalline aluminosilicicates composed of three-dimensional network of alumina and silicon tetrahedral (Si04) that are linked together by shared oxygen atom. The dehydration of zeolites generates pores or channels in them and the dehydrated zeolites are used for separation of compounds from complex mixtures based on subtle differences in shapes and sizes. Therefore, dehydrated zeolites or molecular sieves of different shapes and sizes are utilized for effective separation and concentration of selected biomarkers from petroleum. In an exemplary embodiment of the present invention, the different types of molecular sieves or zeolite molecular sieves utilized for biomarker compounds separation from petroleum crude oil includes, but are not limited to, molecular sieve 5A° (Linde 5A°) , Zeolite NaX (13X) and Zeolite ultra-stable-Y (US-Y).
[0020] In an embodiment of the present invention, the petroleum from which biomarkers are separated may comprise compounds of n-alkanes and branched and cyclic alkanes. In an exemplary embodiment of the present invention, petroleum crude oil is first converted to a saturated fraction comprising oil

and essential oil mixture (EOM) for separating n-alkane hydrocarbons and branched and cyclic alkane hydrocarbons compounds. The saturated fraction comprising both oil and EOM is dissolved in cyclohexane and soxhletted with activated 5A° zeolite molecular sieve for a preferred time period of 24 hrs. The molecular sieve 5A° is utilized for separating n-alkanes from petroleum as n-alkanes comprises the major proportion of petroleum crude oil. The n-alkanes are separated for retrieving branched and cyclic alkane compounds for further analysis. The structure of the molecular sieve 5A° (Linde 5A°) provides a selective adsorption of n-alkane from saturated fraction of petroleum. In an embodiment of the present invention, the saturate of petroleum crude oil fraction comprising oil and EOM solvent present in a round bottom flask is soxheletted and evaporated with the 5A° molecular sieve in a water bath to separate the branched and cyclic alkane compounds and the n-alkanes. The branched and cyclic alkane compounds are retrieved and subsequently evaporated for removal of excess solvent. The n-alkane compounds are adsorbed in the 5A° molecular sieve. The n-alkanes show cross-sectional diameter near 5A° and diffuses or occludes into 5A° molecular sieve when heated thereby leaving a fraction comprising branched and cyclic alkanes. The n-alkanes adsorbed in the 5A° molecular sieve are retrieved by exhaustive soxhelet extraction of the 5A° zeolite for geochemical analysis. The branched and cyclic alkanes fraction is further analyzed for biomarker identification. The evaporated fraction comprising branched and cyclic alkanes are collected and thereafter identified and analyzed utilizing gas chromatography-mass spectrometry (GC-MS) technique. FIG. la illustrates a representation of a Total Ion Chromatogram (TIC) applying m/z 191 with branched and cyclic alkane after appropriate separation from n-alkanes utilizing activated 5A° molecular sieve, in accordance with an embodiment of the present invention. Therefore, TIC illustrates proper removal of n-alkanes from the saturate fraction of oil. The branched and

cyclic alkanes are identified utilizing m/z 183 wherein the essential peaks are identified based on their retention time for the particular GC-MS technique. FIG. lb illustrates a representation of a chromatogram of branched and cyclic alkane after separation with activated 5A° molecular sieve applying m/z 183, in accordance with an embodiment of the present invention. The chromatogram in FIG. lb therefore accurately illustrates only branched and cyclic compounds which have been effectively separated in a short time interval utilizing the 5A° molecular sieve.
[0021] In an exemplary embodiment of the present invention, the collected branched and cyclic alkane fractions are further soxhletted for fast and effective separating of hopane and sterane fractions from branched and cyclic compounds after separating n-alkane and branched and cyclic alkane compounds from a saturated fraction of petroleum crude oil. In a preferred embodiment of the present invention, the branched and cyclic alkane fractions are treated with activated Zeolite 13X for separation of hopane and sterane fractions. The Zeolite 13X molecular sieve has channel dimensions of 8A° which effectively separates, by adsorption, and enrich polycyclic hydrocarbons such as tetra-cyclic sterane, penta-cyclic hopane etc. from the branched and cyclic alkane fraction of crude oil/EOM.
[0022] In a preferred embodiment of the present invention, the separated branched and cyclic alkane hydrocarbon fraction are further treated with freshly activated Zeolite 13X and refluxed with 100 ml n-pentane solvent preferably for a time period of 3 hrs. A mixture I is formed comprising n-pentane and sterane biomarkers, extracted from branched and cyclic compounds of the crude oil. The mixture I is cooled, filtered and washed with 50 ml of n-pentane and thereafter a solvent I is formed. The formed solvent I is further heated until evaporation and an evaporated solvent II is collected. The collected solvent II is further adsorbed on freshly activated

Zeolite 13X molecular sieve. The remaining mixture is further refluxed with 100 ml n-pentane solvent preferably for a time period of 2 hrs. Consequently, mixture II is formed which is thereafter collected and filtered. The Zeolite 13X molecular sieve is further washed with 20 ml n-pentane in small aliquots. A solvent III is formed after washing with 20 ml n-pentane. The formed solvent III, comprising n-pentane, diahopane, non-hopanoids and steranes biomarkers of the crude oil, is collected and thereafter evaporated. The evaporated solvent is referred to as a fraction 1' solvent and analyzed for biomarker analysis using GC-MS and GC-MS-MS techniques. The constituents of the fraction 1 may comprise diahopane, non-hopanoids and sterane compounds. FIG. 2a illustrates a representation of a chromatogram of GC-MS m/z 191 showing fraction 1 with dominant presence of diahopane and non-hopanoids like oleanane, moretane, etc., in accordance with an embodiment of the present invention. FIG. 2b illustrates a representation of a chromatogram of GC-MS m/z 217 showing fraction 1 with better separation of C27, C28 and C29 sterane and good resolution of S-, R- and (3(3 peaks, in accordance with an embodiment of the present invention. Further, the hopane compounds are sorbed into the Zeolite 13X molecular sieve.
[0023] In an embodiment of the present invention, the Zeolite 13X molecular sieve is retrieved after evaporation of the fraction 1 solvent and subjected to exhaustive extraction for a preferred time period of 36 hrs for desorbing and recovering the hopane compounds absorbed in it. The exhaustive extraction is carried out utilizing preferably iso-octane solvent. Further, after the exhaustive extraction a solvent IV comprising iso-octane and hopane hydrocarbon compounds is formed, which is thereafter evaporated. The evaporated solvent IV is referred to as a ^fraction 2' solvent. The fraction 2 solvent therefore, comprises hopane compounds. The fraction 2 solvent is then analyzed for biomarker analysis using GC-MS and GC-MS-MS. FIG. 3a illustrates a representation of a

chromatogram of GC-MS m/z 191 showing only prominent hopanes and norhopane peaks, in accordance with an embodiment of the present invention. Further, FIG. 3b illustrates a representation of a chromatogram of GC-MS m/z 217 showing absence of sterane peaks, in accordance with an embodiment of the present invention.
[0024] In an embodiment of the present invention, the fraction 1 and fraction 2 formed are further separately treated with freshly activated ultra-stable-Y (US-Y) Zeolite molecular sieve. The US-Y Zeolite molecular sieve is used to separate structurally similar petroleum hydrocarbons, for example hopanoid hydrocarbons and bicadinane. In an exemplary embodiment of the present invention, a column chromatography technique is utilized for treating the fraction 1 and fraction 2 wherein the column is formed from the US-Y Zeolite molecular sieve. In a preferred embodiment of the present invention, the molecular sieve column utilized is prepared by using a 25 ml glass column plugged with a small piece of cotton wool and dry packed with 2g of US-Y Zeolite molecular sieve. The column is washed twice with 5 ml of n-pentane. Further, the fraction 1 comprising diahopane, non-hopanoids, sterane compounds is first dried and thereafter dissolved in minimum volume of n-pentane in a 20 ml vial depending upon the quantity of the fraction 1. The minimum volume of n-pentane utilized is preferably in the range of 2 to 5 ml. The dissolved fraction 1 is transferred from the vial and is placed on the column and thereafter allowed to stand preferably for a time period of 5 minutes subsequent to elution process. Further, the column is slowly eluted with n-pentane preferably at a rate of 10 drops/minute. Further, two consecutive fractions are collected comprising 0.5 ml and 1 ml eluent respectively. The two fractions may be referred to as a fraction A and a fraction B. The collected fractions are analyzed using GC-MS and GC-MS-MS. The fraction A comprises diahopane and non-hopanoids like oleanane and the fraction B comprises sterane compounds

such as 26-norcholestane, nordiacholestane, dinosteranes, 24-isopropylcholestane, diasterans, cadinane, bicadinane, 2&3 methylsteranes etc. FIG. 4a illustrates a representation of a chromatogram of sterane after separation by activated US-Y molecular sieve by applying m/z 217, in accordance with an embodiment of the present invention. The chromatogram in FIG. 4a illustrating sterane in GC-MS distinctly provides well resolved S, R, (3(3 isomers of C27, C2>3 and C29. FIG. 4b illustrates a representation of a chromatogram of bicadinane after separation by activated US-Y molecular sieve by applying m/z 412, in accordance with an embodiment of the present invention. The chromatogram in FIG. 4b effectively provides enriched bicadinane which was surprisingly apparent after adsorption of fraction 1 in US-Y molecular sieve. Furthermore, m/z 217 shows enrichment of bicadinane which is further corrugated by m/z 412.
[0025] In an embodiment of the present invention, the fraction 2 comprising hopane compounds is firstly dried and is thereafter dissolved in a minimum volume of n-pentane in a 20 ml vial depending upon the quantity of the fraction 1. The minimum volume of n-pentane utilized is in the range of 2 and 5 ml depending on the quantity of the fraction 2. The dissolved fraction 2 is transferred from the vial and placed on the column and thereafter allowed to stand for 5 minutes subsequent to elution process. Further, the column is slowly eluted with n-pentane preferably at a rate of 10 drops/minutes. Further, two consecutive fractions are collected comprising 0.5 ml and 1 ml eluent respectively. The two fractions may be referred to as fraction C and fraction D. The collected fractions are analyzed using GC-MS and GC-MS-MS. The fraction C comprises norhopanes and tricyclic terpanes and the fraction D comprises homohopanes. FIG. 4c illustrates a representation of a chromatogram of hopane and enrichment of Oleanane (OL) after separation by activated US-Y Zeolite molecular sieve by applying m/z 191, in accordance

with an embodiment of the present invention. Oleanane is an essential source specific and age specific biomarker.
[0026] Advantageously, in accordance with various embodiments of the present invention, the present invention provides an exhaustive and efficient method for biomarker compounds separation, extraction, identification and enrichment from petroleum. The present invention unexpectedly provides for complete separation and identification of individual biomarker compounds which occur in traces that are specific for age and source determination of oil and rocks, such as, but not limited to, hopanes and steranes, for example, 24-norcholestane, dinosterane, C30 24-iso-propyl cholestane, 2&3-methyl steranes, cadinane etc. The present invention provides for fast isolation of branched and cyclic alkanes from n-alkanes. The present invention provides for proper isolation and enrichment of hopane, diahopane and non-hopanoid compounds. The invention provides for further quantification and enrichment of sterane and unmasking of its important peaks as illustrated in the chromatogram identified using GC-MS analysis techniques. Further, the invention provides for recycling of solvents utilized for biomarker studies, thus making the biomarker extraction process cost effective. For example, the isolation process of sterane and hopanes utilizing 13X molecular sieves involves extraction with solvent, therefore the solvent utilized is recycled by distillation process which is further utilized in analytical processes. Further, the present invention provides for unique combination of molecular sieves such as, molecular sieve 5A°, Zeolite 13X and US-Y Zeolite molecular sieve having different structural variations which can be further retrieved and re¬used. The molecular sieves utilized have different diameter, size and shape which separates and concentrates different biomarker compounds effectively and efficiently based on their adsorbing capabilities. Therefore, the present invention

provides for user friendly, less time consuming and economical biomarker separation, identification and enrichment method.
[0027] While the exemplary embodiments of the present invention are described and illustrated herein, it will be appreciated that they are merely illustrative. It will be understood by those skilled in the art that various modifications in form and detail may be made therein without departing from the scope of the invention.

We claim:
1 . A method for one or more biomarker compounds separation, identification and enrichment, the method comprising the steps of:
soxhletting a saturated fraction of petroleum crude oil with 5A° zeolite molecular sieve for extraction of one or more n-alkane hydrocarbons from one or more branched and cyclic alkane hydrocarbons present in the saturated fraction of crude oil;
soxhletting the branched and cyclic alkane hydrocarbons with zeolite 13X molecular sieve for extracting one or more hopane hydrocarbon compounds and one or more sterane hydrocarbon compounds from the branched and cyclic alkane hydrocarbons, wherein the sterane hydrocarbon compounds and the hopane hydrocarbon compounds are separately extracted as a fraction 1 and a fraction 2 respectively; and
treating the fraction 1 and the fraction 2 separately with ultra-stable-Y (US-Y) zeolite molecular sieve.
2. The method as claimed in claim 1, wherein the saturated fraction of petroleum crude oil is dissolved in cyclohexane prior to soxhletion with the 5A° zeolite molecular sieve.
3. The method as claimed in claim 1, wherein the step of extraction of the one or more n-alkane hydrocarbons from the one or more branched and cyclic alkane hydrocarbons based on the 5A° zeolite molecular sieve comprises the steps of:
soxheletting and evaporating in a water bath a saturated solvent of petroleum crude oil in a round bottom flask with the 5A° zeolite molecular sieve for retrieving the branched

and cyclic hydrocarbon compounds from the solvent; evaporating the retrieved branched and cyclic hydrocarbon compounds for removal of excess solvent; and retrieving by exhaustive soxhelet extraction of the 5A° zeolite molecular sieve the n-alkane hydrocarbon compounds present in the saturate solvent of petroleum crude oil which are adsorbed into the 5A° zeolite molecular sieve.
4. The method as claimed in claim 1, wherein the step of extraction of the one or more hopane hydrocarbon compounds and the one or more sterane hydrocarbon compounds from the branched and cyclic alkane hydrocarbons based on the zeolite 13X molecular sieve comprises the steps of:
refluxing branched and cyclic alkane hydrocarbon fraction with 100 ml n-pentane solvent in the presence of the zeolite 13X molecular sieve for 3 hours, wherein a mixture I comprising n-pentane and sterane biomarkers is formed;
cooling, filtering and washing the formed mixture I with 50 ml of n-pentane, wherein a solvent I is formed after cooling, filtering and washing;
heating the solvent I until evaporation and collecting a solvent II, wherein the collected solvent II is adsorbed on the freshly activated Zeolite 13X molecular sieve;
refluxing, after adsorption, the remaining mixture with 100 ml n-pentane solvent for 2 hours, wherein a mixture II is formed which is collected and filtered;
washing the zeolite 13X molecular sieve with 20 ml n-pentane in small aliquots, wherein a solvent III comprising n-pentane, diahopane, non-hopanoids and steranes biomarkers is formed which is collected and evaporated, wherein the evaporated solvent III is a fraction 1 solvent,

and wherein the fraction 1 comprises diahopane, non-hopanoids and sterane compounds;
retrieving and subjecting the zeolite 13X molecular sieve to exhaustive extraction utilizing iso-octane solvent for 36 hours, wherein exhaustive extraction desorbs and recovers absorbed hopane hydrocarbon compounds from the zeolite 13X molecular sieve; wherein subsequent to exhaustive extraction a solvent IV comprising iso-octane and hopane is formed; and
evaporating the solvent IV for separating hopane hydrocarbon compounds from iso-octane, wherein the evaporated solvent IV is a fraction 2 solvent, and wherein the fraction 2 comprises hopane hydrocarbon compounds.
5. The method as claimed in claim 4, wherein the fraction 1 and the fraction 2 are analyzed for biomarker identification using gas chromatography - mass spectrometry (GC-MS) and gas chromatography mass spectrometry ■- mass spectrometry (GC-MS-MS) techniques.
6. The method as claimed in claim 1, wherein the step of treating the fraction 1 with ultra-stable-Y (US-Y) zeolite molecular sieve comprises the steps of:
forming a 25 ml glass column plugged with a cotton wool and dry packed with 2g of ultra-stable-Y (US-Y) zeolite molecular sieve;
washing the column twice with 5 ml of n-pentane;
drying the fraction 1 and dissolving the fraction 1 in 2 to 5 ml of n-pentane in a 20 ml vial;

transferring the fraction 1 from the vial and placing on the US-Y zeolite molecular sieve column for 5 minutes;
eluting the column comprising the fraction 1 with n-pentane at a rate of 10 drops/minute; and
collecting a 0.5 ml eluent as a fraction A and a 1 ml eluent as a fraction B, wherein the fraction A comprises diahopane and one or more non-hopanoids and the fraction B comprises one or more sterane compounds, and wherein the one or more non-hopanoids comprise oleanane and the one or more sterane compounds comprises 26-norcholestane, nordiacholestane, dinosteranes, 24-isopropylcholestane, diasterans, cadinane, bicadinane and 2&3 methylsteranes.
7. The method as claimed in claim 1, wherein the step of treating the fraction 2 with ultra 3table Y (US Y) zeolite molecular sieve comprises the steps of:
forming a 25 ml glass column plugged with a cotton wool and dry packed with the 2g of ultra-stable-Y (US-Y) zeolite molecular sieve;
washing the column twice with 5 ml of n-pentane;
drying the fraction 2 and dissolving the fraction 2 in 2 to 5 ml of n-pentane in a 20 ml vial;
transferring the fraction 2 from the vial and placing on the US-Y zeolite molecular sieve column for 5 minutes;
eluting the column comprising the fraction 2 with n-pentane at a rate of 10 drops/minute; and
collecting a 0.5 ml eluent as a fraction C and a 1 ml eluent as a fraction D, wherein the fraction C comprises

norhopanes and tricyclic terpanes and the fraction D comprises homohopanes.