Method for separating and analyzing biomarkers from sediments

Abstract

To provide a method for separating and analyzing biomarkers from sediments, source rocks or reservoir rocks in one step.SOLUTION: The method includes extracting organic substances from a sample (step a), removing inorganic substances from the sample obtained in step a (step b), removing asphaltene from the sample obtained in step b (step c), classifying the sample obtained in step c into a group including saturated hydrocarbons, a group including aromatic hydrocarbons, and a group including NSO compounds (step d), and detecting components included in each of the group including saturated hydrocarbons, the group including aromatic hydrocarbons, and the group including NSO compounds obtained in step d (step e).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to methods for the isolation and analysis of geological biomarkers, and more particularly to methods for the isolation and analysis of biomarkers from sediments, source rocks or reservoir rocks. [Background technology]

[0002] The term geological biomarker was first defined by Eglinton and Calvin (1967). Geological biomarkers are typically like fingerprints present in sediments and are actively used to study the origin, depositional environment, maturity, and biodegradation of sediments. The principle behind geological biomarkers as such powerful tools is simple. Sediments contain organic matter, which is defined as compounds connected by carbon. Organic matter present in sediments, composed of carbon compounds, originates from organisms and plants present at the time of deposition. For example, sterane is a modified form of sterol, and porphyrin is a modified form of chlorophyll after deposition. Therefore, studying organic compounds present in sediments, especially biomarkers, can help us understand the origin of the organic matter and the environment that affected it after deposition. Organic matter can be broadly classified as insoluble organic matter and soluble organic matter. Kerogen is a type of insoluble organic matter. Among these, soluble organic matter is a substance known as a biomarker. Soluble organic matter is classified into aliphatic hydrocarbons, aromatic hydrocarbons, and NSO compounds based on the carbon bond structure and type of compound.

[0003] Conventional methods for isolating and analyzing biomarkers from sediments, source rocks, or reservoir rocks have been inconvenient because different separation methods must be used depending on the type of biomarker to be isolated (aliphatic hydrocarbons (especially saturated hydrocarbons), aromatic hydrocarbons, or NSO compounds).

[0004] For this reason, the present inventors developed a method for simultaneously separating and analyzing multiple biomarkers from sediments, source rocks, or reservoir rocks in a single step, confirmed its usefulness, and completed the present invention. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Republic of Korea Patent Publication No. 10-1694994 (Announced on January 11, 2017) Summary of the Invention [Problem to be solved by the invention]

[0006] The object of the present invention is to provide a method for simultaneously isolating and analyzing multiple biomarkers from sediments, source rocks, or reservoir rocks in a single step.

[0007] The problems that the present invention aims to solve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0008] In order to achieve the above object, the present invention provides a method for separating and analyzing biomarkers, which includes extracting organic matter from a sample (step a); removing inorganic matter from the sample obtained in step a (step b); removing asphaltenes from the sample obtained in step b (step c); classifying the sample obtained in step c into a group containing saturated hydrocarbons, a group containing aromatic hydrocarbons, and a group containing NSO compounds (step d); and detecting components contained in each of the groups containing saturated hydrocarbons, aromatic hydrocarbons, and NSO compounds obtained in step d (step e).

[0009] The sample in step a above may comprise one or a mixture of two or more selected from soil, rock, sediment, marine sediment, sedimentary rock, source rock, or reservoir rock.

[0010] Step a above may include grinding the sample before extracting the organic matter.

[0011] The step a) above may involve distilling a sample containing rock, sediment, or a mixture thereof that does not contain oil or bitumen with an organic solvent.

[0012] The step a) may involve adding an organic solvent to a sample containing oil, bitumen, or a mixture thereof, and then subjecting the sample to ultrasonic pulverization.

[0013] The step b may involve filtration.

[0014] The above step c may be carried out by mixing a non-polar organic solvent with the sample.

[0015] The step d may involve first separating a group containing saturated hydrocarbons from the sample using a first solvent, second separating a group containing aromatic hydrocarbons from the sample using a second solvent, and third separating a group containing NSO compounds from the sample using a third solvent.

[0016] The first solvent may be an organic solvent having a polarity index of 0 to 0.5, the second solvent may be an organic solvent having a polarity index of 1 to 3, and the third solvent may be an organic solvent having a polarity index of 4 to 5.

[0017] The above step e may be carried out using GC or GC / MS. [Effects of the Invention]

[0018] The present invention has the advantage that multiple biomarkers can be simultaneously separated and analyzed from sediments, source rocks, or reservoir rocks in a single step.

[0019] It should be understood that the effects of the present invention are not limited to the effects described above, but include any effects that can be inferred from the configuration of the invention described in the detailed description of the present invention or the claims. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 shows a process flow diagram of a method for separating and analyzing biomarkers according to one embodiment of the present invention. [Figure 2] 10A and 10B are photographs showing a method for cleaning an agate mortar used in a sample preparation step and crushing a sample according to an embodiment of the present invention. [Figure 3] 1 is a graph for determining the weight of a sample used for extraction in a sample preparation step according to an embodiment of the present invention (X-axis: EOM content, Y-axis: sample weight×S1). [Figure 4] 1 shows a Soxhlet apparatus used in the extraction of organic matter from rock and sediment samples according to one embodiment of the present invention. [Figure 5] 1 is a photograph showing a copper wire and 10% hydrochloric acid stored in MeOH solution used to remove free sulfate in a rock and sediment sample during an organic matter extraction step according to an embodiment of the present invention. [Figure 6]1 is a photograph showing an ultrasonic grinder and a centrifuge used in the step of extracting organic matter from oil and bitumen samples according to an embodiment of the present invention. [Figure 7] 10 is a photograph showing a filter using glass wool and an evaporator used in the inorganic matter removal step according to an embodiment of the present invention. [Figure 8] 10 is a photograph showing a solvent before and after filtering in the inorganic matter removal step according to an embodiment of the present invention. [Figure 9] 1 is a photograph showing asphaltene precipitation and coagulation after pentene is mixed with a sample in the asphaltene removal step according to an embodiment of the present invention. [Figure 10] 1 is a photograph showing that a sample is eluted into each component during the biomarker classification step according to an embodiment of the present invention. [Figure 11] 1 is a photograph showing a GC / MS according to an embodiment of the present invention. [Figure 12] FIG. 1 shows GC / MS results from m / z 191 fragmentogram according to one embodiment of the present invention. [Figure 13] FIG. 1 shows the chemical structural formulas of Hopane and 25norHopane according to an embodiment of the present invention. [Figure 14] FIG. 1 shows GC / MS results from m / z 177 fragmentogram according to one embodiment of the present invention. [Figure 15] FIG. 1 shows GC / MS results from m / z 217 fragmentogram according to one embodiment of the present invention. [Figure 16] FIG. 1 shows chemical structural formulas of various naphthalenes according to an embodiment of the present invention. [Figure 17] FIG. 1 shows GC / MS results from m / z 128, 142, 156, 170, and 184 (fragmentogram) according to an embodiment of the present invention. [Figure 18] FIG. 1 shows chemical structural formulas of various phenanthrenes according to an embodiment of the present invention. [Figure 19] FIG. 1 shows GC / MS results from fragmentograms of m / z 178, 192, and 206 according to one embodiment of the present invention. [Figure 20] FIG. 1 shows chemical structural formulas of various benzothiophenes according to an embodiment of the present invention. [Figure 21] FIG. 1 shows GC / MS results from fragmentograms of m / z 184, 198, and 212 according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0021] It should be noted that in the following description, only the parts necessary for understanding the embodiments of the present invention will be described, and the description of other parts will be omitted to the extent that the gist of the present invention is not obscured.

[0022] The terms and phrases used in the following specification and claims should not be interpreted in a limited way to their ordinary or dictionary meanings, but should be interpreted in a way that is consistent with the technical idea of ​​the present invention, in accordance with the principle that an inventor may appropriately define the concept of a term in order to explain his / her invention in the best possible way.

[0023] Therefore, the embodiments described in this specification and the configurations shown in the drawings are merely preferred embodiments of the present invention and do not represent the technical ideas of the present invention, and it should be understood that there may be various equivalents and modifications that can be substituted for them at the time of filing this application.

[0024] The present invention will be described in detail below.

[0025] The present invention provides a method for separating and analyzing biomarkers, which includes extracting organic matter from a sample (step a), removing inorganic matter from the sample obtained in step a (step b), removing asphaltenes from the sample obtained in step b (step c), classifying the sample obtained in step c into a group containing saturated hydrocarbons, a group containing aromatic hydrocarbons, and a group containing NSO compounds (step d), and detecting components contained in each of the saturated hydrocarbons, aromatic hydrocarbons, and NSO compounds obtained in step d (step e). The method will be described below with reference to FIG. 1.

[0026] Step a: Extract organic matter from the sample (S100). The sample may be soil or rock, and in particular may contain one or a mixture of two or more selected from sediments or marine sediments, sedimentary rocks or sedimentary structures formed therefrom, source rocks or reservoir rocks.

[0027] This step may further comprise grinding the sample prior to extracting the organic matter. In the case of samples containing oil or bitumen, it is preferred that the average particle size is 100 mesh or less.

[0028] The method may involve extracting organic matter from a sample containing rock, sediment, or a mixture thereof that does not contain oil or bitumen by distillation with an organic solvent, specifically by using a Soxhlet extractor, and may further include removing free sulfate from the sample.

[0029] The method may involve adding an organic solvent to a sample containing oil, bitumen, or a mixture thereof, followed by ultrasonic pulverization to extract organic matter, and then further centrifuging the sample to extract the organic matter.

[0030] The organic solvents used in the two types of samples may both have a polarity of 3 to 5, preferably 3.5 to 4.5. Specifically, they may be a mixture of DCM:MeOH at a ratio of 3:7 to 7:3 (v / v), 4:6 to 6:4 (v / v), or 1:1 (v / v).

[0031] Step b: Inorganic substances are removed from the sample obtained in S100 (S200). This step may involve removing inorganic matter, particularly clay minerals, from the organic matter-containing solvent obtained in S100 by filtration. Specifically, the filtration may be performed using glass wool.

[0032] The method may further comprise distilling the solvent filtered as described above.

[0033] Step c: Asphaltene is removed from the sample obtained in S200 (S300). Asphaltene is a highly polar and large molecular weight substance, which significantly interferes with the analysis of biomarkers, and therefore it is necessary to effectively remove it.

[0034] Therefore, in this step, asphaltene is removed from the sample from which inorganic substances have been removed in S200 above.

[0035] Asphaltene has high polarity, it can be removed using an organic solvent with very low polarity, preferably one with a polarity index of 0.5 or less, or 0. For example, heptane, hexane, pentane, etc., which have a polarity index of 0, are preferred for removal. In particular, when pentane is mixed as the organic solvent, asphaltene precipitates or coagulates as an insoluble substance, making it easy to separate and remove.

[0036] Step d: The sample obtained in S300 is classified into a group containing saturated hydrocarbons, a group containing aromatic hydrocarbons, and a group containing NSO compounds (S400). In this step, the sample from which asphaltene has been removed in S300 is classified into a saturated hydrocarbon group, an aromatic hydrocarbon group, and an NSO compound group. In this specification, saturated hydrocarbons refer to compounds in which all covalent bonds between carbon and carbon or carbon and hydrogen are single bonds, and include alkane or paraffin compounds, particularly hydrocarbon compounds containing cycloalkanes. In this specification, aromatic hydrocarbons refer to compounds containing stable cyclic conjugated molecules, such as benzene, which contain stable ring compounds with alternating double bonds, and include single-ring allene compounds, aromatic ring aggregates, fused cyclic aromatic compounds, and compounds containing these substituents. In this specification, NSO compounds refer to organic compounds containing nitrogen (N), sulfur (S), or oxygen (O).

[0037] Specifically, the method may include using a first solvent to perform a primary separation of a group containing saturated hydrocarbons on a sample, using a second solvent to perform a secondary separation of a group containing aromatic hydrocarbons, and using a third solvent to perform a tertiary separation of a group containing NSO compounds.

[0038] The first solvent may be an organic solvent having a polarity index of 0 to 0.5, the second solvent may be an organic solvent having a polarity index of 1 to 3, and the third solvent may be an organic solvent having a polarity index of 4 to 5. Specifically, hexane may be used as the first solvent for separating saturated hydrocarbons, a mixture of hexane and DCM (dichloromethane) in a ratio of 8:2 to 4:6 (v / v) may be used as the second solvent for separating aromatic hydrocarbons, and a mixture of chloroform and methanol in a ratio of 90:10 to 99:1 (v / v) may be used as the third solvent for separating NSO compounds. The flow rate of the first solvent may be 0.1 to 1 drop / sec, the flow rate of the second solvent may be 0.5 to 1.5 drop / sec, and the flow rate of the third solvent may be 1.5 to 2.5 drop / sec (Table 1).

[0039] As described above, the polarity can be adjusted by the type and mixing ratio of the solvent, and the sample can be classified into saturated hydrocarbons, aromatic hydrocarbons, and NSO compounds by adjusting the volume and speed of the solvent.

[0040] The above classification can be achieved by using an alumina activation method, specifically, an alumina column (FIG. 10).

[0041] Step e: The components contained in each of the samples classified in S400 are detected (S500). This step can be performed using GC, GC / MS, or both GC and GC / MS. Biomarkers in the sample can be detected and identified from the graphs of the GC and / or GC / MS results.

[0042] Specifically, biomarkers detected in samples classified into the saturated hydrocarbon group include triterpenes, 25 norhopane, and steranes, while biomarkers detected in samples classified into the aromatic hydrocarbon group include naphthalenes, phenanthrene, and benzothiophenes. Biomarkers detected in samples classified into the NSO compound group include pyrrolidine and carboxylic acids.

[0043] Example The present invention will be described in more detail below with reference to specific examples. The scope of the present invention is not limited to the examples presented below, and various modifications are possible.

[0044] 1. Sample Preparation The prepared samples were finely crushed using an agate mortar. Before using the agate mortar, the mortar was washed with methanol and dichloromethane (DCM) to completely remove any remaining organic matter (Figure 2). Samples 1, 2, and 3 were all collected from a borehole in the Devonian Grosmont Formation in Alberta, Canada.

[0045] 2. Extraction of organic matter The amount of sample used for extraction was determined based on the results of geochemical analysis (Fig. 3).

[0046] (1) Samples containing rocks and sediments For rock and sediment samples, organic matter is extracted using a Soxhlet filter (Figure 4). The Soxhlet filter consists of a round flask (distillation flask) with an extraction tube and a cooling system running above it. A cylindrical filter paper (thimble) is placed in the extraction tube, and the solvent in the round flask is heated. The solvent vapor travels through the tube and condenses in the condenser. It accumulates in the extraction tube and dissolves the soluble organic components in the sample. When the accumulated solvent reaches the top via the siphon on the right, it all returns to the round flask, and new solvent accumulates in the extraction tube again. In this way, extraction is repeated with new solvent. The extract accumulates in the solvent flask and dissolves the soluble organic components in the sample. A 1v:1v mixture of DCM:MeOH is used to extract soluble organic matter using organic solvents. A copper wire is inserted to remove free sulfate from the sample. Free sulfate may affect the chromatography in future GC or GC-MS analysis, so it is preferable to remove it in the initial process of extracting organic matter.

[0047] Cu+H2S--->CuS(s)+H2

[0048] In the case of copper wire to remove free sulfate, the surface is coated with CuO, so the CuO must be removed first. To remove the CuO, use 10% HCl.

[0049] CuO+HCl--->H2O+CuCl

[0050] Immerse the copper wire in the HCl solution for approximately 1 minute, then neutralize it with distilled water. Then, completely remove any remaining organic matter using DCM and MeOH. Store the prepared copper wire in the MeOH solution, completely blocking contact with air (Figure 5).

[0051] (2) Samples containing oil and bitumen Oil and bitumen samples contain a high content of organic matter that is soluble in organic solvents, making it easy to extract the organic matter using an ultrasonic grinder (Figure 6). The solvent used is a 1v:1v mixture of DCM and MeOH, similar to the Soxhlet method. The sample is placed in a centrifuge container, which is then filled to the brim with the solvent mixture. The organic matter is extracted using an ultrasonic grinder for 30 minutes. To effectively separate the organic matter dissolved in the organic solvent from the remaining sample, the sample is centrifuged at 1000 rpm for 10 minutes. The organic solvent is then transferred to a round flask. This process is repeated three times.

[0052] 3.Removal of inorganic matter The extracted organic matter is filtered to remove clay minerals. Glass wool is used for the filter. After creating a three-stage filter using glass wool, the solvent is filtered. After filtering, the solvent is completely distilled in an evaporator at approximately 37°C (Figure 7).

[0053] At this time, if the filtered solvent emits a clear light, it can be determined that the filtering has been effective (Figure 8).

[0054] 4. Asphaltene removal Asphaltene is a highly polar and large molecular weight substance. Therefore, it causes significant interference in biomarker analysis and must be effectively removed. As asphaltene is highly polar, it can be effectively removed using pentane, a non-polar organic solvent. Pentane is a completely non-polar organic solvent with a polarity index of 0, and when asphaltene comes into contact with pentane, a precipitation effect occurs.

[0055] Asphaltene+Pentane→ Precipitation

[0056] When pentane was added to the sample to completely dissolve it, and then the sample was stored in a freezer at -21°C for 12 hours or more, it was confirmed that the asphaltene had settled and solidified (Figure 9).

[0057] Extracted Organic Matter(EOM)+Pentane →(1)Soluble Fraction: Moltene →(2)Insoluble Fraction: Asphaltene

[0058] At this time, the substance dissolved in pentene is classified as moltene, and the undissolved solidified substance is classified as asphaltene. The dissolved upper layer is transferred to a round flask, and the solidified lower layer is dissolved using DCM and then transferred to another flask.

[0059] 5. Classification of biomarkers by component Fractionation is the process of separating the molten material from which asphaltene has been removed into saturated hydrocarbons (Saturated HC), aromatic hydrocarbons (Aromatic HC), and NSO compounds. Using the polarity differences between saturated hydrocarbons, aromatic hydrocarbons, and NSO compounds, the less polar saturated hydrocarbons are separated, and finally the most polar NSO compounds are separated (Figure 10). For this purpose, an alumina column is used.

[0060] The process for making an alumina column is as follows: After filling a 1 cm diameter glass tube with hexane, measure out 7.5 g of alumina and fill the glass tube. The height of the filled alumina column is ideally 6.5 to 7 cm. The solvents used to separate each substance are listed in Table 1 below.

[0061] Approximately 35 mg of the concentrated molten material is eluted with a solvent and adsorbed onto an alumina column (Table 4). First, 17 mL of hexane solvent is added to the alumina column prepared as described above to separate saturated hydrocarbons. Next, 50 mL of a 7:3 v / v mixture of hexane and DCM (solvent) is added to the alumina column to separate aromatic hydrocarbons. Finally, 50 mL of a 98:2 v / v mixture of chloroform and MeOH (solvent) is added to the alumina column to separate NSO compounds.

[0062] The elution patterns of the individual components when fractionated according to the above analytical method are shown in FIG.

[0063] [Table 1]

[0064] 6. Detection After separation, each component of the material is confirmed via GC / MS (Figure 11). The injection amount is approximately 3 mg / ml (Table 5).

[0065] The GC / MS was performed using a Thermo instrument. The MS was equipped with a quadrupole ion trap mass spectrometer. The column was the same as that used for the GC, but with a DB-1. The oven temperature program consisted of maintaining the temperature at 40°C for 1.5 minutes, then increasing the temperature to 300°C at a rate of 4°C / min, and maintaining the temperature at 300°C for 34 minutes.

[0066] The GC / MS analysis was performed in SIM mode. The ion groups were set to 66.0, 83.0, 85.0, 87.0, 110.0, 123.0, 124.0, 133.1, 134.1, 177.3, 183.3, 191.3, 205.3, 217.3, 218.3, 231.3, 232.3, and 253.3 m / z for the saturated hydrocarbon fraction, and 253.3 m / z for the aromatic hydrocarbon fraction. fraction) were assigned m / z values ​​of 77.1, 99.1, 125.1, 106.1, 119.1, 128.1, 142.2, 156.2, 170.2, 178.2, 183.2, 184.2, 188.2, 192.2, 197.2, 198.2, 206.2, 211.2, 212.2, 216.3, 219.2, 219.3, 220.3, 228.3, 230.3, 231.3, 233.3, 234.3, 244.3, and 245.3.

[0067] The samples obtained at each stage and their concentrations are shown in Tables 2 to 5.

[0068] [Table 2]

[0069] [Table 3]

[0070] [Table 4]

[0071] [Table 5]

[0072] 7.Analysis (1) Saturated hydrocarbons (1-1) Triterpanes (m / z 191) Triterpanes are found primarily in oils and bitumens, originating from triterpenoids synthesized by microorganisms. They can be identified from m / z 191 fragmentograms. C19-C25 tricyclic terpanes, C 28 -C 35 ab hopanes, gammacerane, etc. are detected (Figure 12, Table 6).

[0073] (1-2)25norHopane(Demethylated Hopane)(m / z 177) 25norHopane is an organic compound in which the methyl group at position 25 of Hopane has been removed (Figure 13). This can be confirmed from 177 fragmentograms (Figure 14, Table 6).

[0074] [Table 6]

[0075] (1-3)Steranes (m / z 217) Sterane is a tetracyclic saturated hydrocarbon (composed of one 5-ring and three 6-rings) compound found in most organisms higher than cyanobacteria. Sterane compounds can be identified at m / z 217 (Figure 15, Table 7).

[0076] [Table 7]

[0077] (2) Aromatic hydrocarbons (2-1) Naphthalenes (m / z 128, 142, 156, 170, 184) Naphthalenes are the simplest polycyclic aromatic hydrocarbons. The structure of naphthalene consists of a pair of benzene rings (Figure 16). Naphthalene is typically detected at m / z 128, and methyl naphthalene at m / z 142 fragmentation. C2-naphthalenes are typically detected at m / z 156, C3-naphthalenes at m / z 170, and C4-naphthalenes at m / z 184 (Figure 17).

[0078] (2-2)Phenanthrene(m / z 178, 192, 206) Phenanthrene is a polycyclic aromatic hydrocarbon consisting of three benzene rings (Figure 18). Phenanthrene is typically detected at m / z 178, methyl phenanthrene at m / z 192, and dimethyl phenanthrene at m / z 206 fragments (Figure 19).

[0079] (2-3)Benzothiophenes(m / z 184, 198, 212) Benzothiophene has the molecular structure C8H6S, which is similar to naphthalene (Figure 20). Benzothiophene occurs naturally in sedimentary environments, primarily associated with petroleum.

[0080] So far, a specific example of a method for separating and analyzing biomarkers from sediments according to one embodiment of the present invention has been described, but it is clear that various modifications are possible without departing from the scope of the present invention.

[0081] Therefore, the scope of the present invention should not be limited to the described embodiments, but should be determined not only by the claims described below, but also by equivalents to these claims.

[0082] In other words, it should be understood that the above-described embodiments are illustrative in all respects and not limiting, and the scope of the present invention is indicated by the claims below rather than the detailed description, and all modifications or variations derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. Extracting organic matter from the sample (stage a); removing inorganic substances from the sample obtained in step a above (step b); removing asphaltene from the sample obtained in step b above (step c); The sample obtained in step c is classified into a group containing saturated hydrocarbons, a group containing aromatic hydrocarbons, and a group containing NSO compounds (step d); Detecting components contained in each of the group containing saturated hydrocarbons, the group containing aromatic hydrocarbons, and the group containing NSO compounds obtained in step d (step e), Biomarker isolation and analysis methods.

2. The sample of step a comprises one or more mixtures selected from soil, rock, sediment, marine sediment, sedimentary rock, source rock, or reservoir rock; The method for separating and analyzing biomarkers according to claim 1.

3. Step a involves grinding the sample before extracting the organic matter; The method for separating and analyzing biomarkers according to claim 1.

4. Step a) comprises distilling a sample containing rock, sediment, or a mixture thereof that does not contain oil or bitumen with an organic solvent; The method for separating and analyzing biomarkers according to claim 1.

5. In step a, an organic solvent is added to a sample containing oil, bitumen, or a mixture thereof, and then ultrasonic pulverization is performed; The method for separating and analyzing biomarkers according to claim 1.

6. Step b uses filtration; The method for separating and analyzing biomarkers according to claim 1.

7. Step c is carried out by mixing the sample with a non-polar organic solvent; The method for separating and analyzing biomarkers according to claim 1.

8. Step d involves first separating a group containing saturated hydrocarbons from the sample using a first solvent, second separating a group containing aromatic hydrocarbons using a second solvent, and third separating a group containing NSO compounds using a third solvent. The method for separating and analyzing biomarkers according to claim 1.

9. The first solvent is an organic solvent having a polarity index of 0 to 0.5, the second solvent is an organic solvent having a polarity index of 1 to 3, and the third solvent is an organic solvent having a polarity index of 4 to 5. The method for separating and analyzing biomarkers according to claim 8.

10. Step e is carried out using GC or GC / MS; The method for separating and analyzing biomarkers according to claim 1.

Images