Small-Scale Column Chromatography Separates Hopanes in Crude Oil and Rock Extracts with Higher Purity for GC–MS Analysis

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The reliability of column chromatography for GC–MS analysis of hopanes was tested in oil samples of three different types, using neutral alumina as a solid-phase adsorbent and a Pasteur pipette as a separation device.

Research by Zhongdeng Lu, of China University of Geosciences in Wuhan, China, and Zulin Chen and others, of Yangtze University in Wuhan and Jingzhou, China, has demonstrated the effects of solid-phase adsorbent type and column length on the separation of hopanes using small-scale column chromatography, showing the superior effectiveness of this method in comparison to more widely used approaches.

For the study, published in the Journal of Chromatography A, three different types of oil samples (normal crude oil, degraded crude oil, and rock extracts), were selected with the intention of separating their hopanes, an essential class of biomarkers present in petroleum and other geological samples, which commonly diagnose paleoenvironmental changes (1). Gas chromatography–mass spectrometry (GC–MS) has been used in the past to detect hopanes in saturated hydrocarbons, but challenges remain in separating high-purity hopanes even with gas chromatography coupled to isotope ratio mass spectrometry (GC–IRMS).

Hopanes are cyclic organic compounds that contain a tetracyclic structure with four six-membered rings and one five-membered ring. They are found in petroleum and are considered to be one of the most important biomarkers in petroleum geochemistry. Hopanes are derived from the biological material present in the source rocks and can provide valuable information about the depositional environment, thermal maturity, and the origin of the petroleum. They are resistant to thermal and biodegradative processes, which makes them ideal for studying the history of petroleum. The distribution and abundance of hopanes can also help in identifying the source of oil spills and in oil exploration.

Previous research has reported two predominant separation methods for hopanes. One is molecular sieve adsorption, in which ultrastable, Y-type molecular sieves are used as a stationary phase for separating steranes and hopanes using high performance liquid chromatography (HPLC). The other method, urea complexation, is quicker, more convenient, and therefore more widely used, but tends to be less pure and contain a higher quantity of steranes. The researchers in this study sought an even faster, straightforward pretreatment method that could also stand up to carbon isotope detection.


The method of small-scale column chromatography examined in this study was characterized by 100-200 µm neutral alumina as a solid-phase adsorbent and a Pasteur pipette with a length of 180 mm. After the three oil samples were selected, they were eluted with mixed reagents (V hexane: V petroleum ether = 8:2), with ten fractions being collected at intervals of 0.5 mL and tested for compounds using GC–MS. The elution order of biomarkers in the chromatographic column was n-alkanes, steranes, and hopanes, with the fourth and fifth fractions demonstrating a higher purity of separated hopanes compared to urea complexation.

Further, the concentration of n-alkanes was reduced using the small-scale method from 1.99 to 4.83 mg/mL to 0.79 to 0.94 mg/mL, and the concentration of steranes was reduced from 12% to 0.45%. Residual n-alkanes and steranes were also not visible in the original GC–MS detection. Regarding the carbon isotopes of hopanes, the study found that when column length was increased, the C29Ts/C29αβ ratio of hopanes decreased gradually from 1.63 to 0.73. The researchers speculated that the reasons for the variation were maturity combined with oil and gas migration.

Because studying carbon isotopic composition of hopanes can provide clues to the depositional environment of sediments and their biological origin, this new approach to compound purification using small-scale column chromatography may be more effective than other techniques for analyzing stable isotopes.


(1) Lu, Z.; Chen, Z.; Liu, Y.; et al. A small-scale neutral alumina column chromatography method for carbon isotope determination of hopanes in crude oil or rock extracts. J. Chromatogr. A 2023, 1689, 463729. DOI: 10.1016/j.chroma.2022.463729