The analysis of organic biomarkers in chert samples offers key information about the environmental conditions in which these samples were formed, and this information can be used to track the lithic materials of many archaeological artifacts. Since the content of the organic fraction is very low and the complexity of the organic extracts is quite high, we have optimized the GC×GC separation of these mixtures. Making use of mixture of C(16)H(34)-C(44)H(90)n-alkane standards, a central composite design was carried out taking into account the carrier flow in the first and second columns, the modulation period and the discharge time. Regarding the measured responses, though the initially considered one was the peak volume, we have also evaluated the effects on the number of modulated peaks per analyte, the symmetry of the modulated peaks and the number of detected compounds. The final optimum conditions were defined as follows: a hydrogen flow of 1.2 mL/min in the first column and 18 mL/min in the second one, a modulation period of 1.4 s and a discharge time of 0.1 s and under these conditions all the response variables showed optimum values. Based on this optimized method several chert samples obtained from different stratigraphic levels in an ancient quarry were studied and we were able to distinguish them on the basis of the different constituents of organic biomarkers, such as mono-methylated alkanes, cyclic n-alkanes, branched alkanes, steranes and hopanes.
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