Gas-chromatograph analysis

Quali-quantitative analysis of n-alkanes in the organic extracts was performed with an Agilent Technologies gas-chromatograph (GC) 6890N (Figure 34A) equipped with a HP-5 capillary column 30m x 0,250 mm (Figure 34B) and a flame ionization detector (FID). The gas-chromatograph analysis is a common type of chromatography used in analytical chemistry to separate components of a mixture, which can be vaporized without decomposition, thanks to their different affinities with a stationary or mobile phase. The mobile phase is usually an inert (non-reactive) carrier gas, while the stationary phase is a stable substance inside the chromatography column that consists in a long capillary tube wrapped on itself and placed inside the thermostat oven of the GC.

Figure 34: Agilent Technologies gas-chromatograph 6890N (A) equipped with a HP-5 capillary column (B).

The samples, consisting of the solvent extracts, are placed into 2 mL GC vials and loaded into the autosampler of the instrument. Autosampler carousel and robotic arm provide the sample transport to the inlet, which injects it into the chromatographic column through the continuous flux of gas carrier (nitrogen). Mobile phase and sample, mixed together, passing through the column come in contact with the stationary phase. During this step the components present in the mobile phase are separated according to their different affinity with the stationary phase and consequently leaving the column at different times (retention time): components with lower affinity come out first, while those with higher affinity have a greater retention time. The flame ionization detector (FID) burns the sample as it comes off the column; in fact the gas from the column is mixed with hydrogen and air and passing through a flame is subjected to a combustion reaction. This creates ions that produce an electrical current representing the signal provided by the FID. Several factors

A

B

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affect separation efficiency in GC, including column temperature and carrier gas flow rate. The main parameters, used in the oil hydrocarbons analysis, are reported in the following table (Table 6).

Gas chromatograph parameters Injection volume 1 l

Inlet

Temperature 270 °C

Pressure 19 psi

Gas flow rate 51.7 mL/min Column Initial pressure 19 psi

Detector

Temperature 320 °C H2 flow rate 30.0 mL/min Air flow rate 300 mL/min

Make up flow N2

Table 6 Gas chromatograph parameters for the analysis of n-alkanes.

The gas chromatograph provides a graph said chromatogram (Figure 35), which reports on the x-axis the retention time (in minutes), and on the y-axix the electrical signal (in mV) given by the detector. This graph is characterized by several peaks, which indicate the compounds present in the mixture; in this specific study the n-alkanes C10-C40.

Figure 35 Example of a chromatogram: n-alkanes (C13-C27), pristane (Pr) and phytane (Ph)

The chromatogram allows an immediate qualitative analysis by identifying the components present in the mixture, given the correspondence of their retention times with those of a standard mixture C10-C40.

Furthermore, it is possible to obtain a quantitative analysis through a calibration curve by considering the area of the individual peak proportional to the concentration of each n-alkane. A set of standard solutions of known concentration is prepared and GC-analyzed producing a series of measurements. The calibration curve is a graph where concentrations are plotted along the x-axis and the detected areas of each GC peak are plotted along the y-axis. The plot of instrument response (area) versus concentration shows a linear relationship. The data (concentrations of the analyte and the area for each standard) can be fit to a straight line, using linear regression analysis.

This yields a model described by the equation:

𝐴 = 𝑚𝐶 + 𝑞,

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where A = area of the chromatographic peak;

C = concentration;

m = slope (also called the regression coefficient);

q = intercept.

The peak area of the unknown sample is measured and using the calibration curve, it is interpolated to find the concentration. The analyte concentration (C) of unknown samples may be calculated from this equation.

A standard mixture of n-alkanes from C10 to C40 and a mixture containing C17, n-C18, pristane and phytane were used to obtain 6-point calibration curves (0.125, 0.25, 0.5, 1, 2.5 e 5 mg/L). Calibration curves were verified constantly.

Figure 36 shows the calibration curve of C10, as example.

Figure 36 Calibration curve of C10

Attention was placed on pristane (Pr) and phytane (Ph), two aliphatic hydrocarbons having 19 and 20 carbon atoms, respectively. Based on their retention times, the pristane comes off the chromatography column immediately after the C17, while the phytane immediately after the C18. These two molecules are recalcitrant to degradation under anaerobic conditions, so their concentration should remain almost constant over time. The concentration of pristane and phytane was used for the normalization of the results in anaerobic conditions (Nikolopoulou and Kalogerakis, 2009). Conversely, because under aerobic conditions these two compounds are more easily biodegraded, the normalization of the results for the aerobic sets was made using another more recalcitrant compound, the decamethyl anthracene (DMA), which was amended during the microcosms sets up. A standard sample of DMA was used to obtain 5-point calibration curve (1, 2.5, 5, 10, 20 mg/L).

For each sample, after the identification of the peaks of the n-alkanes (from C10 to C40), Pr and Ph (for anaerobic set) and DMA (for aerobic sets), the corresponding areas were detected in order to calculate the concentrations (mg/L) through the

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The n-alkanes concentrations (g n-alkanes/kg sediment) were obtained using the following formula:

𝐶(_𝑘𝑔^𝑔) = 𝐶(^𝑔_𝐿) ∙ 𝑉_{𝑠𝑜𝑙𝑣𝑒𝑛𝑡}(𝐿) ∙_{𝐷𝑊 (𝑘𝑔)}¹

where, Vsolvent= volume of solvent hexane:DCM used during the extraction (L);

DW = dry weight of the sediment (kg),

C (g/L) = hydrocarbon concentration (g/L) obtained from the calibration curve.

The concentration g n-alkanes/kg sediment was normalized using the concentration of Pr and Ph (for anaerobic test) and DMA (for aerobic sets) with the formula:

𝐶_{𝑛𝑜𝑟𝑚}= _{𝑃𝑟+𝑃ℎ}^𝐶 ∙ 𝐴(𝑃𝑟, 𝑃ℎ) and 𝐶_{𝑛𝑜𝑟𝑚}= _𝐷𝑀𝐴^𝐶 ∙ 𝐴(𝐷𝑀𝐴) , where C = concentration of n-alkanes (g/kgsediment);

Pr = concentration of pristane (g/kgsediment);

Ph = concentration of phytane (g/kgsediment);

A (Pr, Ph) = average concentration of Pr and Ph obtained from the triplicate sterile microcosms in the first sampling (g/kgsediment);

DMA = concentration of DMA (g/kgsediment);

A (DMA) = average concentration of DMA obtained from the triplicate sterile microcosms in the first sampling (g/kgsediment).

For each sample, the normalized concentrations of each n-alkane (C10-C40) were added, obtaining the total concentration of n-alkanes, normalized to Pr and Ph (anaerobic set) or DMA (aerobic sets).

Moreover, for each microcosm were calculated the C/C0 ratio, where C is the normalized concentrations at a determined sampling time and C0 the concentration at the first sampling (time zero) or in the sterile un-amended control. For the different experimental conditions, it was so possible to evaluate the changes of the hydrocarbons concentration (C/C0) over time. All the data presented are the mean of replicate microcosms (± standard deviation).