2.4.5. Gas Chromatography-Olfactometry (GC-O) Analysis of HEO

GC–O is a hyphenated technique that allows the simultaneous analysis of volatile fractions by an instrumental detector and the human nose. This allows us to separate and recognize the “odor active” fractions, which contribute to the odor of the extract. The result of this analysis is a double plot called “aromagram” (Figure 1), which represents the response of both detectors, chromatographic peaks and sensory responses, indicated as OE (odor event). GC-O analyses were performed at CREA-IT laboratory, located in Milan. The system used was composed of an Agilent 6890 N GC equipped with an FID and a DB-1 capillary column (60 m × 0.25 mm i.d., 0.25 μm film thickness). Helium was used as carrier gas (1.3 mL min⁻¹). The injector and FID temperatures were set at 250 °C and the following column temperature program was applied: 40 °C for 5 min, 2.5 °C min⁻¹ up to 160 °C held for 5 min (duration: 58 min). The eluate was split 1:1 at the column outlet, linked to an olfactometric system that included the Olfactory Detector Port ODP2 Gerstel (Gerstel GmbH) equipped with the ODPneumatics module to control humidification and make up gas flows. The analyses were performed by using a direct intensity method described in [14]. The olfactometric data (intensity, duration and area of each odor event, OE) were collected through a potentiometer with the ODP recorder integrated with the GC software Chemstation Rev A 10.02. The area of each OE was calculated by the software from the intensity and duration values and shown as a chromatographic peak. The GC-O results were expressed as OE areas (A, average) and maximum intensity (Imax, median). The panel was composed of 7 panelists (1 male and 6 female), aged between 35 and 50 years, who were familiarized with the products. Before the analysis of the samples, all panelists attended two training sessions to learn to identify the main odor categories present in hop and beer products, using one representative compound for each of them. Solutions at different concentrations of the following standards were used: ethyl hexanoate (fruit), citronellol (citrus, flower), β-myrcene (resinous), α-humulene (woody), mesifurane (caramel). For each GC–O analysis, 2 panelists were involved, sniffing was divided into two parts of 25 min and each panelist participated in the sniffing of both parts. For hop analysis, oil was diluted in hexane (1:2 v/v) and 1.5 µL were injected in split mode (split flow: 12 mL min⁻¹, split ratio 1:10). GC-O results were related to composition data resulting from the GC-MS of HEO (Table 2).

A representative aromagram from Gas chromatography-olfactometry (GC-O) analysis of hop samples analyzed.

GC-O analysis of hop samples: odour descriptors, average peak areas, and maximum odour intensities.

¹ OE: odorous event; ² Kováts Index calculates using a linear series of n-alkanes. Differences between mean area values followed by different letters on the same row are significant (p < 0.05, LSD test); -: not detectable.

GC-MS was performed using an Agilent 5973N MSD connected to an Agilent 6890 GC, with the same column and chromatographic conditions of GC-O. The MS settings were as follows: filament voltage, 70 eV; scan range, m/z 45–800; scan speed, 1.4 scan/s; injector and interface temperature, 250 °C. Identification was performed by comparing mass spectra with those stored in databases (NIST 08 and Wiley 7 libraries), and comparing their Kováts indices, calculated using n-alkanes reference hydrocarbons, with tabulated Kováts indices.