Chapter 11 – General Methods 234
Chapter 11
General Methods
11.1 Extraction tecniques
- HS-SPMEThe analysis of volatile compounds performed using the Solid-Phase Micro-Extraction (SPME) technique, using a Supelco SPME devices coated with polydimethylsiloxane (PDMS, 100 μm). Fibres were conditioned prior to analyses, according to the manufacturer recommendations. A portion of the fresh plant material was put into a 100mL flask and allowed to equilibrate for 30 minutes at room temperature. The fibre was maintained over the sample for the necessary time (seconds or few minutes), according to the analyzed material. After the sampling time, the fibre was withdrawn into the needle, then transferred immediately to the injection port of the GC, and GC-MS systems, where the fiber was desorbed. The GC operating conditions were the same of those used for the identification and the quantification of the essential oil constituents (see 11.2), apart from the splitless injection mode and the injector temperature (250 °C).
- Hydrodistillation
The fresh or dry plant material was insert into a 2 liter balloon, 1 liter of water was added and the mixture boiled for 2 h using a Clevenger-type apparatus, according to the method recommended by the Italian Pharmacopoeia. After the hydrodistillation, the obtained essential oils were collected in vials and stored at -4 °C until analysis.
- Squeezing
This tecnique was used only for the cold extraction of the Citrus essential oils from their peels. The fruits were peeled, then the peels were squeezed manually inside a glass becker. The essential oil was recovered
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washing the inner walls of the becker using n-hexane. The recoveder essential oil wer collected in vials and stored at -4 °C unitl analysis.
- Direct Immersion
This technique was used for the evaluation of the Citrus juices previously obtained by squeezing of the fruits. Each juice sample was filtered with normal filter paper before the analysis. The DI technique were performed with Supelco SPME devices coated with DVB/CAR/PDMS 50/30um, Stableflex 24Ga, to evaluate the polar compounds the represent the aroma of the juice. The fibre was maintained inside each sample for the necessary time, according to the analyzed material.(usually 30 seconds). After the sampling time, the fibre was withdrawn into the needle, then transferred immediately to the injection port of GC. The same GC operating conditions were used for the identification and the quantification of the constituents (see 11.2), apart from the splitless injection mode and the injector temperature.
11.2 Analysis and evaluation
- GC-FID Analysis
The GC-FID analyses were accomplished using an HP-5890 Series II instrument equipped with FID and two cap. columns, an HP-WAX and an HP-5 (30 m0.25 mm i.d., film thickness 0.25 mm). The oven temp. was programmed isothermal at 608 for 10 min and then rising from 60 to 2208 at 58/min; injector and detector temp., 2508; carrier gas, N2 (2 ml/min); detector, dual FID; split ratio, 1:30; injection volume, 0.5
ml. For both columns, the identification of the components was performed by the comparison of i) their retention times (tR) with those of pure authentic samples and ii) their linear retention indices (LRIs,
determined relative to the tR of a series of n-alkanes) with those of the literature [28–33]. The relative
contents of the essential oil constituents, expressed as percentages, were obtained by FID peak-area normalization, all relative response factors being taken as one.
- GC/MS Analysis
The GC/MS analyses of the essential oils were carried out with a Varian CP-3800 gas chromatograph equipped with a DB-5 cap. column (30 m 0.25 mm i.d., film thickness 0.25 mm) and a Varian Saturn 2000 ion-trap mass selective detector. The oven temp. was programmed rising from 60 to 2408 at 38/min; injector temp., 2208; transfer-line temp., 2408; carrier gas, He (1 ml/min); injection volume, 0.2 ml (10% hexane solns.); split ratio, 1:30.
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- Caracterization of compounds
The identification of the constituents was based on the comparison of i) the retention time (tR)with those
of authentic samples, comparing their linear indices relative to a series of n-hydrocarbons, and on computer matching against commercial (NIST 98 and ADAMS) and also made possible by the use of a homemade library of mass spectra built up from pure substances and components of known oils, and MS literature data. Moreover, the molecular weights of all the identified substances were confirmed by GC-CIMS, using methanol as CI ionizing gas.
11.3 Statistical analysis
Data were analyzed in matrix taxa x chemical compounds (values under 1.5% were excluded for the purpose of statystical analysis). Before the Cluster Analysis (CA) and NMDS (Non-metric multidimesional scaling), each matrix were subjected to square-root transformation to underestimate the most abundant components, then were applied the Bray-Curtis index of similiraty (proper to matrix with biological data, caracterized by the presence of many zero).
The groups obtained with the CA were analyzed with the Good Clustering Level (GCL) method, analyzing the variability among and inside the groups. The statystical multivariate analysis were made with R 2.14.0 software (vegan package, J. Oksanen, F.G. Blanchet, R. Kindt, P. Legendre, P.R.Minchin, R.B. O’harar, G.L. Simposon, P. Solymos, M.H.H. Stevens, H. Wagner, vegan: Community Ecology Package, R package version 2.0-2, http://cran.r-project.org/package=vegan, 2011).
