Research vineyards are located at NE Hansen research farm at South Dakota State University (SDSU, Brookings, SD, USA) and Iowa State University Horticulture Farm (ISU, Ames, IA, USA). Cultivars were grown in a randomized complete block design in both test vineyards in 2008 as part of the NE-1020 Cold Hardy Wine Grape Cultivar Trial. Grape clusters were randomly selected and tagged, so the same clusters were sampled from veraison to harvest for VOCs emitted in vivo from ripening grapes. Two in vivo sampling methods utilizing SPME were used, namely, extractions from whole air chambers enveloping ripening clusters in the 2012 season and vacuum-assisted extractions from single berries using modified glass vials in the 2013 season, respectively. Volatiles present in the headspace of crushed berries (i.e., skin, pulp, and seeds) were also investigated for the 2012 and 2013 growing seasons for comparisons with in vivo sampling. Berries from each cultivar were collected on the same day as in vivo sampling, collected from untagged clusters, crushed, and analyzed in comparison. These berries were kept in the dark and frozen at −20 °C for batch analysis after harvest.
A 65 µm polydimethylsiloxane/divinylbenzene (PDMS/DVB) fused silica SPME fiber (P/N 57310-U, Sigma-Aldrich, St. Louis, MO, USA) was conditioned according to the manufacturer’s specifications. Cleaned fibers were stored and transported in aluminum foil packets and sealed in mason jars. Foil and mason jars had been thermally cleaned overnight in an oven (at 110 °C) to minimize interferences from the environment. After sampling, described in the following sections, SPME fibers are wrapped in clean aluminum foil, placed and sealed in clean glass mason jars, and transported on ice packs to the lab for analysis on the same day. Samples of the volatiles collected on SPME fibers in Brookings, SD were prepared and used in the same manner and sent to Ames, IA via an overnight carrier for analyses. Volatiles emitted from whole clusters of Frontenac and Marquette cultivars were investigated for the 2012 growing season in both Iowa and South Dakota. Volatiles emitted from single berries of St. Croix and La Crescent cultivars were investigated for the 2013 growing season.
3.5. Volatiles Released from Crushed Grape Berries
Berries were collected from each cultivar on the same day and time of in vivo sampling. Five berries were collected from clusters adjacent to the cluster tagged for in vivo sampling (i.e., from the same vine but a different cluster than the in vivo sampled berries). Collected berries were frozen prior to analysis and stored in a −20 °C freezer. Berries collected in South Dakota were also frozen and shipped on blue ice blocks overnight for analysis in Iowa. Frozen berries were hand-crushed in the lab, placed into 20 mL amber screw top vials (P/N: 16-6000, Wheaton, Millville, NJ USA) with PTFE/silicone septa. A CTC CombiPal (LEAP Technologies, Carrboro, NC, USA) was used for automated SPME sampling. Briefly, the vials were agitated and heated to 50 °C for 10 min, followed by 30 min agitated headspace sampling using a 65 μm PDMS/DVB SPME fiber. The fiber was thermally desorbed under a flow of helium prior to each sample exposure. Optimized SPME parameters (sampling time, extraction temperature) were determined, data not shown.
3.6. Data Acquisition and Analysis
A custom multidimensional GC was used (Microanalytics, a part of Volatile Analysis Corporation, Round Rock, TX, USA), built on a standard Agilent 6890 platform (Agilent Technologies, Santa Clara, CA, USA). System automation and data acquisition software were MultiTrax v. 6.00.1 (Microanalytics, Round Rock, TX, USA) and ChemStation E.01.01.335 (Agilent Technologies, Santa Clara, CA, USA). Chromatography was performed on two capillary columns connected in series. The use of conventional retention indexes (e.g., Kovats retention index) is not appropriate for identification in this type of column configuration. The first column was 5% phenyl polysilphenylene-siloxane (30 m × 0.53 mm inner diameter × 0.5 μm thickness, Trajan Scientific, Austin, TX, USA) with a fixed restrictor pre-column. The second polar column was bonded polyethylene glycol in a Sol-Gel matrix (30 m × 0.53 mm inner diameter × 0.5 μm thickness, Trajan Scientific, Austin, TX, USA). The midpoint between the two columns was maintained at a constant pressure of 0.39 atm by a pneumatic switch. In this research, all effluent from the first column was directed into the second analytical column, that is, no heartcutting was performed. True multidimensional analyses were not performed (i.e., the system was used in full heartcut mode), meaning separation was performed on both columns in series. Effluent from the second polar column was simultaneously directed to a single quadrupole mass spectrometer (MS) (Model 5973N, Agilent Technologies, Santa Clara, CA, USA) and an olfactometry (sniff) port via an open spit interface at atmospheric pressure. The sniff port was equipped with a purge flow controller and supplied with humidified air at 0.54 atm. Flow to the MS and sniff port was determined by fixed restrictor columns, one part to the MS and three parts to the sniff port. Olfactometry was not utilized in the research. The GC inlet was operated in splitless mode at 250 °C. GC oven parameters started with an initial temperature of 40 °C, held for 3.0 min, followed by a 7 °C per min ramp to 240 °C, held for 8.43 min. Total run time was 40 min. Carrier gas was ultra-high purity (UHP) helium (99.999%, Airgas, Des Moines, IA, USA). Temperatures of the sniff port and MS transfer lines were 240 °C and 280 °C, respectively. MS full scan range was set from 34 m/z to 350 m/z. Scans were collected in electron ionization (EI) mode with an ionization energy of 70 eV. MS heated zones for quadrupole and source were 150 °C and 230 °C, respectively. Daily tuning of the MS was performed with perfluorotributylamine (PFTBA) before each analysis.
Identification of compounds was performed using the Automated Mass Spectral Deconvolution & Identification System (AMDIS) target library search with at least 80% mass spectral match [8
]. Target libraries included (a) the 6 libraries that are included with the AMDIS program, (b) an onsite library created from analysis of pure standards (200+ compounds), and (c) NIST11 mass spectral library. Retention times were also verified with pure standards.