# Effect of Seawater Irrigation on the Sugars, Organic Acids, and Volatiles in 'Reliance' Grape
## 2 Materials and Methods
### Extraction of Aromatic Compounds and Analysis  
The extraction and determination of volatile compounds followed the method described by [PERSON] [7] with minor modifications. Briefly, 100 g of de-seeded grape berries were ground into powder in liquid nitrogen followed by the addition of 0.5 g of PVPP (Crosslinked Polyvinylpyrrolidone, to inhibit oxidation of phenols) and 0.5 g of D-glucose acid lactone (to inhibit glycosidase activity). The mixture was left to stand for 4 h at 4 \({}^{\circ}\)C and then centrifuged at 4000 rpm at 4 \({}^{\circ}\)C for 15 min to collect the supernatant (juice). Subsequently, 10 mL of juice was mixed with 1 g of NaCl powder (to prevent sample browning) and 3 \(\mu\)L of the internal standard (2-octanol, 0.822 g L\({}^{-1}\)) in a 20 mL vial capped with a PTFE-silicon septum. \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline
**Na\({}^{+}\) (mg L\({}^{-1}\))** & **Ca\({}^{2+}\) (mg L\({}^{-1}\))** & **Mg\({}^{2+}\) (mg L\({}^{-1}\))** & **Cl\({}^{-}\) (mg L\({}^{-1}\))** & **SO4\({}^{2-}\) (mg L\({}^{-1}\))** & **Degree of Mineralization (g L\({}^{-1}\))** & **pH** \\ \hline
11,041 & 300 & 1627 & 19,241 & 2498 & 1200 & 7.9 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Physical and chemical properties of original concentration seawater. \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline
**Date** & **May** & **May** & **Jun.** & **Jun.** & **Jun.** & **Jun.** & **Jul.** & **Jul.** & **Jul.** \\  & **29 th** & **31 st** & **12 th** & **14 th** & **26 th** & **28 th** & **5 th** & **12 th** & **19 th** \\ \hline initial irrigating time & Control, S1 & & S2 & & S3 & & & & \\ sampling time & & EL 31 & & EL 33 & & EL 35 & EL 36 & EL 37 & EL 38 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Specific date of initial irrigation time and sampling time in 2018. An auto headspace solid-phase microextraction (HS-SPME) was then used to extract volatile compounds on an AOC-6000 autosampler (Shimadzu, Kyoto, Japan). The SPME fiber (50/30 \(\upmu\)m CAR/DVB/PDMS, Supelco, Bellafonte, PA, USA) was preconditioned in the injection port of the GC before the extraction following the manufacturer's instructions. The vial contents were continuously stirred for 30 min at 35 \({}^{\circ}\)C followed by exposure of the SPME fiber to the headspace for 40 min while maintaining the sample at 35 \({}^{\circ}\)C. After sampling, the SPME fiber was then inserted into the GC injector after sampling and left for 5 min at 250 \({}^{\circ}\)C to allow for thermal desorption of the analytes. A GCMS-TQ8050 system (Shimadzu, Kyoto, Japan) was employed to analyze the volatile compounds. A capillary column (RTX-5 MS, 60 m \(\times\) 0.25 mm \(\times\) 0.25 \(\upmu\)m, Shimadzu, Kyoto, Japan) was used to separate the volatiles at a1 mL min\({}^{-1}\) flow rate of the carrier gas (Helium). The injection mode was splitless at an injector temperature of 250 \({}^{\circ}\)C. The oven temperature was set at 40 \({}^{\circ}\)C for 2 min, then heated to 230 \({}^{\circ}\)C at a speed of 5 \({}^{\circ}\)C min\({}^{-1}\), and finally maintained at 230 \({}^{\circ}\)C for 5 min. The mass spectrometer interface and ion source temperature was set at 250 \({}^{\circ}\)C and 230 \({}^{\circ}\)C, respectively. The mass spectra were acquired using an electron ionization mode (EI) at an electron multiplier voltage of 70 eV and a full mass scan of \(m/z\) 30-450. Each compound was identified using the NIST and Wiley 2 libraries. A series of C7-C27 n-alkane standards (Supelco, Bellefonte, PA, USA) were determined under similar chromatographic conditions to calculate their retention indices (RI). Volatile compounds were identified by comparing their RI and mass spectra with the NIST2011 library. A synthetic juice matrix (pH = 3.2, citrate-phosphate buffer solution) was prepared for the qualification of volatile compounds. Standards were first dissolved in methanol, mixed, and then added into the synthetic juice matrix.