# Analytical Methods to Assess Polyphenols, Tannin Concentration, and Astringency in Hard Apple Cider
## 2 Materials and Methods
### Polyphenol, Astringency, and Tannin Analyses
\begin{table}
\begin{tabular}{c c c c c c} \hline
**Cultivar Name** & **Sugar Juice [°Bx]** & **Titatable Acidity [g/L]** & **pH Juice** & **pH Cider** & **Alcohol Cider [\%vol]** \\ \hline Black Limbertwig (JCA) & 12.7 \(\pm\) 0.6 & 6.0 \(\pm\) 0.3 & 3.40 \(\pm\) 0.17 & 3.44 \(\pm\) 0.10 & 6.99 \(\pm\) 0.07 \\ Black Winesap (DA) & 15.5 \(\pm\) 0.8 & 5.6 \(\pm\) 0.3 & 3.50 \(\pm\) 0.18 & 3.56 \(\pm\) 0.11 & 8.53 \(\pm\) 0.09 \\ Chestnut Crab (CFA) & 16.0 \(\pm\) 0.8 & 6.2 \(\pm\) 0.3 & 3.71 \(\pm\) 0.19 & 3.75 \(\pm\) 0.11 & 8.80 \(\pm\) 0.09 \\ Fugi (DA) & 14.1 \(\pm\) 0.7 & 3.6 \(\pm\) 0.2 & 3.76 \(\pm\) 0.19 & 3.81 \(\pm\) 0.11 & 7.76 \(\pm\) 0.08 \\ Geneva Crab (CFA) & 8.4 \(\pm\) 0.4 & 6.5 \(\pm\) 0.3 & 3.20 \(\pm\) 0.16 & 3.22 \(\pm\) 0.09 & 4.62 \(\pm\) 0.05 \\ Golden Delicious (DA) & 12.3 \(\pm\) 0.6 & 1.9 \(\pm\) 0.1 & 3.93 \(\pm\) 0.19 & 3.40 \(\pm\) 0.10 & 6.77 \(\pm\) 0.07 \\ Golden Russet (JCA) & 16.4 \(\pm\) 0.8 & 5.2 \(\pm\) 0.3 & 3.58 \(\pm\) 0.18 & 3.60 \(\pm\) 0.11 & 9.02 \(\pm\) 0.09 \\ Gragg (JCA) & 11.2 \(\pm\) 0.6 & 4.3 \(\pm\) 0.2 & 3.62 \(\pm\) 0.18 & 3.44 \(\pm\) 0.10 & 6.16 \(\pm\) 0.06 \\ Hewes Crab (CFA) & 16.8 \(\pm\) 0.8 & 7.4 \(\pm\) 0.4 & 3.36 \(\pm\) 0.17 & 3.42 \(\pm\) 0.10 & 9.24 \(\pm\) 0.09 \\ [PERSON] (JCA) & 13.1 \(\pm\) 0.6 & 6.2 \(\pm\) 0.3 & 3.36 \(\pm\) 0.17 & 3.34 \(\pm\) 0.10 & 7.21 \(\pm\) 0.07 \\ [PERSON] (JCA) & 12.6 \(\pm\) 0.6 & 3.0 \(\pm\) 0.2 & 3.36 \(\pm\) 0.19 & 3.65 \(\pm\) 0.11 & 6.93 \(\pm\) 0.07 \\ Nevertown Pippin (DA) & 14.6 \(\pm\) 0.7 & 6.6 \(\pm\) 0.3 & 3.39 \(\pm\) 0.17 & 3.42 \(\pm\) 0.10 & 8.03 \(\pm\) 0.08 \\ Notely P. (JCA) & 10.9 \(\pm\) 0.5 & 7.8 \(\pm\) 0.4 & 3.16 \(\pm\) 0.16 & 3.13 \(\pm\) 0.09 & 6.00 \(\pm\) 0.06 \\ Red Delicious (DA) & 12.1 \(\pm\) 0.6 & 2.6 \(\pm\) 0.1 & 3.62 \(\pm\) 0.18 & 3.56 \(\pm\) 0.11 & 6.66 \(\pm\) 0.07 \\ [PERSON] (DA) & 12.2 \(\pm\) 0.6 & 2.7 \(\pm\) 0.1 & 3.73 \(\pm\) 0.19 & 3.40 \(\pm\) 0.10 & 6.71 \(\pm\) 0.07 \\ Royal Limbertwig (JCA) & 11.1 \(\pm\) 0.6 & 5.4 \(\pm\) 0.3 & 3.41 \(\pm\) 0.17 & 3.42 \(\pm\) 0.10 & 6.11 \(\pm\) 0.06 \\ [PERSON] (DA) & 16.4 \(\pm\) 0.8 & 7.2 \(\pm\) 0.4 & 3.38 \(\pm\) 0.17 & 3.49 \(\pm\) 0.11 & 9.02 \(\pm\) 0.09 \\ Summer Banana (DA) & 14.3 \(\pm\) 0.7 & 4.0 \(\pm\) 0.2 & 3.66 \(\pm\) 0.18 & 3.52 \(\pm\) 0.11 & 7.87 \(\pm\) 0.08 \\ Summer Rambo (JCA) & 12.1 \(\pm\) 0.6 & 8.0 \(\pm\) 0.4 & 3.19 \(\pm\) 0.15 & 3.25 \(\pm\) 0.09 & 6.66 \(\pm\) 0.07 \\ \hline \end{tabular}
\end{table} 
Table 1: Apple cultivars included in this study, in alphabetic order with all relevant juice and cider data (JCA: Juice/cider apple; DA: Dessert apple; CrA: Crab apple). phase A (10 mM KH\({}_{2}\)PO\({}_{4}\)/H\({}_{3}\)PO\({}_{4}\) buffer at pH 1.5 in 5% acetonitrile in water; mobile phase B: 10 mM KH\({}_{2}\)PO\({}_{4}\)/H\({}_{3}\)PO\({}_{4}\) buffer at pH 1.5 in 50% acetonitrile in water), decreased to 85% by 7 min, from 85% to 70% after 15 min, from 70% to 60% by 20 min, from 60% to 40% by 24 min, and to 10% after 27 min. After 30 min, it was then set back to initial conditions (100% mobile phase A) for three minutes to reach equilibrium (total analysis time 33 min). For the detection of various phenolic compounds in cider, the detection wavelengths were set to 280 nm and 320 nm. Selected compounds were quantified with an external calibration using pure standards (all purchased from VWR, Radnor, PA, USA). The limits of quantification were determined by setting the threshold to a signal to noise ratio of 10:1. Unknown peaks were identified based on the retention time of known standards and spectral information from the literature. Following the recommendation of [PERSON], [PERSON], [PERSON], [PERSON], [PERSON] and [PERSON] (2015), unknown compounds were appointed to known molecules if they demonstrated spectral similarities; for example, a compound was labelled as \(p\)-coumaric acid derivative if the spectrum matched that of \(p\)-coumaric acid but the retention time did not.