# Single-Stage Fractionation of Vine Shoots Using Microwave Heating
## 2 Materials and Methods
### Analytical Procedures  
The compositions of the feedstock and processed solid samples were assayed according to the following standard methods: extractive content, NREL/TP-510-42619; polysaccharides and Klason lignin, NREL-510-42618; and ash, NREL/TP-510-42622 [31; 32; 33]. The method NREL-510-42618 consisted of a two-step quantitative acid hydrolysis, performed with 72% and 4% H\({}_{2}\)SO\({}_{4}\), separately, and led to an insoluble lignin residue (Klason lignin, KL) and to a liquid phase. KL was measured gravimetrically after oven drying at 105 \({}^{\circ}\)C. Acid soluble lignin (ASL) was determined spectrophotometrically at 205 nm. Samples from the liquid phase were analyzed by HPLC using an Agilent 1200 series instrument (Agilent Technologies, Santa Clara, CA, USA), fitted with a refractive index detector (RID). Aliquots were assayed for monosaccharides, acetic acid, and furans (furfurral and hydroxymethylfurral, HMF) using a 300 \(\times\) 7.8 Aminex HPX-87H column (BioRad Life Science Group, Hercules, CA, USA) kept at 50 \({}^{\circ}\)C and eluted with 0.003 N H\({}_{2}\)SO\({}_{4}\) at 0.6 mL\(\cdot\)min\({}^{-1}\). Additional aliquots were neutralized with BaCO\({}_{3}\) and assayed for monosaccharides (glucose, xylose, galactose, arabinose and mannose) using a CARBOSep CHO-682 column(Transgenomic Inc. Omaha, NE, USA) kept at 80 \({}^{\circ}\)C, using water as a mobile phase (flow rate, 0.4 mL\(\cdot\)min\({}^{-1}\)). Analyses were performed in triplicate.  
Aqueous samples from the fractionation, membrane and enzymatic stages were subjected to quantitative post-hydrolysis (4% of H\({}_{2}\)SO\({}_{4}\) at 121 \({}^{\circ}\)C). The OS concentrations and their degree of substitution with acetyl groups (AG) were calculated from the increases in the amounts of monosaccharides and acetic acid upon post-hydrolysis. The content of total non-volatile compounds (NVC) was measured by oven-drying at 105 \({}^{\circ}\)C. The content of the fraction named \"other non-volatile compounds\" (ONVC) was calculated as the difference between NVC and the non-volatile compounds identified by HPLC. Analyses were performed in triplicate.  
Hemicellulose-derived products in the aqueous phase from fractionation, membrane processing and enzymatic treatments were characterized using the following methods:  
* HPLC, to assess the type and amount of OS structural units and substituents;
* High performance size exclusion chromatography (HPSEC), to assess the DP distribution of OS;
* High performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), to provide a quantitative assessment on the small-DP OS derived from hemicelluloses;
* Matrix-assisted laser desorption and ionization time of flight mass spectrometry (MALDI-TOF-MS) to elucidate the DP and substitution pattern of OS composed of backbones containing between 3 and 7 anhydro xylose units.  
HPLC was performed using the method indicated above. HPSEC was conducted with two TSK Gel G3000 PWXL and G2500 PWXL columns in series combined with a PWX-guard column (Tosoh bioscience, Stuttgart, Germany) [34]. HPAEC-PAD was conducted using an ICS3000 chromatographic system (Dionex, Sunnyvale, CA, USA) equipped with a CarboPac PA guard column and a CarboPac PA-1 column [35]. MALDI TOF-MS analyses were performed using an Autoflex III Smartbeam Mass Spectrofotometer (Bruker Daltonics, Bremen, Germany). The spectra were acquired and treated using the Flex control 3.0 and Flex-Analysis 3.0 software (Bruker Daltonics) [36].