# Production of Cellulosic Ethanol from Enzymatically Hydrolysed Wheat Straws
## 2 Materials and Methods
### Methods
#### 2.2.6 HPLC Instrumentation and Separation Conditions  
The individual phenolic compounds, organic acids and individual carbohydrates were analysed using a high-performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan) equipped with a LC-20 AD liquid chromatograph, SIL-20A autosampler, CTO-20 AC coupled with a SPD-M20A diode array detector (DAD) and RID-10A refractive index detector (RID) respectively. The separation of phenolic compounds, organic acids and individual carbohydrates was performed in a column specific to each constituent analysed. The standards of phenolic compounds, organic acids and individual carbohydrates were determined based on the retention times and quantified based on their calibration curves (all the curves had R\({}^{2}\) higher than 0.98). For samples, the limits of detection (LOD) and limits of quantification (LOQ) were calculated according to [PERSON] et al. (2018) [36; 37; 38]. Determination of Individual Phenolic Compounds  
For the analysis of the individual phenolic compounds resulting from the acid pretreatment and from the enzymatic hydrolysates, the following was performed: 1 mL of solution from each sample pretreated with acid and respectively 1 mL of solution from the enzyme-hydrolysed samples for 24 h were filtered through PTFE membrane with 0.45-\(\mu\)m dimension of pores and were stored at \(-\)20 \({}^{\circ}\)C until analysis. The obtained samples were analysed using HPLC-DAD. The separation of individual polyphenols was performed into a Phenomenex Kinetex(r) 2.6 \(\mu\)m Biphenyl 100 A column, LC Column 150 \(\times\) 4.6 mm and thermostated at 25 \({}^{\circ}\)C (Column oven). The utilised method of analysis was described by [PERSON] et al. (2011) and used by [PERSON] et al. (2020) with some modifications [39; 40; 41; 42]. The identification of the 12 phenolic compounds from pretreated and enzyme-hydrolysed samples of WS was performed at 280 nm for gallic, protocatechuic, vanillic and p-hydroxybenzoic acid; at 320 nm for chlorogenic, caffeic, p-coumaric and rosmarinic acid, quercetin, luteolin and kaempferol. The 12 phenolic compounds were injected individually to identify their retention time, then a mix of them was made. The final concentration of each identified phenolic compound was expressed in mg/L. Determination of Organic Acids  
In order to identify the organic acids resulting from the acid pretreatment and from the enzymatic hydrolysates, the following was performed: 1 mL of solution pretreated with acid samples and 1 mL of solution enzyme-hydrolysed samples for 24 h were filtered through PTFE membrane with 0.45 um dimension of pores and were stored at -20 \({}^{\circ}\)C until analysis. The obtained mixtures were analysed using HPLC-DAD equipment. The separation of organic acids was performed into a Phenomenex Kinetex(r) 5 um C18 100 A HPLC Column 250 \(\times\) 4.6 mm. The utilised method of analysis was described by [PERSON] et al. (2012, 2013). The standards of individual organic acid (gluconic, acetic, formic, succininic, propionic, lactic and butyric acid) and mixed solution of them were prepared to determine the concentration of organic acids, expressed in mg/L. Determination of Individual Carbohydrates After Enzymatic Hydrolysis  
After 24 h of enzymatic hydrolysis, 1 mL of each sample was filtered through PTFE 0.45 um dimension of pores and analysed for determination of carbohydrates using HPLC-RID. The separation of carbohydrates was performed in a Phenomenex Luna(r) Omega 3 um de SUGAR 100 A column, 150 \(\times\) 4.6 mm. The utilised method of analysis was described by [PERSON] et al. (2002) (2002). Before analysis, the inactivation of enzymes was achieved by exposing the samples for 5 min at 121 \({}^{\circ}\)C (2013).