Residual sugars (glucose and fructose), glycerol, ethanol and acetic acid from the fermentation end point samples were determined by HPLC (Thermo Fisher Scientific, Waltham, MA. USA) using a refraction index detector and a HyperREZTM XP Carbohydrate H+ 8 μm column (Thermo Fisher Scientific) equipped with a HyperREZTM XP Carbohydrate Guard (Thermo Fisher Scientific). Samples were diluted 3-fold, filtered through a 0.22-μm nylon filter (Symta, Madrid, Spain) and injected in duplicate. The analysis conditions were: eluent, 1.5 mM of H2SO4; 0.6 ml min-1 flux and a 50°C oven temperature.
Hyperrez xp carbohydrate h 8 μm column
Manufactured by Thermo Fisher Scientific
Sourced in United States
The HyperREZTM XP Carbohydrate H+ 8 μm column is designed for the separation and analysis of carbohydrates. It has a particle size of 8 μm and is compatible with aqueous mobile phases.
Automatically generated - may contain errors
6 protocols using hyperrez xp carbohydrate h 8 μm column
1
Quantification of Fermentation Byproducts
2
Quantification of Fermentation Byproducts
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Residual sugars (glucose and fructose), glycerol, ethanol and acetic acid from the fermentation end point samples were determined by HPLC (Thermo Fisher Scientific, Waltham, MA. USA) using a refraction index detector and a HyperREZTM XP Carbohydrate H+ 8μm column (Thermo Fisher Scientific) equipped with a HyperREZTM XP Carbohydrate Guard (Thermo Fisher Scientific). Samples were diluted to maintain our target compounds within the allowed range of detection, filtered through a 0.22 μM nylon filter (Symta, Madrid, Spain) and injected in duplicate. The analysis conditions were: eluent, 1.5 μM of H2SO4; 0.6 mL/min flux and a 50°C oven temperature.
3
HPLC Analysis of Sugars and Organic Acids
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Sugars and organic acids were extracted and determined essentially as described in Zacarías-García et al. [31 (link)]. Sugars and organic acids content was measured by HPLC (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a refraction index detector. For glucose, fructose, citric acid, malic acid, quinic acid, and succinic acid, separation and determination were employed in a HyperREZTM XP Carbohydrate H+ 8 μm column (Thermo Fisher Scientific). For sucrose determination, a Pb column was used (Hi-Plex Pb, 300 × 7.7 mm, Agilent Technologies). Chromatographic conditions used are described in Pérez-Través et al. [48 (link)]. The identification and quantification of sugars and organic acids were carried out by comparison with the retention times of authentic standards (Sigma-Aldrich, Barcelona, Spain). Two independent replicates of each sample were analyzed, and the results are expressed as mean ± standard deviation.
4
HPLC Analysis of Fermentation Supernatants
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The supernatants recovered from micro-fermentations were filtered by 0.22 µm nylon pore (Symta, Madrid, Spain) and poured in vials suitable for high performance liquid chromatography HPLC analysis. The chromatograph is equipped with a refraction index detector (Thermo Fisher Scientific, Waltham, MA, USA) and a HyperREZTM XP Carbohydrate H + 8 μm column protected with a HyperREZTM XP Carbohydrate Guard (Thermo Fisher Scientific). The analysis was performed with a mobile phase composed of 1.5 mmol/L H2SO4 aqueous solution, pH 2.5 ± 0.1, at 0.6 mL/min and 50 °C. The analysis was performed by duplicate.
5
Evaluating Carbon Source Utilization in Y. lipolytica
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Y. lipolytica strain precultures were grown overnight, then centrifuged and washed two times with YNB supplemented with NH4Cl and phosphate buffer (YNB N and P). They were then resuspended in 1 mL of YNB N and P. Cultures were inoculated at OD600 nm = 0.1 in 50 mL of YNB medium supplemented with 50, 100, or 200 mmol/L of phosphate buffer (pH = 6.8) depending of the quantity of substrate (1, 2, or 4) and 170 mmol/L each of fumarate, lactate, malate, and succinate, then incubated at 28°C with agitation (160 rpm). To analyze the consumption of carbon sources, a sample of each culture was centrifuged for 1 min at 13 000 rpm; the resulting supernatant was analyzed with HPLC (UltiMate 3000; Dionex-Thermo Fisher Scientific, UK) using a HyperREZ XP carbohydrate H+ 8-μm column (Thermo Fisher Scientific, Villebon sur Yvette, France) coupled to a UV (210 nm) detector. The column was eluted with 0.01 N H2SO4 at room temperature and a flow rate of 0.6 mL min−1. Standard samples were used to determine the quantity of each compound. Prior to HPLC analysis, samples were filtered on 0.45-μm pore-size membranes.
6
Microvinification Metabolite Analysis
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The glucose, fructose, glycerol, and ethanol at the end point of microvinifications were determined by HPLC (Thermo Fisher Scientific, Waltham, MA, USA) using a refractive index detector and a HyperREZ ™ XP Carbohydrate H + 8 μm column (Thermo Fisher Scientific), equipped with HyperREZTM XP Carbohydrate protection (Thermo Fisher Scientific). Samples were diluted 5 times, filtered through a 0.22 mm nylon filter (Symta, Madrid, Spain) and injected in duplicate. The analysis conditions were: eluent, 1.5 mM H 2 SO 4 ; flow of 0.6 ml min -1 and the stove temperature was 50 °C.
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