Ketoreduction reactions were modified from a method described previously (Piasecki et al., 2011 ) with 5 mM 3-ketopentanoyl-S-NAC and 5 µM ketoreductase in a 250 mM NaCl, 10% v/v glycerol, 125 mM HEPES pH 7.5 buffer containing an NADPH-regeneration system (200 mM d -glucose, 200 µM NADP+, 1 µM B. subtilis glucose dehydrogenase). The reactions were incubated overnight at 22 °C, extracted with ethyl acetate (3 × 500 µL) and dried in a speedvac. All samples were resuspended in ethanol prior to chromatographic analysis. Chiral chromatography was performed with a ChiraCel OC-H column (250 × 4.6 mm) with a Beckman Coulter System Gold 126 pump and a System Gold 166 PDA detector equipped with a 20 µL loop. Absorbance was monitored at 235 nm. The solvent system used was 7% ethanol in hexanes at 0.8 mL/min. Enantiomeric excess was determined from peak area integrations.
System gold 166
Manufactured by Beckman Coulter
Sourced in United States, Germany
The System Gold 166 is a high-performance liquid chromatography (HPLC) system manufactured by Beckman Coulter. It is designed to provide accurate and reliable separation and detection of a wide range of compounds in complex samples. The System Gold 166 features a modular design, allowing for customization to meet specific analytical requirements.
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Lab products found in correlation
4 protocols using system gold 166
1
Enantioselective Ketoreduction Reactions
2
Separation of Ketoreduction Products
3
Thermosensitive Nanoparticle Drug Release
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2 g of thermosensitive LSs-loaded-MT and 35 mg of thermosensitive LSs-based MT-loaded nanoparticles were injected into vials. The thermosensitive LSs samples were then thermostated at 37 °C for 5 min to form gels, and nanoparticles were suspended in 1 mL PBS buffer (pH = 7.00 ± 0.05). Following that, the materials were immersed in dialysis membrane (3500 Da), placed in 10.0 mL of preheated PBS buffer (pH = 7.00 ± 0.05), and shaken at 130 rpm and 37 °C. After predefined time intervals, the release medium was withdrawn for further testing and entirely replaced with 10.0 mL of preheated fresh PBS buffer. The obtained samples were stored at 18 °C prior to HPLC analysis. The drug release experiments lasted 12 h.
The HPLC apparatus (Beckman Coulter, Miami, Florida, USA) was equipped with an autosampler (Triathlon 900, Spark Holland B.V., Emmen, Netherlands), pomp (Beckman Coulter System Gold® 125NM Solvent Module, Fullerton, CA, USA) and UV/VIS detector (Beckman Coulter System Gold® 166, Fullerton, CA, USA). The analysis was performed at 274 nm with a C18 column (Nucleodur C18 Gravity 150 × 4.6 mm, 5 um, Macherey-Nagel). Solvents A and B (75:25), H2O + 0.05% TFA and acetonitrile (ACN), were used to make the mobile phase. The flow rate was set to 1.0 mL/min, the injection volume was set to 20 µL, and the column temperature was set to 35 °C.
The HPLC apparatus (Beckman Coulter, Miami, Florida, USA) was equipped with an autosampler (Triathlon 900, Spark Holland B.V., Emmen, Netherlands), pomp (Beckman Coulter System Gold® 125NM Solvent Module, Fullerton, CA, USA) and UV/VIS detector (Beckman Coulter System Gold® 166, Fullerton, CA, USA). The analysis was performed at 274 nm with a C18 column (Nucleodur C18 Gravity 150 × 4.6 mm, 5 um, Macherey-Nagel). Solvents A and B (75:25), H2O + 0.05% TFA and acetonitrile (ACN), were used to make the mobile phase. The flow rate was set to 1.0 mL/min, the injection volume was set to 20 µL, and the column temperature was set to 35 °C.
4
Quantifying Metabolite Production in Fermentation
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To follow glucose consumption and production of gluconate and propionate during fermentation, samples of 10 mL were withdrawn from the cultivation, from which 1 mL was immediately centrifuged at 13,000 rpm for 1 min, and the supernatant stored at 20°C until further analysis. Quantification was done via high‐performance liquid chromatography (HPLC) using a Beckman System Gold 126 Solvent Module with an organic acid resin column (Polystyrene divinylbenzene copolymer (PS DVB), 300 × 8.0 mm, CS‐Chromatographie). Analytes were eluted with 5 mM H2SO4 at a flow rate of 0.6 mL h−1 for 30 min at 40°C. Detection was realised with a System Gold 166 UV detector (Beckman Coulter), and a Smartline RI Detector 2300 (KNAUER Wissenschaftliche Geräte, Berlin, Germany) set to 25°C.
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