The riboflavin content analysis was performed using high-performance liquid chromatography (HPLC) combined with a fluorescence detector (Agilent-1100 Series, Palo Alto, CA, USA), as well as the C-18 column Unison UK-C18 (4 × 100 mm, 3 μm, Imtakt, Portland, OR, USA). The SGS culture medium was boiled at 100 °C for 15 min and then centrifuged at 13,000 rpm and 4 °C for 15 min. The supernatant was collected and filtered through a 0.45 μm membrane filter (Millipore, Burlington, MA, USA). Methanol (mobile phase A) and a 10 mM sodium dihydrogen phosphate (Na2HPO4) solution (pH 5.5, mobile phase B) were used as the mobile phases according to the following gradient conditions: 0 min—A 90%, B 10%; 10 min—A 60%, B 40%, 10.1 min A—90%, B 10%; 20 min—A 90%, B 10%. The column temperature, injection volume, flow rate, and excitation and emission wavelengths were 40 °C, 10 µL, 0.8 mL/min, 445 nm, and 530 nm, respectively. Supelco (Bellefonte, PA, USA), a riboflavin standard reagent, was used for riboflavin analysis, and the retention time and peak area was compared to quantify the riboflavin concentration of the sample.
Unison uk c18
Manufactured by Imtakt
Sourced in United Kingdom
The Unison UK-C18 is a reverse-phase high-performance liquid chromatography (HPLC) column. It is designed for the separation and analysis of a wide range of organic compounds. The column features a C18 stationary phase, which is commonly used for the separation of non-polar or moderately polar analytes.
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3 protocols using unison uk c18
1
Quantifying Riboflavin in SGS Culture
2
HPLC Analysis of Ascorbic Acid in Tomatoes
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Ascorbic acid measurements were performed using the Shimadzu Prominence HPLC system (Shimadzu) according to Brause et al. (2003) (link). The extraction solution was freshly prepared by dissolving 56 g of metaphosphoric acid in 1 L of water on the day of extraction. Plastic tubes (50 mL) were filled into racks and 35 mL of extraction solution was added to each tube. Frozen tomato samples (5 g) were placed into tubes and an additional 10 mL of extraction solution was added. Samples were homogenized at 200×g for 30 s and then filtered through a 110-mm filter paper. The filtrate was further filtered with a 0.45-μm sterile filter connected to a syringe and collected into a 1.5-mL tube. The HPLC syringe was used to collect 0.05 mL of the filtrate and injected into the machine to measure concentrations. During the analysis, the fitted column was a Unison UK-C18 (3 μm, 4.6×150 mm) ODS column (Imtakt); the mobile phase was 2 mM HClO4 and 100 mM NaBH4, which were pumped at flow rates of 1.0 mL min–1 and 0.5 mL min–1, respectively; the injection volume was 20 μL; the column temperature was maintained at 40°C; and UV detection was performed at 300 nm.
3
Analytical Characterization of Protein Sample
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The HPLC system consisted of a Unison UK-C18 (4.6 × 250 mm; Imtakt Corp., Kyoto, Japan), a JASCO Intelligent System Liquid Chromatograph (Jasco Corp., Tokyo, Japan), and detection at 300 nm. The bound material was eluted in 20 % methanol at a flow rate of 0.75 mL/min at 40 ºC. MALDI-TOF mass spectra were acquired using an AutoFlex spectrometer (Bruker Daltonics GmbH, Bremen, Germany) in positive reflection mode with 20 kV ion acceleration and without post acceleration. The spectra were recorded using a detector voltage of 1.65 kV and averaged from at least 300 individual laser shots. A solution of 65 mM 2,5-dihydroxybenzoic acid in 30 % ethanol was used as the matrix. Samples were dissolved in water and mixed with the matrix (1:4 v/v). Each mixture (1 µL) was spotted onto a stainless steel platform and allowed to crystallize at room temperature. The spectrometer was calibrated with peptide calibration standard II (Bruker Daltonics). 500 MHz 1H NMR spectra and 125 MHz 13C NMR spectra were recorded using a JEOL ECX-500 II spectrometer (Jeol Ltd., Tokyo, Japan). Chemical shifts were expressed in ppm relative to the methyl resonance of the external standard sodium 3-(trimethylsilyl)propionate. HEWL concentration was calculated from a standard curve by measuring the absorbance at 280 nm.
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