Analysis of MDA, 4-HNE, and 4-HNE concentrations was then conducted according to a previously described method (20 (link)). The preparation of 10 mg/ml-MDA was procured through the hydrolysis of TEP in a 5%-TCA solution. The standard working solutions of (0.02 ~ 10 mg/L for MDA, 4-HNE, and 4-HNE) were prepared using ethanol/water 1:1 (v/v). Then 2 g oil was mixed with 2.5% (w/v) TCA and DNPH [0.05 mol/L in ethanol/HCl 12 M 9:1 (v/v)]. After preparing derivatives, the separation was used with a CBM-20A HPLC system (Shimadzu, Shanghai, China) equipped with an SPD-M20A detector and an ODS HYPERSIL column (4.6 × 250 mm, 5 μm) from Thermo Scientific. The UV wavelength was set at 378 and 310 nm to detect 4-HHE, 4-HNE, and MDA derivatives. The flow rate was set at 1.0 ml/min, whereas the injection volume was set at 20 μl. Furthermore, the mobile phase was acetonitrile and water (solvent A and B). The gradient elution program was 45% solvent A maintained for 0–18 min, then from 45 to 70% solvent A in 5 min, and then 70% solvent A for 15 min.
Hplc cbm 20a system
Manufactured by Shimadzu
Sourced in Japan
The HPLC CBM-20A system is a communication bus module that provides communication functions for Shimadzu HPLC systems. It enables connection and control of various HPLC components, such as pumps, detectors, and autosamplers, through a unified interface.
Automatically generated - may contain errors
Lab products found in correlation
Spd 20a, by Shimadzu (3 mentions)
Rf 10axl, by Shimadzu (2 mentions)
Amino columns, by Phenomenex (2 mentions)
Ods hypersil column, by Thermo Fisher Scientific (1 mentions)
Hydro rp column, by Phenomenex (1 mentions)
C18 column, by Phenomenex (1 mentions)
Luna 5 μm nh2 column, by Phenomenex (1 mentions)
Lc 20at pump, by Shimadzu (1 mentions)
Zorbax nh2, by Agilent Technologies (1 mentions)
11 protocols using hplc cbm 20a system
1
HPLC Analysis of Lipid Oxidation Products
2
Quantification of Acrylamide in Samples
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Samples were analysed on a Shimadzu CBM-20A HPLC system (Shimadzu Corporation, Kyoto, Japan) that consists of a degasser, quaternary pump, autosampler, and temperature controlled oven coupled to a Shimadzu LCMS-8050 triple quadrupole MS detector (Shimadzu Corporation, Kyoto, Japan). Separation of acrylamide was carried out on a Synergi Hydro-RP column (150 mm × 3 mm, 4 μm particle size) with a SecurityGuard Catridges C18 guard column (4 mm × 2 mm i.d.) (Phenomenex Inc., California, USA). The mobile phase was ultrapure water and methanol (99.5:0.5, v/v) at a flow rate of 0.2 mL/min. The oven temperature was 40°C and the injection volume was 10 μL. The Electrospray Ionization (ESI) source was operated in positive mode with an interface temperature of 300°C, desolvation line temperature of 250°C, and heat block temperature of 400°C. The nebulizing gas flow was 1 L/min, whereas the gas flow for heating and drying was 10 L/min. The transitions monitored for acrylamide was 72 > 55 and 72 > 27.1 while the transitions monitored for labelled acrylamide was 75 > 58.1 and 75 > 30.1. The dwell time for each transition was 247 ms. All samples and spiked samples were injected in duplicates. The calibration curve was constructed by plotting the response ratio (peak area for acrylamide: 72 > 55/peak area for labelled acrylamide: 75 > 58.1) against acrylamide concentration.
3
HPLC Analysis of Glycan Derivatives
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A Shimadzu HPLC CBM-20A system with UV detector SPD-20A and fluorescence
detector RF-10Axl was used for HPLC analysis. UV absorption at 330 nm or
fluorescence at 330 nm excitation (Ex) and 420 nm emission (Em) was used for
detection of anthranilic acid (AA), 2-aminobenzamide (AB), and
2-amino-N-(2-aminoethyl)benzamide (AEAB) tags. For Fmoc-protected glycans, UV
absorption at 330 nm or fluorescence at 330 nm excitation (Ex) and 420 nm
emission (Em) was also used. Agilent amino columns were used for normal phase
HPLC. Both C18 and PGC columns were used for reverse phase HPLC separation.
Detailed methods are described in individual experiments. The mobile phases were
acetonitrile, water, and aqueous ammonium acetate buffer at pH 4.5. A linear
gradient from 20 mM ammonium acetate in 80% acetonitrile to 200 mM ammonium
acetate in 10% acetonitrile in either 25 or 50 minutes was used.
detector RF-10Axl was used for HPLC analysis. UV absorption at 330 nm or
fluorescence at 330 nm excitation (Ex) and 420 nm emission (Em) was used for
detection of anthranilic acid (AA), 2-aminobenzamide (AB), and
2-amino-N-(2-aminoethyl)benzamide (AEAB) tags. For Fmoc-protected glycans, UV
absorption at 330 nm or fluorescence at 330 nm excitation (Ex) and 420 nm
emission (Em) was also used. Agilent amino columns were used for normal phase
HPLC. Both C18 and PGC columns were used for reverse phase HPLC separation.
Detailed methods are described in individual experiments. The mobile phases were
acetonitrile, water, and aqueous ammonium acetate buffer at pH 4.5. A linear
gradient from 20 mM ammonium acetate in 80% acetonitrile to 200 mM ammonium
acetate in 10% acetonitrile in either 25 or 50 minutes was used.
4
HPLC Characterization of Anthranilic Acid Derivatives
5
HPLC Analysis and Semipreparation of Glycans
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Analysis and semipreparation of the AEAB- and F-MAPA–glycan and the reaction product by GNPTAB were performed on a Shimadzu HPLC CBM-20A system, equipped with a UV detector (SPD-20AV) and a fluorescence detector (RF-20A). For analysis, the samples were chromatographed on a Luna 5-μm NH2 column (250 × 4.6 mm, Phenomenex) with a concentration of 16 to 40% H2O and 4 to 50% 250 mM ammonium acetate over 60 min. The GNPTAB products had later elution time in comparison to the substrate. For semipreparation, the separation was on a Luna 5-μm NH2 column (250 mm by 10 mm, Phenomenex) with either the same gradient or a shallower gradient to resolve the Fmoc-labeled Man4-GlcNAc2-Asn to Man9-GlcNAc2-Asn using 16 to 24% H2O and 4 to 21% 250 mM ammonium acetate over 50 min. Detection of the AEAB-glycans was at UV 330 nm, fluorescence at excitation (Ex) = 330 nm, and emission (Em) = 420 nm; detection of F-MAPA-glycans was at UV 254 nm, fluorescence Ex = 254, and Em = 340, respectively. LNnT-AEAB and LNnT-F-MAPA conjugates with known concentrations were used for quantification.
6
Analytical and Preparative HPLC Protocols
7
Anaerobic Clostridium Metabolism Analysis
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Clostridium sp. ZJ6 (ZJ6) was isolated from the caecum of 15-day-old chicks and cultured for 24 h in Reinforced Clostridium Medium (RCM) under anaerobic conditions (10% H2, 10% CO2, and 80% N2) at 37°C. Next, these cells were sub-cultured 1:10 in fresh RCM with 40 μM DZN and dihydrodaidzein (DHD) for 24 h. The fermentation products of C1 were then subjected to analysis by HPLC. First, 1 mL of the fermentation supernatant was mixed with 0.7 mL of ethyl acetate by vigorous shaking and left to sit for 5 min, centrifuged for 5 min at 5,000 × g, and the supernatant was transferred to a new centrifuge tube. Each sample was extracted twice following the same procedure. Subsequently, all collected supernatants were dried by vacuum centrifugation and then dissolved in 0.2 ml of methanol. Finally, a Shimadzu HPLC CBM-20A system (Kyoto, Japan) with UV detector SPD-20A was used for HPLC analysis, and separations were performed on a SunFireTM C18 (5 μm, 4.6 × 250 mm) column. Elution was performed using a 0.1% trifluoroacetic acid aqueous solution (A) as well as methanol acetonitrile solution (B, 4:6, V/V), at a flow rate of 0.8 mL/min and a solution B gradient of 50% for 15 min. The detection wavelengths were 205 and 254 nm, the detection temperature was 30°C, and the injection volume was 5 μL.
8
HPLC Analysis of BHA and AEAB
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HPLC profiles were acquired using a Shimadzu HPLC CBM-20A system equipped with a UV detector SPD-20A (Shimadzu Co., Kyoto, Japan). UV absorption at 254 nm or fluorescence at 330 nm excitation and 420 nm emission was used for the detection of BHA tag or AEAB. Phenomenex amino columns were used for normal-phase HPLC. For normal-phase HPLC, the mobile phases were acetonitrile, water, and aqueous ammonium acetate buffer at pH 4.5. A linear gradient from 20 mM ammonium acetate in 80% acetonitrile to 20 mM ammonium acetate in 10% acetonitrile for 45 min was used.
9
Fmoc-labeled Asn-Glycan HPLC Analysis
10
HPLC Analysis of Glycan Derivatives
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