Molecular mass and sample quality of P4H-TM with and without Ca2+ were analyzed with a miniDAWN MALS device (Wyatt Technology Corporation) connected to a Shimadzu HPLC unit (Shimadzu Corporation) with a Superdex 200 Increase 10/300 GL SEC column (GE Healthcare Life Sciences) at constant 10 °C temperature and equilibrated with the SEC buffer with and without 2-mM CaCl2. The sample concentration was 3.3 mg/ml, and the flow rate 0.5 ml/min. The RID-10A refractive index detector (Shimadzu Corporation) connected to the HPLC system was used as a concentration source for the calculations. ASTRA software (version 7.3.1.) (Wyatt Technology Corporation) was used to calculate the molecular weight and polydispersity of the samples.
Hplc unit
Manufactured by Shimadzu
Sourced in Japan
The HPLC unit from Shimadzu is a high-performance liquid chromatography system used for the separation, identification, and quantification of chemical compounds in complex mixtures. It consists of a pump, an injector, a column, and a detector, which work together to achieve precise, efficient, and reliable analysis of samples.
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Lab products found in correlation
Superdex 200 increase 10 300 gl sec column, by GE Healthcare (2 mentions)
Minidawn mals device, by Wyatt Technology (2 mentions)
Astra software version 7.3.1, by Wyatt Technology (2 mentions)
Rid 10a refractive index detector, by Shimadzu (2 mentions)
Nmr spectrometer, by Bruker (1 mentions)
Ods a column, by Shimadzu (1 mentions)
Catechin, by Merck Group (1 mentions)
Epicatechin, by Merck Group (1 mentions)
Procyanidin b2, by Merck Group (1 mentions)
Procyanidin b1, by Merck Group (1 mentions)
Spd m10avp, by Shimadzu (1 mentions)
Digital 10p, by Bandelin (1 mentions)
5 protocols using hplc unit
1
Molecular Mass Analysis of P4H-TM
2
Molecular Mass Analysis of P4H-TM
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Molecular mass and sample quality of P4H-TM with and without Ca 2+ were analyzed with a MiniDAWN MALS device (Wyatt Technology Corporation, Santa Barbara, USA) connected to a Shimadzu HPLC unit (Shimadzu Corporation, Kyoto, Japan) with a Superdex 200 Increase 10/300 GL SEC column (GE Healthcare Life Sciences) at constant 10°C temperature and equilibrated with the SEC buffer with and without 2 mM CaCl2. The sample concentration was 3.3 mg/ml. The RID-10A refractive index detector (Shimadzu Corporation) connected to the HPLC system was used as a concentration source for the calculations. ASTRA software (version 7.3.1.) (Wyatt Technology Corporation) was used to calculate the molecular weight and polydispersity of the samples.
3
HPLC, NMR, and MS Analysis of AHL Degradation
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AHL degradation products of purified AHL degrading enzyme reaction and AHLs were analyzed using reverse-phase HPLC, NMR and electron impact ionization-mass spectrometry (EI-MS) [11 (link),22 (link)]. For the reverse-phase HPLC analysis, the sample was dissolved in 0.1 mL 50% (v/v) methanol (in MiliQ water) and analyzed using a C18 reverse-phase column (250×4.6mm S-4μm, 80A, YMC co. ltd. JH08S04-2546WT). The fractions were separated by isocratic gradient (1–100%) of water: formic acid (0.1%, pump A) and acetonitrile: formic acid (0.1%, pump B) at a flow rate of 0.3 mL min-1 detected by SPD-10UV detector at 210nm in HPLC unit (Shimadzu Corp., Japan). The EI-MS was performed using a DSQII GC/MS with TRACE GC Ultra unit (Thermo scientific), and high resolution 1H and 13C NMR was performed using Bruker NMR spectrometer. For MS analysis the sample was dissolved in 50% (v/v) methanol (in MiliQ water) and ionized by a positive-ion electron impact, while for NMR samples was dissolved in D2O. As a control standard, 5 mM of synthetic HHL and HSL (Sigma-Aldrich, India) dissolved in 50% (v/v) methanol (in MiliQ water) were used as AHL standards while the uninoculated medium was used as an additional control. The percentage of AHL degradation was determined by estimation of AHL with respect to known concentration that was loaded.
4
Quantification of Açaí Seed Flavan-3-ols by HPLC
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The quantification of flavan-3-ols in açaí seed extracts was carried out using an analytical Shimadzu HPLC unit, equipped with a Shimadzu ODS-A column (4.6 mm × 250 mm, 5 μm) and a photodiode array detector (DAD) (SPD-M10AVp, Shimadzu Co., Kyoto, Japan). Açaí seed extracts (crude or digested) were filtered using a 0.22 μm PTFE membrane filter and aliquots of 20 μL were injected into the HPLC system at a flow rate of 1.0 mL/min at a constant temperature of 28 °C. The mobile phase used was composed of water/formic acid (99.9/0.1, v/v) (A) and acetonitrile/formic acid (99.9/0.1, v/v) (B). The elution gradient started with 5% B and increasing to 7% B (7 min), reaching 20% B (50 min), 45% B (70 min), 100% B (85 min), held at 100% until 95 min, and finally decreasing to 5% B (100 min). The authentic standards procyanidin B1, procyanidin B2, catechin, and epicatechin (Sigma–Aldrich, St. Louis, MO, USA) were examined. A standard curve was prepared for each standard to perform the quantification of the compounds in the samples. The Limits of Detection (LOD) and Limits of Quantification (LOQ) of the standards were as follows: procyanidin B1 (LOD = 4.6 × 10−6; LOQ = 1.4 × 10−5); procyanidin B2 (LOD = 5.9 × 10−6; LOQ = 1.8 × 10−5); catechin (LOD = 3.8 × 10−5; LOQ = 1.1 × 10−4); Epicatechin (LOD = 8.7 × 10−6; LOQ = 2.6 × 10−5).
5
HPLC Analysis of Shrimp Shell Extracts
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The eluents used in the HPLC unit (Shimadzu, Kyoto, Japan) equipped with a photodiode array detector (PDA) model SPD-M20A were filtered through a 0.22 μm nylon membrane filter (Fioroni Filters, Ingré, France) using a vacuum pump (Dinko D-95, Barcelona, Spain) and degassed for 15 min in an ultrasonic bath (Sonorex Digital 10P, Bandelin DK 255P, Germany). Twenty μL of each shrimp shell extract were injected on an analytical HPLC with a low-pressure quaternary pump (model LC-20AT), a degasser (model DGU-20A5R), an auto-sampler (model SIL-20AT), and a column oven (model CTU-20AC). The gradient and column were previously described [15 (link)] and were used with some modifications. Briefly, compound separation was achieved using a C30 YMC Carotenoid S-5µm (25.0 × 0.46 cm; 5 μm particle size) column (Kyoto, Japan). The solvent system consisted of methanol (A) and MTBE (B) starting with 95% A, using a gradient to obtain 70% A at 30 min, 50% A at 60 min, and again 95% A at 65 min. The solvent flow rate was 0.9 mL/min. Chromatograms were recorded at 450 nm, and data were processed on LabSolutions software. Compounds were identified by comparing their UV–vis spectra and retention times with standards injected in the same conditions.
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