Honey solutions were prepared following the steps presented in Section 2.3.3 [30 (link)]. The samples were filtered through 0.45 µm PTFE membrane filters and then injected (with a volume of 10 µL) into the HPLC instrument (Schimadzu, Kyoto, Japan) for analysis using an SPD-M-20A diode array detector. The separation was carried out on a Phenomenex Kinetex 2.6 μm Biphenyl 100 Å HPLC Column 150 × 4.6 mm thermostated at 25 °C. Elution was carried out with a solvent system consisting of 0.1% acetic acid in water (solvent A) and acetonitrile (solvent B) as previously described by Palacios et al. [32 (link)] with modifications. The solvent flow rate was of 1 mL·min−1. The determined phenolic compounds were gallic acid, vanillic acid, protocatechuic acid and p-hydroxibenzoic acid at 280 nm, and chlorogenic acid, p-coumaric acid, caffeic acid, rosmarinic acid, myricetin, quercetin, luteolin and kaempherol at 320 nm. The obtained standard calibration curves showed high degrees of linearity (R2 > 0.99). Data collection and subsequent processing were performed using the LC solution software version 1.21 (Shimadzu, Kyoto, Japan).
Spd m20a
Manufactured by Schimadzu
Sourced in Japan
The SPD-M20A is a diode array detector for high-performance liquid chromatography (HPLC) systems. It provides simultaneous monitoring of multiple wavelengths, enabling efficient analysis of complex samples. The device offers high-speed data acquisition and a wide dynamic range to support a variety of analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Luna 5μ phenyl hexyl column, by Phenomenex (2 mentions)
Hplc instrument, by Schimadzu (1 mentions)
Lc solution software version 1, by Shimadzu (1 mentions)
Orthophosphoric acid, by Merck Group (1 mentions)
Hplc grade methanol, by Merck Group (1 mentions)
Curcumin, by HiMedia (1 mentions)
Optilab rex detector, by Wyatt Technology (1 mentions)
Dynapro nanostar, by Wyatt Technology (1 mentions)
5 protocols using spd m20a
1
HPLC Analysis of Phenolic Compounds in Honey
2
Quantitative Analysis of Curcumin
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XH was obtained as a gift sample from Siemen. Hop Steiner, Germany. Curcumin was purchased from HiMedia (Mumbai, India). HPLC grade Methanol, Ortho-Phosphoric acid were purchased from Merck (Mumbai, India). Triple distilled water was used throughout the study. UFLC system (Schimadzu LC-20AD, Tokyo, Japan) with a photodiode array detector (SPD-M20A, Tokyo, Japan) and a Rheodyne sample injector loop (20 µL) was used for quantitative analysis. Vortex mixer and cooling centrifuge were from REMI, India.
3
Phenolic and Anthocyanin Profiling by HPLC
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Individual phenolics and anthocyanins in the extracts were detected using a high performance liquid chromatography with a PDA detector (SPD M20A, Schimadzu, Kyoto, Japan) according to our previous work [26 (link)]. For the chromatographic separation of the phenolic compounds, an ACE C18 column (250 mm × 4.6 mm, 3 μm) with a guard column (4.0 mm × 10 mm, 2 μm) (Advanced Chroma-tography Technologies Ltd., Aberdeen, UK) was used. The gradient of mobile phase A (MQ-water/formic acid, 99.9/0.1 v/v) and mobile phase B (acetonitrile) was used.
The chromatographic separation of anthocyanins was performed on a Luna-5μ-Phenyl-Hexyl column (250 mm × 4.6 mm, 5 μm) (Phenomenex, Torrance, CA, USA) using a gradient of MQ-water/formic acid (95:5 v/v) for mobile phase A and acetonitrile for mobile phase B, as previously described [26 (link)].
The chromatographic separation of anthocyanins was performed on a Luna-5μ-Phenyl-Hexyl column (250 mm × 4.6 mm, 5 μm) (Phenomenex, Torrance, CA, USA) using a gradient of MQ-water/formic acid (95:5 v/v) for mobile phase A and acetonitrile for mobile phase B, as previously described [26 (link)].
4
HPLC Analysis of Phenolics and Anthocyanins
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Individual phenolics and anthocyanins in the extracts were detected using a high performance liquid chromatography (HPLC) with a PDA detector (SPD M20A, Schimadzu, Kyoto, Japan). The chromatographic separation of phenolics was performed on an ACE C18 column (250 mm × 4.6 mm, 3 μm) with a guard column (4.0 × 10 mm, 2 μm) (Advanced Chromatography Technologies Ltd., Aberdeen, UK). A gradient of mobile phase A (MQ-water/formic acid, 99.9/0.1 v/v) and mobile phase B (acetonitrile) was used. The flow rate was 0.5 mL/min, and the injection volume was 10 μL for each standard mixture, and the column temperature was set to 40 °C. A 55 min gradient program was used with the gradient profile as follows: 0–5 min: 10% B, 5–45 min: 55% B, 45–48 min: 90% B, 48–55 min: 10%, and 50–55 min: 10% B.
The chromatographic separation of anthocyanins was performed on a Luna 5μ Phenyl-Hexyl column (250 mm × 4.6 mm, 5 μm) (Phenomenex, Torrance, CA, USA). A gradient of mobile phase A (MQ-water/formic acid, 95:5 v/v) and mobile phase B (acetonitrile) was used. The flow rate was 0.5 mL/min, and the injection volume was 10 μL for each standard mixture, and the column temperature was set to 40 °C. A 55 min gradient program was used with the gradient profile as follows: 0–40 min: 5% B, 40 min: 30% B, 55 min: 50% B, 57–59 min: 100% B, and 60–65 min: 5%.
The chromatographic separation of anthocyanins was performed on a Luna 5μ Phenyl-Hexyl column (250 mm × 4.6 mm, 5 μm) (Phenomenex, Torrance, CA, USA). A gradient of mobile phase A (MQ-water/formic acid, 95:5 v/v) and mobile phase B (acetonitrile) was used. The flow rate was 0.5 mL/min, and the injection volume was 10 μL for each standard mixture, and the column temperature was set to 40 °C. A 55 min gradient program was used with the gradient profile as follows: 0–40 min: 5% B, 40 min: 30% B, 55 min: 50% B, 57–59 min: 100% B, and 60–65 min: 5%.
5
SEC-LS Characterization of Proteins
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Size exclusion chromatography (SEC)-Light scattering (LS) experiments were conducted at 4 °C on an HPLC chromatography system consisting of a degasser DGU-20AD, a LC-20AD pump, an autosampler SIL20-ACHT, a communication interface CBM-20A and a UV–Vis detector SPD-M20A (Schimadzu, Kyoto, Japan), a column oven XL-Therm (WynSep, Sainte Foy d’Aigrefeuille, France), a static light scattering miniDawn Treos, a dynamic light scattering DynaPro Nanostar and a refractive index Optilab rEX detectors (Wyatt, Santa-Barbara, USA). The analysis was carried out with the software ASTRA, v5.4.3.20 (Wyatt, Santa-Barbara, USA). Samples of 20 μL were injected at 0.5 mL min−1 on a Superdex 200 10/300 GL (GE Heathcare), equilibrated with 20 mM Tris-HCl pH8, 100 mM NaCl, 5 mM β-mercaptoethanol. Bovine Serum Albumin, at 2 mg mL−1, in PBS buffer was injected as a control.
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