The organic acid and sugar content was determined using the method proposed previously by Wojdyło et al.30 (link) by HPLC–PDA (Waters Co.; Milford, CT, USA) and HPLC-ELSD (PL-ELS 1000; Merck; Hitachi, Japan), respectively. The sample (approx. 3 g of fruits and 1 g of leaves) mixed with distilled water, sonicated (Sonic 6D; Polsonic, Warsaw, Poland) for 15 min and boiled for 30 min, finally sample was centrifuged (MPW-55; Warsaw, Poland) at 12,000xg for 10 min at 4 °C. The supernatant (2.5 mL) was applied onto the Sep-Pak C-18 (1 g, Millipore Waters, Milford, MA, USA) and finally eluted by water to Eppendorf tubes. The extract before analysis was filtered through 0.20 μm hydrophilic PTFE membrane (Millex Simplicity Filter; Merck, Germany). All samples were assayed in triplicate repetition. Results expressed as g per 100 g dry weight (dw).
Hplc pda
Manufactured by Waters Corporation
Sourced in United States
The HPLC-PDA is a high-performance liquid chromatography (HPLC) system equipped with a photodiode array (PDA) detector. The HPLC-PDA is designed to separate, identify, and quantify various chemical compounds in complex mixtures. The PDA detector allows for the simultaneous detection of multiple wavelengths, providing detailed spectral information about the separated compounds.
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Lab products found in correlation
Sonic 6d, by Polsonic (2 mentions)
Mpw 55, by Polsonic (2 mentions)
Pl els 1000, by Merck Group (2 mentions)
Millex simplicity filter, by Merck Group (1 mentions)
Zorbax extend c18 column, by Agilent Technologies (1 mentions)
Luna c18 column, by Phenomenex (1 mentions)
Pda 2996, by Waters Corporation (1 mentions)
Capcell pak c18 mg column, by Shiseido (1 mentions)
Atlantis t3 column, by Waters Corporation (1 mentions)
Rx 5000, by Atago (1 mentions)
11 protocols using hplc pda
1
Determination of Organic Acids and Sugars
2
Phenolic Profiling of Fruit Vinegars
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The phenolic components in 23 fruit vinegars were analyzed by High Performance Liquid Chromatography coupled with Photometric Diode Array detector (HPLC-PDA) (Waters, Milford, MA, USA) based on the literature [17 (link)]. Separation was conducted using an Agilent Zorbax Extend-C18 column (250 × 4.6 mm, 5 μm) (CA, USA) at 40 °C. Mobile phase A was formic acid solution (0.1%, v/v), and B was methanol. The procedure of gradient elution was set as: 0 min, 5% (B); 15 min, 20% (B); 20 min, 30% (B); 25 min, 37% (B); 40 min, 40% (B); 60 min, 50% (B); 65 min, 50% (B); 65.1 min, 5% (B); and 70 min, 5% (B). The spectra were scanned between 200 and 600 nm. Peak area was used to quantify phenolic compounds and the results were expressed as μg/mL.
3
Quantification of Bioactive Compounds in Dried Amburana Extract
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Dried extract of A. cearensis (DEAC) was analyzed by high-performance liquid chromatography–photodiode array (HPLC-PDA) (Waters) according to the method developed previously by our laboratory [10 ]. The analysis of the three chemical markers (coumarin (CM), amburoside A (AMB), and vanillic acid (VA)) was performed through their calibration curves, obtained by injection of external standards. The analysis of DEAC was performed in a C18 reverse phase column (4.6 mm × 250 mm) at a temperature of 45°C. The mobile phase was composed of acetonitrile (A), 0.01% phosphoric acid (B), and n-propanol (C), flow of 1 mL per minute. Detection was performed in a PDA detector using the wavelengths of 219 nm, 277 nm, and 220 nm for the determination of VA, CM, and AMB, respectively. The HPLC-PDA analysis of the DEAC allowed the identification and quantification of vanillic acid, amburoside, and coumarin.
4
HPLC Analysis of Phenolic Compounds
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To prepare the sample for HPLC analysis, 10 mL of FP was filtered with 0.45 μm PVDF syringe filter and evaporated at 37 °C. The FP concentrates were diluted with DW at 20 mg/mL. The phenolic compounds in FP were identified by HPLC-PDA (Waters Co., Milford, MA, USA). After 40 μL of sample injection, separation was carried out with a Phenomenex Luna C18 column (250 × 4.6 m2, 5 μm). The separation process was performed in a ternary mobile phase gradient (solvent A, 0.1% trifluoroacetic acid in water; solvent B, acetonitrile) at a flow rate of 1 mL/min. The composition of solvent A was maintained at 90% for 10 min and gradually decreased to 0% for 90 min.
5
HPLC-PDA Analysis of Fractions
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A Waters™ HPLC-PDA (pump 600, controller 600S, and PDA detector 2998) was used for the chromatographic analysis of fractions FX, FXI, and, FXV. The HPLC conditions were as follows: reverse stationary phase (Waters™ RP-18 Xterra Shield, 125 Å, 3.5 µm, 4.6 × 150 mm); mobile phase: 70% acetonitrile and 30% acidulated water (0.1% TFA); flux: 1 mL/min, λ = 210 nm.
6
Carotenoid Extraction and Analysis
7
Quantitative Analysis of Organic Acids and Carbohydrates
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The analysis of organic acids and carbohydrate was performed as described previously by Wojdyło et al. [14 (link)], using HPLC-PDA (Waters Co.; Milford, CT, USA) and HPLC-ELSD (PL-ELS 1000; Merck; Hitachi, Japan), respectively. The sample (approximately 3 g of fruit) was mixed with distilled water, exposed to ultrasounds (Sonic 6D; Polsonic, Warsaw, Poland) for 15 min, heated at 90–100 °C for 30 min, and finally centrifuged (MPW-55; Warsaw, Poland) at 12,000× g for 10 min at 4 °C. The supernatant (2.5 mL) was injected into a Sep-Pak C-18 cartridge (1 g, Millipore Waters, Milford, MA, USA) and eluted with H2O into Eppendorf tubes. Before analysis, the extract was filtered through a hydrophilic PTFE membrane (0.20 µm; Millex Simplicity filters; Merck, Germany). The organic acids were analyzed on Polymex IEX H column (8 μm, 250 × 8 mm, Watrex; Prague, Czech Republic) using isocratic elution with 0.9 M sulfuric acid in H2O for 20 min. The carbohydrates were analyzed on Alltech® PrevailTM Carbohydrate ES HPLC Column-W 250 × 4.6 mm, 5 µm (Columbia, MD, USA) using isocratic elution with 70% acetonitrile in H2O for 20 min. The results were expressed in g per 100 g of d.w.
8
HPLC Analysis of HX109 Phytochemicals
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High-performance liquid chromatography analysis was employed to validate the quality of HX109. Reference standards for chicoric acid, maltol, dihydrophaseic acid, and isoschaftoside were used for qualitative and quantitative analyses of HX109. Analytical samples of HX109 were studied by HPLC-PDA (Waters, Millford, MA, USA) with Capcell PAK C18 MG column (4.6 mm × 250 mm, 5 µm, Shiseido, Japan). Water (0.05% trifluoroacetic acid) for solvent A and acetonitrile (0.01% trifluoroacetic acid) for solvent B was used for the mobile phase. The mobile phase gradient was 5–27% B (0–10 min), 27–35% B (10–25 min), 35–100% B (25–30 min); the flow rate was 1.0 mL/min, and the injection volume was 5 µL at the concentration of 20 mg/mL. The samples were analyzed at a wavelength of 280 nm and the optimum temperature for HPLC separation was 25 °C.
9
Detailed Protocol for Yijin Capsule (YC) Extraction
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YC is composed of 11 Chinese medicines: Epimedium sagittatum (Siebold and Zucc.) Maxim. [Berberidaceae], Gypsophila vaccaria (L.). Sm. [Caryophyllaceae], Concha Ostreae (calcined), Angelica sinensis (Oliv.) Diels [Apiaceae], Astragalus mongholicus Bunge [Fabaceae], Litchi chinensis Sonn. [Sapindaceae], Placenta Hominis (sheep placenta), Vitex negundo L. [Lamiaceae], Rehmannia glutinosa (Gaertn.) DC. [Orobanchaceae], Phyllolobium chinense Fisch. [Fabaceae], Hirudo. The ratios of these medicines are 13.3: 13.3: 13.3: 10: 10: 6.7: 6.7: 6.7: 6.7: 6.7: 6.7. A total of 100 g YC crude drugs were immersed in 10 times (v/w) of water, heated, and boiled for 60 min. The filtrate was collected, concentrated with rotary evaporation at 60 °C until a final volume of 100 mL, and then lyophilized with a freeze dryer to get the extract (16.3 g). YC extract was diluted into 0.1 or 0.2 g/kg (expressed as gram extract per kilogram body weight) using normal saline for in vivo experiments and diluted into 1.6, 16, and 160 μg extract/mL using PBS solution for in vitro studies. The main components of YC, including ferulic acid (HY-N0820, MCE), catalpol (HY-N0820, MCE), complanatuside (HY-N0820, MCE), arctiin (HY-N0820, MCE), hyperoside (HY-N0820, MCE), and calycosin-7-O-β-D-glucoside (HY-N0820, MCE), were measured by HPLC-PDA (Waters Corporation, United States).
10
Quantification of Bioactive Compounds in Actinidia arguta and Perilla frutescens
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For the qualitative and quantitative analyses of ACTPER, standard solutions of hydroxymethylfurfural, 2-furoic acid, protocatechuic acid, chlorogenic acid, caffeic acid, hyperoside, apigenin 7-O glucuronide and rosmarinic acid were prepared by dissolving the reference compounds in methanol separately. Sample solutions for analysis were prepared by dissolving ACTPER and an extract of each plant (Actinidia arguta and Perilla frutescens) in water at the concentration of 20 mg/mL. All samples for analysis were filtered through a 0.45 μm membrane filter.
Analytical samples were studied by HPLC-PDA (Waters, Millford, MA, USA) with an Atlantis T3 column (4.6 mm × 250 mm, 5 μm, Waters, Millford, MA, USA). The mobile phase was composed of A (Water containing 0.01% trifluoroacetic acid) and B (Acetonitrile containing 0.01% trifluoroacetic acid) with a gradient elution: 5% B (0–5 min), 5–20% B (5–25 min), 20–30% B (25–40 min), 30–45% B (40–50 min), 45–100% B (50–55 min); then keeping 100% B for 5 min to clean the column, and the re-equilibrating step of the column was 5% B isocratic for 5 min. The flow rate was 1.0 mL/min, and the injection volume was 10 μL. The samples were analyzed at a wavelength of 254 nm and the column temperature was maintained at 25 °C.
Analytical samples were studied by HPLC-PDA (Waters, Millford, MA, USA) with an Atlantis T3 column (4.6 mm × 250 mm, 5 μm, Waters, Millford, MA, USA). The mobile phase was composed of A (Water containing 0.01% trifluoroacetic acid) and B (Acetonitrile containing 0.01% trifluoroacetic acid) with a gradient elution: 5% B (0–5 min), 5–20% B (5–25 min), 20–30% B (25–40 min), 30–45% B (40–50 min), 45–100% B (50–55 min); then keeping 100% B for 5 min to clean the column, and the re-equilibrating step of the column was 5% B isocratic for 5 min. The flow rate was 1.0 mL/min, and the injection volume was 10 μL. The samples were analyzed at a wavelength of 254 nm and the column temperature was maintained at 25 °C.
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