Pure standards were purchased from Sigma-Aldrich Chemical Co. (Shanghai, China). Solvents of spectrophotometric grade were obtained from J & K Co. Ltd. (Beijing, China). A DB-Wax gas chromatography column was purchased from J & W Scientific, Rancho Cordova, CA. All the other chemicals were of analytical grade.
Pure standards
Manufactured by Merck Group
Sourced in Germany, United States, Italy
Pure standards are reference materials used to calibrate and validate analytical instruments in laboratory settings. They provide a known concentration of a specific compound or substance, enabling accurate quantification and measurement in various analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Uv 1900 uv vis, by Shimadzu (1 mentions)
Hp 6890, by Hewlett-Packard (1 mentions)
Hp innowax, by Hewlett-Packard (1 mentions)
Spectra system p4000 hplc, by Thermo Fisher Scientific (1 mentions)
Uv 6000 lp, by Thermo Fisher Scientific (1 mentions)
Zorbax ods column, by Agilent Technologies (1 mentions)
Dionex ics 5000, by Thermo Fisher Scientific (1 mentions)
Carbopac pa1 guard, by Thermo Fisher Scientific (1 mentions)
Reversed phase amino acid analysis system, by Waters Corporation (1 mentions)
Accq fluor reagent, by Waters Corporation (1 mentions)
Accq tag method, by Waters Corporation (1 mentions)
Empower software, by Waters Corporation (1 mentions)
4 methyl 2 pentanol, by Merck Group (1 mentions)
Gc 2010, by Shimadzu (1 mentions)
Xevo tq xs, by Waters Corporation (1 mentions)
Acquity uplc 1 class system, by Waters Corporation (1 mentions)
Masslynx software, by Waters Corporation (1 mentions)
Isopropanol, by Avantor (1 mentions)
Chloroform, by Avantor (1 mentions)
Acetonitrile, by Avantor (1 mentions)
17 protocols using pure standards
1
Analytical Grade Chemical Procurement
2
Anthocyanin Quantification in Berry Fruits
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Anthocyanins were measured by the HPLC method described by Ponder and Hallmann [30 (link)]. Freeze-dried berry fruit samples were extracted with 80% methanol. After first centrifugation (conditions: time 10 min, rpm 6000, temperature 30 °C), 2.5 mL of the supernatant was collected in a new plastic tube, and then 2.5 mL of 10 mol HCl and 5 mL of 100% methanol were added. The samples were gently shaken (up and down) and put in a cold place (5 °C, 10 min). Next, 900 µL of the extract was transferred into HPLC vials and analyzed. The anthocyanins were separated under isocratic conditions with a flow rate of 1.5 mL/min. One mobile phase, 5% acetic acid, methanol, and acetonitrile (70:10:20), was used. The HPLC set was performed from modules: two pumps (LC-20AD), one controller (CBM-20A), one column oven (SIL-20AC), one spectrometer (UV–VIS SPD-20 AV). Phenomenx Fusion 80-A (4.6 × 250 mm, practical shape 4 µm), and one column (C18) were used. The analysis time was 15 min at a wavelength of 520 nm. The anthocyanins were identified by using pure standards (Sigma-Aldrich, Poland) and the retention times for the standards. The limit of detection (LOD) and level of quantification (LOQ) for all quantified anthocyanin compounds are presented below (Table 1 ).
3
Quantification of Soluble Sugars and Organic Acids
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Soluble sugars (i.e., D-glucose, D-fructose, and sucrose) were measured using the K-SUFRG commercial kit (Megazyme, Wicklow, Ireland) following the manufacturer’s protocol. After extraction with ethanol 80% (v/v), D-glucose, D-fructose, and sucrose were determined with a spectrophotometer (UV-1900 UV-vis, Shimadzu, Kyoto, Japan) at 340 nm. Organic acids (i.e., citric, malic, shikimic, and quinic acids) were determined according to Eyéghé-Bickong et al. [62 (link)], with minor modifications. After extraction with 100% HPLC-demineralized water, these organic acids were measured with the same UHPLC reported above equipped with a pre-column Repromer H (8 mm internal diameter × 20 mm length, 9 μm particle size) and a Repromer H column (8 mm internal diameter × 300 mm length, 9 μm particle size) using 9 mM sulphuric acid as the eluent and a flow rate of 1 mL min−1. Organic acids were detected using their absorbance at 210 nm with the same detector reported above. To quantify their content, known amounts (0.003–0.5 mg ml−1) of pure standards (Sigma-Aldrich, St. Louis, MO, USA) were injected into the UHPLC system and an equation correlating the peak area to the organic acid concentration was formulated.
4
Quantifying Carotenoids and Chlorophylls
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All carotenoids and chlorophylls were identified by using pure standards (Sigma-Aldrich, Warsaw, Poland) and the retention times for the internal standards. Pure carotenoid (lutein, zeaxanthin, beta-carotene) and chlorophyll (a and b) standards were used for standard solution preparation. From each standard solution, five injections were made. Each time, the chromatographic pick area was determined and calculated based on the standard solution concentration. From standard curves, a mathematical equation was prepared. On the basis of the dilution coefficient and equation, the concentration of individual compounds was calculated.
5
Fatty Acid Profiling from Plasma and Muscle
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Lipids from blood plasma samples and biceps femoris muscle were extracted and methylated using the procedure described by [22 (link)]. Fat extracts were methylated in the presence of sulfuric acid and analyzed by gas chromatography. Previously fatty acid methyl ester (FAME) samples were identified by gas chromatography, as described elsewhere [23 (link)]. GC-MS was performed using an HP-6890 (Hewlett Packard, Avondale, PA, USA) gas chromatograph, equipped with a flame ionization detector and capillary column (HP-Innowax, 30 m by 0.32 mm ID and 0.25 μm polyethylene glycol-film thickness). A temperature program of 170 °C to 245 °C was used. The injector and detector were maintained at 250 °C. The carrier gas (helium) flow rate was 2 mL/min. For the identification of each fatty acid, pure standards were used (Sigma). The concentration of individual fatty acids was calculated as a % of total fatty acids. The results were expressed as grams per 100 g of detected FAMEs.
6
Extracting and Analyzing Photosynthetic Pigments
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Chlorophyll and carotenoids were extracted and analyzed as reported by Castagna et al.39 (link). Carotenoids included β-carotene, neoxanthin, lutein, violaxanthin, antheraxanthin and zeaxanthin. Leaf disks were homogenized under dimmed room light in 100% HPLC-grade acetone with 1 mM sodium ascorbate. The extract was filtered through 0.2-µm filters (Sartorius Stedim Biotech, Goettingen, Germany) and analyzed by a Spectra System P4000 HPLC equipped with a UV 6000 LP photodiode array detector (Thermo Fisher Scientific, Waltham, MA) using a Zorbax ODS column (5 μm particle size, 250 × 4.6 mm Ø, Agilent Technologies, Santa Clara, CA, USA) at a flow rate of 1 mL min−1. Acetonitrile/methanol (85/15) and methanol/ethyl acetate (68/32) were used as solvent A and solvent B, respectively, according to the following gradient: solvent A: 100% (0–15 min), 100–0% (15–17.5 min), 0% (17.5–32 min), followed by 5 min re-equilibration in the initial condition before the next injection. Commercial standards of chlorophyll a, chlorophyll b, lutein and β-carotene were used for external calibration curves. Pigments were detected at 445 nm and quantified by injecting known amounts of pure standards (Sigma-Aldrich, Milan, Italy).
7
Honey Sugars Quantification by HPAEC-PAD
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The sugars from the honey varietals (glucose, fructose, and sucrose) were quantified using high-performance anion exchange chromatography with pulsed amperometry detection (HPAEC-PAD, Dionex ICS-5000; Thermo-Fisher, USA) in conjunction with CarboPac PA1 guard (4 mm x 50 mm; Thermo-Fisher, USA) and analytical (250 mm x 4 mm) column. The sugars were eluted at 25˚C in 10 mM NaOH for 15 min, followed by 100 mM NaOH for 30 min at a 1 mL/min flow rate. Honey samples were diluted in deionized water and filtered through a 0.45 μm nylon filter injection into the chromatographic system. Calibration curves were constructed from pure standards (Sigma-Aldrich, St. Louis, MO) and were used to quantify honey sugars.
8
Quantitative Analysis of Polyphenols
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All polyphenols were identified by using pure standards (Sigma-Aldrich, Warsaw, Poland) and the retention times for the internal standards. Standard curves prepared for all phenolic compounds are presented in Figure 1 and Figure 2 . Pure phenolic standards were used for standard solution preparation. From each standard solution, five injections were made. Each time, the chromatographic pick area was determined and calculated based on the standard solution concentration. From standard curves, a mathematical equation was prepared. On the basis of the dilution coefficient and equation, the concentration of individual compounds was calculated.
9
Amino Acid Profiling of Zebrafish Embryos
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To evaluate the amino acid incorporation efficiency of sonophoresis technique the FAAs profile of the zebrafish embryos was analysed. FAAs profile of the larvae at the end of the experiment (22 dpf) were also analysed to evaluate the larval metabolic modulation of FAAs along the experimental period. Prior to analysis all samples were freeze-dried. FAAs analysis of zebrafish embryos and larvae was performed after homogenization in 0.1 M HCl on ice, centrifugation at 1500g at 4°C for 15 min and deproteinization of the supernatant by centrifugal ultrafiltration (3 kDa cut-off, 13500g at 4°C for 20 min). Samples were precolumn derivatized with Waters AccQ Fluor Reagent (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) using the AccQ Tag method (Waters, Milford, MA). Analyses were performed by ultra-high-performance liquid chromatography (UPLC) on a Waters Reversed-Phase Amino Acid Analysis System, using norvaline as an internal standard. Amino acids were identified by retention times of standard mixtures (Waters) and pure standards (Sigma, Madrid, Spain). Instrument control and data acquisition and processing were achieved through Waters Empower software.
10
Tissue Vitamin Analysis by HPLC
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The research was carried out using the equipment of the Core Facility of the Karelian Research Centre of the Russian Academy of Sciences. All analyses were performed within a month after sampling of tissues. The concentrations of retinol and α-tocopherol in tissues were determined by high performance liquid chromatography.
The samples of tissues (100 mg) were homogenized in 0.9 ml of 0.25 M sucrose solution (pH 7.4); then 0.25 ml of homogenate was mixed with ethanol containing an antioxidant (butylated hydroxytoluene) to precipitate proteins. After that, n-hexane was added. The mixture was vortexed for 5 min for the extraction of vitamins, centrifuged at 3000 × g for 10 min, and kept for 40 min at 4 °C. The hexane layer was injected into the HPLC system. Chromatographic separation was carried out by microcolumn chromatography with a UV detector with n-hexane and isopropanol as an eluent (98.5:1.5). The eluate was monitored at 292 nm for α-tocopherol and at 324 nm for retinol, and the vitamins were identified by retention time compared with pure standards (Sigma-Aldrich). Quantification was performed using Uni-Chrom software by the external standard method. All samples were analyzed in triplicate and the mean value was used.
The samples of tissues (100 mg) were homogenized in 0.9 ml of 0.25 M sucrose solution (pH 7.4); then 0.25 ml of homogenate was mixed with ethanol containing an antioxidant (butylated hydroxytoluene) to precipitate proteins. After that, n-hexane was added. The mixture was vortexed for 5 min for the extraction of vitamins, centrifuged at 3000 × g for 10 min, and kept for 40 min at 4 °C. The hexane layer was injected into the HPLC system. Chromatographic separation was carried out by microcolumn chromatography with a UV detector with n-hexane and isopropanol as an eluent (98.5:1.5). The eluate was monitored at 292 nm for α-tocopherol and at 324 nm for retinol, and the vitamins were identified by retention time compared with pure standards (Sigma-Aldrich). Quantification was performed using Uni-Chrom software by the external standard method. All samples were analyzed in triplicate and the mean value was used.
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