Carotenoid were extracted from fresh petals at the flowering stage and detected following the methods of Cao et al.51 (link). Carotenoid analysis was performed using LC-2010AHT HPLC (Shimadzu, Kyoto, Japan) with C30 column (YMC, Kyoto, Japan). Carotenoids were identified by the typical retention time of the standard compounds, including violaxanthin (Sigma-Aldrich, Saint Louis, America), lutein (Solarbio, Beijing, China), α-carotene and β-carotene (Wako, Osaka, Japan). The identification of prolycopene was performed based on reported the typical retention time and relative order of carotenoid compound peaks22 (link),43 (link),51 (link). Carotenoid content was quantified according to Morris’ method52 (link). The total carotenoid content was the sum of all the detected carotenoid compound contents. Three biological replicates were used for all analyses and the calculation of means and standard deviations were conducted. The significant difference between 92S105 and 15S1040 was analyzed by t-test.
C30 column
Manufactured by YMC
Sourced in Japan, United States, Germany
The C30 column is a chromatography column designed for the separation and purification of a wide range of organic compounds. It features a stationary phase made of a highly cross-linked polymer material, providing efficient and reliable separation performance.
Automatically generated - may contain errors
Lab products found in correlation
1200 hplc, by Agilent Technologies (2 mentions)
1100 hplc system, by Agilent Technologies (2 mentions)
Agilent 1100 system, by Agilent Technologies (2 mentions)
Analyst 1, by AB Sciex (2 mentions)
Hplc instrument, by Shiseido (2 mentions)
Violaxanthin, by Merck Group (1 mentions)
Lutein, by Solarbio (1 mentions)
α carotene, by Fujifilm (1 mentions)
Lc 2010a ht hplc, by Shimadzu (1 mentions)
β carotene, by Fujifilm (1 mentions)
Hplc system, by Shimadzu (1 mentions)
2695 hplc system, by Waters Corporation (1 mentions)
Beckman system gold, by Beckman Coulter (1 mentions)
Agilent 1200, by Agilent Technologies (1 mentions)
Malvern zeta sizer apparatus, by Malvern Panalytical (1 mentions)
Capsanthin, by Merck Group (1 mentions)
Zeaxanthin, by Merck Group (1 mentions)
Agilent 1260 hplc system, by Agilent Technologies (1 mentions)
E2695 separation module, by Waters Corporation (1 mentions)
Alliance 2695, by Waters Corporation (1 mentions)
40 protocols using c30 column
1
Carotenoid Extraction and Quantification from Flowering Petals
2
Quantitative Analysis of Carotenoids by HPLC
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The residue of carotenoids was dissolved in 1.5 mL methyl tert-butyl ether (MTBE) that contained β-apo-8′-carotenal as an internal standard (5 μg/mL) [51 (link)]. The carotenoid content analysis was performed by using a high-performance liquid chromatography (HPLC) system (Shimadzu Corporation, Kyoto, Japan). This system consists of an LC-20AT infusion pump, CTO-10AS VP column oven and SPD-M20A spectrophotometric detector. A C30-column (YMC Co. Ltd. Japan, 4.6 × 250 mm, 5 μm) was used at 25 °C, with a flow rate of 0.8 mL/min, and the data were recorded from 200 to 800 nm and measured at 450 nm. An amount of 10 μL analytes was injected. The elution solvents were solvent A (methanol) and solvent B (MTBE), and elution was performed as follows: 0 min, 0% B; 18min, 46% B; 35 min, 70% B; 37min, 70% B; 40min, 0% B; and 47min, 0% B.
3
Comprehensive Plasma Antioxidant Analysis
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Plasma malondialdehyde was measured by HPLC-fluorescence (FL) following thiobarbituric acid derivatisation and butanol extraction as we described (4) on a Shimadzu LC-20XR system equipped with a RF-20AXL FL detector set to 532/553 nm (excitation/emission). Plasma ascorbic acid and uric acid were measured from perchloric acid-treated plasma, and vitamin E (as αand γ-tocopherol) was measured following saponification and hexane extraction using our separate HPLCelectrochemical detection procedures (33) . Plasma carotenoids were measured as described (34) with minor modification. In brief, 200 µl of plasma was mixed with 200 µl of ethanol, 50 µl of β-apo-8-carotenal (60 nM, internal standard) before being extracted three times with 3 ml of methanol-tetrahydrofuran (1:1) and 4 ml of hexane. The extract was evaporated under N 2 gas and reconstituted in 150 µl methanol and 150 µl methyl tert-butyl ether before being filtered (0•22 µm) and injected (50 µl) onto the HPLC system. Carotenoids (lutein, zeaxanthin, αand β-carotene, αand β-cryptoxanthin, lycopene) were quantified at 450 nm on a Waters 2695 HPLC system equipped with a 2996 photodiode array detector. Separation was performed isocratically (1•0 ml/min) using a YMC C30 column (150 × 4•6 mm, 3•0 µm) and a mobile phase consisting of methanol-methyl tert-butyl ether-water (96:2:2).
4
Reversed-phase HPLC Analysis of Astaxanthin
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Reversed-phase HPLC of astaxanthin-containing samples was performed with a Beckman System Gold (Beckman Coulter, Brea, CA, USA) on a C30 column (4.6 mm × 250 mm, particle size 5 µm) (YMC Europe, Schermbeck, Germany) following a previously described method [58 (link)] with minor modifications. The absorbance was monitored at 480 nm by a Beckman 168 diode array detector. The injection volume was 50 µL. The elution was carried out at a flow rate of 1 mL/min using acetone (solvent A) and water (solvent B) as follows: isocratic elution at 84:16 (A:B) for 10 min and a gradient to 97:3 (A:B) for 100 min.
5
Phytohormone Profiling in Citrus Fruits
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The standards of abscisic acid (ABA), jasmonic acid (JA), indole-3-acetic acid (IAA) and salicylic acid (SA) were purchased from OlChemImm (OlChemIm, Olomouc, Czech Republic). Phytohormones were extracted according to previously method [16 (link)]. A 50 mg portion of lyophilized juice sacs were homogenized with 0.5 mL extraction solvent (isopropanol: water: HCl = 100: 50: 0.1). After extraction for 12 h at 0 °C, the samples were dipped in 0.5 mL extraction solvent, shaken for 1 h at 230 r/min, followed by the addition of 2 mL dichloromethane and shaking for another 1 h. After 10 min of centrifugation at 4000 g under 4 °C, the layers were transferred into a new 1.5 mL tube and dried with a gentle stream of nitrogen. The resulting residue was re-dissolved in 0.15 mL methanol, and then subjected to 15 min of centrifugation at 4000 g under 4 °C. 10 μL of the supernatant was taken out for analysis by using a UPLC-ESI-MS (Shimadzu Corporation, Kyoto, Japan) equipped with a C30 column (4.6 mm × 150 mm, 5 μm, YMC) as described before [12 (link)]. 0.02% acetic acid was prepared as mobile phase A, and 0.02% acetic acid-acetonitrile was prepared for mobile phase B. Gas flow was set to 250 μL min−1.
6
Carotenoid Quantification in Carrot Extracts
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The identification and quantification of extracts was carried out using HPLC-DAD consisting of a HPLC Agilent 1200 equipped with a UV/vis detector (DAD). Separation was attained at a temperature of 25 °C on a YMC C30 column (250 × 4.6 mm and 3 µm ID). The mobile phase in this case consisted of a combination of A (methanol/MTBE/water, 81/15/4) and B (methanol/MTBE/water, 10/90/4). The linear gradient was from 100% to 0% B (v/v) within 90 min. The pumping flow rate was 1.0 mL/min. The volume used for the sample volume was 5 µL, while the quantitative detection was measured at 450 nm. To establish carotenoids calibration curves, standard carotenoids were used with concentrations ranging from 5, 10, 25, 50, to 100 mg/L. Identification of the main carotenoids in carrot extracts was carried out in triplicate by comparing the retention times and absorption spectra.
7
Quantification of Vitamin E in Plasma and Tissue
8
Quantifying Lycopene in Nanospheres
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The content of lycoene in nanospheres was determined by high-performance liquid chromatography (HPLC) as previously described.[24 (link)] Briefly, 20 mg of prepared nanospheres were dissolved in 0.5 ml of dimethyl sulfoxide (DMSO) and mixed with 0.98 ml of methanol: Water (50:50 vol./vol.) solution. Then, the 50 μl of mixture was injected into the HPLC column. In the HPLC analysis, a C30 column (3 μm, 150 × 4.6 mm, YMC, Wilmington, NC, USA) was used. The mobile phase for the determination of the lycopene was methanol/methyl-tert-butyl ether/water (83:15:2, v/v/v, with 1.5% ammonium acetate in the water; solvent A and methanol/methyl-tert-butyl ether/water (8:90:2, v/v/v, with 1% ammonium acetate in the water; solvent B at a flow rate of 1.2 ml/min and then the percentage of lycoene loading was then calculated as:
Amount of lycoene in nanospheres × total volume tested × 100/total sample volume × initial amount of nanospheres.
The particle size, zeta-potential, and polydispersity index of the lycopene-loaded nanospheres were evaluated using Malvern Zetasizer apparatus (Malvern Instrument, Worcestershire, UK), as reported previously.[22 (link)] Briefly, the particle size and zeta-potential of nanospheres were measured at angles of 90° and 120° at 25°C, respectively. These experiments were done in triplicate.
Amount of lycoene in nanospheres × total volume tested × 100/total sample volume × initial amount of nanospheres.
The particle size, zeta-potential, and polydispersity index of the lycopene-loaded nanospheres were evaluated using Malvern Zetasizer apparatus (Malvern Instrument, Worcestershire, UK), as reported previously.[22 (link)] Briefly, the particle size and zeta-potential of nanospheres were measured at angles of 90° and 120° at 25°C, respectively. These experiments were done in triplicate.
9
HPLC Analysis of Photosynthetic Pigments
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A high-performance liquid chromatograph (1200 HPLC, Agilent Technologies, Santa Clara, CA, USA) was used to analyze samples grown under either GMF or NNMF condition. The molecules were separated, identified, and quantified as previously described [77 (link)]. The mobile phases (Solvent A [90% (v/v/v) MetOH, 3% (v/v/v) MTBE, and 5% (v/v/v) H2O]; Solvent B [88% (v/v/v) MTBE, 10% (v/v/v) MetOH, and 2% (v/v/v) H2O]) were fluxed in a thermally (25 °C) equilibrated C30 column (250 mm 2.1 mm i.d., 3 m, YMC America, Devens, MA, USA). In order to separate the pigments of interest, Solvent A and Solvent B were flushed at a constant flow rate of 0.2 mL/min following the ratio reported in Supplementary Table S2 . The compounds eluted from the column at different retention times (RT) were detected by a diode array detector (DAD) set at the following wavelengths: 661 nm (Chl a, Chl a′), 642 nm (Chl b, Chl b′), 667 nm (Pheo 566 a, Pheo a′) and 460 nm (carotenoids). Pigment identification and quantification were performed based on injections of pure standards.
10
HPLC Analysis of Chlorophylls and Carotenoids
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Chlorophylls, Chl degradation products, and CARs extracted from samples were analyzed with a high-performance liquid chromatography (1200 HPLC, Agilent Technologies, Santa Clara, CA, USA). Chromatographic separation, identification, and quantifications of the compounds were performed as previously described [20 (link)]. Briefly, in a thermally (25 °C) equilibrated C30 column (250 mm × 2.1 mm i.d., 3 μm, YMC America, Devens, MA, USA), the mobile phase consisting of 95% (v/v) Solvent A [90% (v/v/v) MetOH, 3% (v/v/v) MTBE, and 5% (v/v/v) H2O] and 5% (v/v) Solvent B [88% (v/v/v) MTBE, 10% (v/v/v) MetOH, and 2% (v/v/v) H2O] was fluxed. To both solution A and solution B, 50 mM Ammonium Acetate was added. In order to separate the pigments of interest, the various proportions of Solvent A and Solvent B shown in Supplementary Table S4 were flushed at a constant flow rate of 0.2 mL/min. Chromatograms were integrated for quantification at the following wavelengths: 661 nm (chlorophyll a and a′), 642 nm (chlorophyll b and b′), 667 nm (pheophytin a and a′), 655 nm (pheophytin b and b′) and 460 nm (carotenoids). Pigment identification was according to the literature data [50 (link)] and injection of pure standards.
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