Carotenoids were extracted from lyophilized and homogenized chilli pepper powder. A 10 mg aliquot of tissue was used in the extraction process, employing chloroform (500 µl) and HPLC-grade methanol (250 µl). The suspension was incubated on ice, in the dark, before HPLC-grade water (250 µl) was added. A phase separation was created, and the organic layer was collected. Chloroform (500 µl) was again added to the material for extraction, a phase separation was created, and the organic layer was pooled with the initial organic phase. Separation and detection of carotenoids was performed by HPLC with photodiode array (PDA) detection, using a C30 reverse-phase column (250 × 4.6 mm), purchased from YMC, Wilmington, NC, USA. The solvent system used has been detailed previously (Fraser et al., 2000 (link)). Carotenoids isolated from pepper fruit exocarp discs (1 cm) were extracted by washing discs in chloroform and methanol (1:1 ratio; 10 ml) in dark conditions, on a rotator. Wash solution was replaced every 24 h, and all solvent used for extraction were pooled and evaporated. Exocarp carotenoids were analysed by HPLC-PDA, as described.
C30 reverse phase column
Manufactured by YMC
Sourced in United States
The C30 reverse-phase column is a laboratory equipment used for chromatographic separation and analysis. It is designed to separate and purify a wide range of compounds based on their hydrophobicity. The column features a stationary phase composed of C30 alkyl chains bonded to silica particles, which provides high-resolution separation capabilities.
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4 protocols using c30 reverse phase column
1
Carotenoid Extraction and Analysis from Chili Peppers
2
Carotenoid Extraction and HPLC Analysis
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Carotenoids were extracted with acetone from ca. 20 mg lyophilized mycelia samples in a Fast-Prep-24 homogenizer (MP Biomedicals LLC Europe, France) using sea sand (Panreac Química SAU, Barcelona, Spain) and two pulses of 30 s at 6 m s -1 . In each pulse, 1 ml acetone was added and the extracted fractions were collected and vacuum dried. Total amounts of colored carotenoids were estimated from absorption maxima in hexane, using an average maximal ε (1 mg ml -1 cm -1 ) of 0.2.
For in vitro analyses, HPLC separations were performed in a Waters System (Waters, Eschborn, Germany) equipped with a photodiode array detector (model 996) and a C30 reverse-phase column (YMC Europe, Schermbeck, Germany), using solvent A (acetonitrile:tetrahydrofuran:NH 4 acetate 1% H 2 O 4:1:5, v/v/v) and B (acetonitrile: tetrahydrofuran:H 2 O 50:40:6, v/v/v). The column was developed at a flow rate of 1 ml min -1 , with a linear gradient from 100% A to 100% B within 20 min, followed by 5 min at a flow rate of 2 ml min -1 and another 10 min to 100% A.
For in vitro analyses, HPLC separations were performed in a Waters System (Waters, Eschborn, Germany) equipped with a photodiode array detector (model 996) and a C30 reverse-phase column (YMC Europe, Schermbeck, Germany), using solvent A (acetonitrile:tetrahydrofuran:NH 4 acetate 1% H 2 O 4:1:5, v/v/v) and B (acetonitrile: tetrahydrofuran:H 2 O 50:40:6, v/v/v). The column was developed at a flow rate of 1 ml min -1 , with a linear gradient from 100% A to 100% B within 20 min, followed by 5 min at a flow rate of 2 ml min -1 and another 10 min to 100% A.
3
Quantification of Broccoli Sprout Phenolics
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Identification and quantification of individual phenolic compounds were performed as described by Sánchez et al. [51 (link)]. Briefly, 25 µL of clarified ethanolic extracts, previously filtered using 0.45 µm nylon membranes (VWR, Radnor, PA, USA), were injected in the HPLC-DAD system (1260 Infinity, Agilent Technologies, Santa Clara, CA, USA). Carotenoids and chlorophylls were separated on a C30 reverse phase column (4.6 mm × 150 mm, 3 µm particle size) (YMC, Wilmington, NC, USA), coupled to a corresponding C30 guard cartridge. The temperature of the vial chamber was 4 °C, and column temperature was 30 °C. The mobile phase consisted of 50% methanol, 45% MTBE, and 5% water. The system was isocratic. Total elution time was 35 min at a constant flow rate of 0.5 mL/min. Carotenoids and chlorophylls were detected at 450 nm, and identified by comparing their retention times and absorption spectra with reference standards or previous reports [18 (link),49 (link),50 (link),51 (link)]. Xanthophylls were quantified using a calibration curve of lutein standard (0–12 ppm), and expressed as mg of lutein equivalents per kg of broccoli sprouts DW. For the quantification of chlorophylls, a standard curve of chlorophyll b was prepared in the range of 10–175 ppm. The concentration of total and individual chlorophylls was expressed as mg of chlorophyll b equivalents per kg of broccoli sprouts DW.
4
Serum Carotenoids and Tocopherols Analysis
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