For pigment extraction, 50 mg of leaf material from WT and kvc was ground in liquid nitrogen directly after 6 h CL and FL treatments. The pigments were extracted using alkaline acetone: 0.2 M NH4OH 9:1 (v/v) and incubation for 30 min at −20°C followed by centrifugation for 30 min (15,000 × g, 4°C). The HPLC analysis of the pigment‐containing supernatant was performed on Agilent LC systems (Agilent, USA) equipped with a DAD detector (440 nm, Agilent 1100 series) using a Prontosil column (200‐3‐C30, 3 μm; Bischoff Chromatography, Germany). For gradient elution, the composition of the mobile phase (A:B, v/v) with A = 90% acetonitrile, 10% H2O, 0.1% triethylamine, and B = 100% ethyl acetate was changed as follows: t0min 100:0, t1min 60:40, t19min 0:100, t20min 0:100, t2.1–23min 100:0. The mobile phase flow rate was 1.6 mL min−1. Peaks were quantified using authentic standards.
Agilent lc systems
Manufactured by Agilent Technologies
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Agilent LC systems are high-performance liquid chromatography (HPLC) instruments designed for analytical and preparative separations. They provide precise control of mobile phase flow, temperature, and other parameters to achieve efficient and reproducible separation of complex mixtures. Agilent LC systems offer a range of configuration options to meet the diverse needs of various applications in the pharmaceutical, chemical, and life sciences industries.
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2 protocols using agilent lc systems
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Pigment Extraction and Quantification
2
Quantifying Leaf Tetrapyrroles and Heme
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Tetrapyrroles were extracted from homogenized leaves using 300 µL of acetone:0.2 M NH4OH (9:1), incubated for 30 min at −20°C and centrifuged for 30 min (16,000 × g, 4°C). The supernatant was analyzed by HPLC for (Mg) porphyrins and Chls. Noncovalently bound (ncb) heme was extracted from the remaining pellet by resuspension in 200 µL of AHD (acetone:HCl:DMSO, 10:0.5:2) and incubated for 15 min at RT. After centrifugation for 15 min (16,000 × g, RT), the amount of ncb heme in the supernatant was quantified by HPLC. HPLC analyses were performed on Agilent LC systems following previously described methods (Richter et al. 2019 (link)) and using authentic standards for peak quantification.
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