Pigments were extracted twice in 100% methanol (HPLC grade, VWR Czech Republic) from wet cells or purified complexes. The extracted pigments were centrifuged at top speed for 3 min in the Eppendorf centrifuge and the supernatant evaporated to dryness under a stream of nitrogen. The dried sample was dissolved in 50 μl of HPLC grade methanol mixed with 5 µl of 25% of ammonium acetate buffer and 20 μl was injected into a SCL-40 HPLC (Nexera series, Shimadzu, Japan) equipped with SPD-M40 PDA detector. The pigments were separated using a reverse-phase analytical C8 column (Kinetex, 2.6 µm, 100 Å, 150 × 4.6 mm column, Phenomenex, USA) heated at 40 °C and the following mobile phase was employed: 25% 28 mM ammonium acetate in water with 75% methanol (solvent A) and 100% methanol (solvent B) at a flow rate of 0.8 ml/min. Gradient is as follows: A/B 0/100 (23 min), 0/100 (25 min),100/0 (27 min), and 100/0 (28 min). Eluting pigments peaks were monitored at spectral range of 250–800 nm and was collected manually. Samples were handled in ice under the dim light condition to minimize its oxidation and isomerization.
Scl 40 hplc
Manufactured by Shimadzu
Sourced in Japan
The SCL-40 is a high-performance liquid chromatography (HPLC) system designed and manufactured by Shimadzu. It is a comprehensive and versatile instrument capable of performing a wide range of HPLC applications. The SCL-40 provides precise control and monitoring of various parameters, including flow rate, temperature, and pressure, to ensure the reliable and reproducible separation and analysis of chemical compounds.
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2 protocols using scl 40 hplc
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Extracting and Analyzing Pigments via HPLC
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Extraction and HPLC Analysis of Pigments
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Pigments were extracted twice in 100% methanol (HPLC grade, VWR Czech Republic) from cells or purified complexes. The extracted pigments were centrifuged at top speed for 3 min in the Eppendorf centrifuge and the supernatant evaporated to dryness under a stream of nitrogen. The dried sample was dissolved in 50 μl of HPLC grade methanol mixed with 5 µl of 25% of ammonium acetate buffer and 20 μl was injected into a SCL-40 HPLC (Nexera series, Shimadzu, Japan) equipped with SPD-M40 PDA detector. The pigments were separated using a reverse-phase analytical C8 column (Kinetex, 2.6 µm, 100Å, 150 x 4.6 mm column, Phenomenex, USA) heated at 40°C and the following mobile phase was employed: 25% 28 mM ammonium acetate in water with 75% methanol (solvent A) and 100% methanol (solvent B) at a flow rate of 0.8 ml/min. Gradient is as follows: A/B 0/100 (23 min), 0/100 (25 min),100/0 (27 min), and 100/0 (28 min). Eluting pigments peaks were monitored at spectral range of 250-800 nm and was collected manually. Samples were handled in ice under the dim light condition to minimize its oxidation and isomerization.
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