Spd m20a photodiode array detector

Under dim light, pigments were extracted twice from cells with 1 ml of acetone/methanol (7:2, v/v) immediately before HPLC analyses. After centrifugation, extracts were dried with a rotary evaporator. The residue was dissolved in chloroform/methanol (3:1, v/v) and then analyzed with an HPLC system equipped with Mightysil RP-18 GP analytical column (4.6 mm × 150 mm, 5 μm particles, Kanto Chemical, Tokyo, Japan) and guard column (4.6 mm × 5 mm, 5 μm particles, Kanto Chemical, Tokyo, Japan). The elution conditions were as follows: 0–10 min, linear gradient from 90% methanol/H₂O (v/v) to 100% methanol; 10–50 min, isocratic 100% methanol at 1.0 ml min⁻¹. Absorbance spectra (250–700 nm, 1.2 nm resolution) and retention times were recorded with an SPD-M20A Photodiode Array Detector (Shimadzu, Kyoto, Japan).
The composition of the major carotenoids was calculated from these molar absorption coefficients and areas under the peak in the chromatogram of absorbance at 445 nm. Relative carotenoid content per cell was calculated by normalizing molar ratios of major carotenoid species to chlorophyll a (carotenoids/chlorophyll a) based on absorbance at 445 nm in HPLC analysis with chlorophyll a content per cell (mol cell⁻¹) determined as described above.

A reverse phase HPLC system composed of the following modules was used: LC-6AD pumps, CBM-20A communication module, SIL-20A HT autosampler, CTO-20A column oven, and SPD-M20A Photodiode Array detector (Shimadzu, Columbia, MD, USA). The reverse-phase column selected was a 250 × 21.2 mm Luna pentafluorophenyl column with 5 µm particle size and 100 Å pore size (Phenomenex, Torrance, CA, USA). Samples were filtered prior to injection with a Phenex RC 0.45 µm, 15 mm membrane syringe filter (Phenomenex, Torrance, CA, USA). With a flow rate of 10 mL/min and a run time of 30 min, peaks were separated and collected. An isocratic system with the following solvents were used: 11:89 (Solvent A: Solvent B v/v) with Solvent A being 4.5% formic acid in HPLC grade water and Solvent B was HPLC grade acetonitrile. Elution of peaks was monitored at 500 nm. Peaks were manually collected. The two collected peaks were diluted with distilled water and again subjected to SPE semi-purification to remove formic acid and acetonitrile. Rotary evaporation was used to remove methanol (40 °C, under vaccuum), and the pigments were stored in 0.01% HCl in acidified water.

All analyses were performed using a Shimadzu Prominence series HPLC system (Shimadzu Corporation, Kyoto, Japan), equipped with an LC-20AB binary pump (Serial: L20124200883), SIL-20A HT autosampler (Serial: L20345256104), CTO-20AC temperature-controlled column oven (Serial: L2021525077), SPD-M20A photodiode array detector and CBM-20A communications bus (Serial: L20235154327). All equipment were controlled by Shimadzu Lab Solutions Lite software version 5.71 SP2. For separation, an Ultra C18 column, 3 μm, 50 × 4.6 mm (RESTEK Corporation, Bellefonte, PA) was used. Dynantin samples were analyzed at a constant solvent flow rate of 0.7 mL/min at 35 °C using a binary gradient (Table 1). Solvent A consisted of a 25% solution of acetonitrile (HPLC grade, Fisher Scientific, Fairlawn, NJ, USA) in ddH₂O (0.2 μm filtered) and solvent B consisted of acetonitrile with each solvent containing 0.1% trifluoroacetic acid (v/v, protein sequencing grade, Sigma Aldrich, Fairlawn, NJ, USA).

A prominence UFLC system from Shimadzu (Kyoto, Japan) equipped with a CBM-20A Communication Bus Module, SPD-M20A Photodiode Array Detector, LC-20AD Pump, SIL-20A Autosampler, and CTO-20A Column Oven was used. After a 10 μL injection, the analytes were separated through a Hector-M C18 column (4.6 × 250 mm, 5 μm) from RStech (Daejeon, Korea) using mobile phase (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile at a flow rate of 0.3 mL/min. The elution started at 0% B for 20 min and then was increased to 100% B for 110 min. The eluents were then detected by the LCMS-8040 system (Shimadzu) with an ESI interface source in positive and negative mode at 3.5 and −3.5 kV interface voltage, respectively. The MS source conditions were as follows: nebulizing gas flow rate, 3 L/min; desolvation line temperature, 250 °C; drying gas flow, 15 L/min; and heat block temperature, 400 °C.

The photosynthetic pigments were extracted from cell pellets in 90% (v/v) methanol and analyzed by high performance liquid chromatography (HPLC) in a LC-20AT High Pressure Liquid Chromatographer with a SPD-M20A Photodiode Array Detector (Shimadzu, Japan) as described in [15 (link)]. Pigments were identified by comparing retention times and absorption spectra with standard pigments (DHI, Hørsholm, Denmark), and their concentration determined via external authentic standards.

A Shimadzu HPLC system equipped with a size exclusion chromatography (SEC) column was used to determine the molecular weights (number-average molecular weight, M_n, and weight-average molecular weight, M_w), and dispersity (Đ) of ligands 4–6. Measurements were performed using 0.3 M sodium acetate buffer (pH 6.5) as a mobile phase for Superose 6 (10 × 300 mm) at a flow rate of 0.5 mL min⁻¹. Poly(HPMA-co-MA-AP-TT) polymer precursor used for preparing ligands 4–6 was measured in methanol/0.3 M sodium acetate buffer (pH 6.5, 4/1, v/v) using a TSKgel Super SW3000 (4.6 × 300 mm) at a flow rate of 0.3 mL min⁻¹. A multiangle light scattering (MALS) detector (DAWN HELEOS II, Wyatt Technology Co., USA), Optilab-rEX differential refractometer index (RI) detector and SPD-M20A photodiode array detector (Shimadzu, Japan) were employed.