Hplc chromatograph

Porphyrins from cell lines were separated by HPLC analysis on a ODS Hypersil C18 column (5 µm, 3 mm × 200 mm; Thermo Scientific, Waltham, MA, US) in a HPLC chromatograph (Shimadzu, Long Beach, CA, US). Porphyrins were separated with a 60 min gradient elution and a two-component mobile phase consisting of ammonium acetate (1 M, pH 5.16, solvent A) and 100% acetonitrile (solvent B) at a flow rate of 1 mL/min. All analyses were performed at 20 °C, and porphyrins were detected by fluorescence with an excitation wavelength 405 nm and emission wavelength 610 nm.

¹H NMR (in CDCl₃) and FTIR spectra of MMAA were recorded on a Bruker DPX 300 spectrometer (Billerica; MA, USA) and a Perkin Elmer PARAGON 1000 PC spectrometer (Bucks, UK), respectively. Gas and HPLC SEC chromatography was performed on a Clarus 500 Perkin Elmer gas chromatograph (Shelton, CT, USA) and a Shimadzu HPLC chromatograph (Kyoto, Japan) equipped with a mass spectrometry and UV–vis, RID, or DAWN 8 MALS detectors (Wyatt Technology; Santa Barbara, CA, USA), respectively. Agilent DB-35MS GC column (30 m × 0.25 mm id, 0.25 µm film; helium carrier gas) and Chromolith RP-18 e HPLC column (100 mm × 4.6 mm id) with a flow rate of 5 mL acetonitrile/water mixture per min were used. Number- (D_n) and weight-average particle diameter (D_w) and polydispersity index (PDI = D_w/D_n) characterizing the particle size distribution were determined from analysis of at least 500 particles on micrographs from a FEI Tecnai G² Spirit transmission electron microscope (TEM; Brno, Czech Republic). Dynamic light scattering (DLS) was measured on a Zetasizer Nano-ZS Model ZEN3600 (Malvern Instruments; Malvern, UK) providing hydrodynamic diameter D_h and polydispersity PI. UV–vis spectra were recorded on a Specord 250 Plus spectrophotometer (Analytic Jena; Jena, Germany).

The polar fraction of oil was analyzed by HPSEC to determine the contents of oligomers, dimers, monomers, DAG, and free fatty acids (FFA). The polar fraction of the oil was dissolved in tetrahydrofuran and analyzed using a Shimadzu HPLC chromatograph equipped with a SIL-10AD injector, a LC-20AD pump, and a RID-10A refractive index detector. The separation was performed on two 100 and 500 Å phenogel columns (30 × 0.78 cm internal diameter) packed with porous, highly cross-linked styrene–divinyl benzene copolymers (film thickness 5 µm) (Phenomenex, Torrance, CA, USA) connected in series, with tetrahydrofuran (1 mL/min) as the mobile phase. The columns were connected in a series to improve the separation of the oil components. Each group of the polar fraction distributions was quantified using the following equations:




where P_TPC, P_oligomer, P_dimer, P_oxTAG, P_DAG, and P_FFA represent the percentages of total polar compound, TAG oligomers, TAG dimers, monomeric oxidized triacylglycerols, diacylglycerols, and free fatty acids found in the polar fraction of the oil sample, respectively; A_oligomer, A_dimer, A_oxTAG, A_DAG, and A_FFA represent the peak area of each specific fractions; and ∑A represents the sum of all peak areas [7 (link)].