Model lc 20at

¹H and ¹³C NMR, COSY, HMQC, HMBC, and NOESY spectral data were obtained using an Agilent Superconducting FT-NMR 400–500 MHz Spectrometer System. HR-ESI mass spectra were recorded on an Agilent Technologies, 6530 Accurate-Mass Q-TOF LC/MS. The HPLC system (Shimadzu, Japan) used was equipped with a pump (Model LC-20AT), an UV detector (Model SPD-20A at 254 nm), and a data station (Model CBM-20A). Column chromatography was performed using silica gel (230–400 mesh, Merck, Germany) and Sephadex LH-20 gel (25–100 μM mesh, Pharmacia, Sweden).

The chromatographic equipment (Shimadzu-Prominence) consisted of an on-line degasser (Model DGU 20A5), solvent delivery module (Model LC-20AT), autosampler (Model SIL-20AHT), column oven (Model CTO-20A), fluorescence detector (Model RF-10AXL) and system controller CBM-20A. A reverse-phase C8 column Vydac of 5 μm, 4.6 mm × 250 mm (Thermo Fischer Scientific, Massachusetts, USA) with a pre-column of 5 μm, 4.6 mm × 50 mm (Thermo Fischer Scientific, Massachusetts, USA) was used. The mobile phase consisted of a mixture of 0.12 % (m:v) TFA in Milli-Q water (pump A) and methanol (pump B) at 40:60 (v/v) rendering an isocratic phase. Fluorimetric measurements were carried out in a 12-μL flow cell at 610 and 675 nm excitation and emission wavelengths, respectively. The injection volume was 5 μl and the flow rate was 1 ml/min at a working pressure of 135 kgf.cm⁻². Analyses were performed with a column temperature of 30 °C. LCsolution Software (Shimadzu, Tokyo, Japan) was used for data processing.
The calibration curve was generated with AlPc solutions with concentrations ranging from 0.01 to 8 μM. For AlPc quantification in nanoemulsions, sample aliquots were dissolved in ethanol (1:40, v/v), vortexed for 3 min, filtered through 0.22-μm nylon filters (Millex GN, Millipore, Darmstadt, Germany), and injected into the HPLC system.

HPLC system (Model-LC-20AT, Shimadzu Corporation, Kyoto, Japan) equipped with low pressure quaternary gradient pump, column oven, autosampler, and PDA detector was used for analysis. Chromatography data was processed by LabSolution software (Shimadzu, Kyoto, Japan). Analysis was performed on Reverse phase C18 ACE^® (250 × 4.6 mm i.d., particle size 5 μm) column coupled with security guard column (4 × 3 mm, Phenomenex^®) at room temperature. The mobile phase was composed of 0.1 % phosphoric acid (solvent A) and methanol (solvent B) in gradient elution mode as follows: 0–1 min 85% of solvent A; 1–4 min linear gradient to 70% of solvent A; 4–5 min 70% of solvent A; 5–6 min linear gradient to 85% of solvent A; 6–20 min 85% of solvent A to re-equilibrate the system. The mobile phase was degassed before pumping into the HPLC system at a flow rate of 1.0 mL/min. Injection volume of 50 μL was used. The wavelength monitored was 280 nm.