UPLC of Porphyrins
ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY (UPLC) QUANTITATION OF PORPHYRINS
- Inject 10µL of supernatant solution of porphyrins in 1.5M HCl into a Waters Acquity UPLC system which includes a binary solvent manager, sample manager, fluorescence detector, column heater and an Acquity UPLC BEH C18, 1.7 µM, 2.1 x 100 mm column.
- Set the fluorescence detector at 404nm excitation and 618nm emission. Keep the sample chamber dark at ambient temperature. Solvent A is 0.2% aqueous formic acid while Solvent B was 0.2% formic acid in methanol.
- Set the flow rate at 0.40 mL per minute at 60˚C for the total run time of 8 min.
- Use the following successive gradient settings for run time in minutes versus
- A: 0.0, 60%
- 2.0, 20%
- 2.5, 1%
- 3.5, 1%
- 3.6, 60%
- Set the solvent composition gradient from 0.0 to 2.0 min as Waters Gradient 5 (convex with a higher slope at 0.1 min compared to that at 2.0 min).
- Keep all other gradients are linear.
Use solutions of known concentrations of authentic porphyrins dissolved in 1.5M HCl as standards (Porphyrin Acids, Chromatographic Marker, Product # CMK-1A, Frontier Scientific, Logan, Utah).
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