322 hplc pump

All chemicals used were purchased pure from commercially available sources such as Sigma Aldrich, VWR, Fisher or other chemical vendors. ¹H NMR (300 MHz) spectra were recorded on a Bruker Biospin NMR spectrometer. Thin layer chromatography was performed using Whatman silica gel 60 Å plates with florescent indicator and visualized using a UV lamp (254 nm) or KMnO₄ stain. Flash chromatography was performed on Grace with GraceResolve Normal Phase disposable silica columns. High performance liquid chromatography (HPLC) was performed on a Gilson 322 HPLC pump with a Gilson UV/VIS-155 detector and a Phenomenex Gemini C18 column (10 µm, 250 mm × 10 mm). Liquid chromatography electrospray ionization mass spectroscopy (LC–MS/ESI–MS) were acquired on an Agilent LC/MSD-SL with an 1100 HPLC and G1956B mass spectrometer with a Phenomenex Gemini 5 μm C18 110 Å 50 × 3 mm column.

AAI (CAS number 313-67-7) and AAII (CAS number 475-80-9) were purified from the commercially available AA mixture by reverse phase chromatography. Injections (1 mL) of AA mixture, at 10 mg/mL concentration in acetonitrile/25 mM triethylammonium acetate (TEA) mix, were made onto a Gemini column (10 μm, 250 × 21.2 mm, C18, Phenomenex, USA). Chromatographic separation at room temperature was carried out using 322 HPLC pump (Gilson, USA) over a 15-min gradient elution from 25:75 to 40:60 acetonitrile/25 mM TEA at a flow rate of 20 mL/min. Collection was triggered by UV signal (acquired at 254 nm) and collected using a Gilson GX-281 Liquid Handler system (Gilson, USA). The resulting TEA salts of AAI and AAII were converted back to the free acids using HCl. Addition of molar equivalent of NaOH produced the sodium salts which were freeze-dried for use. The purity of AAI and AAII (as sodium salts) was checked using 1D and 2D NMR and high-resolution mass spectrometry (HRMS) which confirmed the compound structures.