Luna pfp c18 column

An Agilent HPLC 1100 series system consisting of a binary pump, a column oven, a degasser, a diode array detector and an autosampler was used for chromatographic analysis. The purity check of 20D₃ analogs was carried out on a Phenomenex Luna-PFP C18 column (5 μm, 250 mm × 4.6 mm; Phenomenex) maintained at 25°C. The flow rate was 1.0 ml/min. The UV absorption at 263 nm was set for displaying the chromatograms. Isocratic elution using water (A) and acetonitrile (B) was as follows: 60% B for compound 10a, 70% B for compound 10b, 70% B for compound 10c, 80% B for compound 10d and 90% B for compound 10e.

Purities (SI Figure S28–S29) of final VD3 products (17a and 17b) were determined by using an Agilent HPLC 1100 system and a Phenomenex Luna-PFP C18 column (5 μm, 250 mm × 4.6 mm, Torrance, CA) at 25 °C and a flow rate of 1.0 mL/min. Acetonitrile and water were used as mobile phases with a gradient comprising 40–70% acetonitrile for 30 min; 263 nm was used to display chromatograms. The purities of 17a and 17b were determined as ≥98%.
For identifying which chemically synthesized product was identical to enzymatic product by HPLC, retention times of chemically synthesized isomer I (17a) and isomer II (17b) were compared with that of enzymatic 20S,23(OH)2D3. HPLC was carried out on a C18 column (Grace Alltima, 25 cm × 4.6 mm, 5 μm), with secosteroids being monitored by a UV detector at 265 nm. Two different solvent systems were used, 64–100% methanol for 20 min, then 100% methanol for 25 min or 45–100% acetonitrile for 25 min, then 100% acetonitrile for 25 min. The flow rate was 0.5 mL/min.