Reverse-phase prep HPLC

Automation of Prep-HPLC. Reverse phase prep-HPLC separation has proven to be a very reproducible technique. For this reason, the process can be automated. In addition to the standard components of an HPLC system, the following are required for automation an autoinjector capable of large volume injections (2.0 to 5.0 ml), a programmable controller (a microprocessor controller), a fraction collector, and a waste valve (3-way valve) controlled by a solenoid (Fig. A). The microprocessor should allow programming of the solvent flow rate, the sequence for solvent gradient formation, the time of injection, advancement of the fraction collector (to collect specific fractions rather than just uniformly incremented advancement), and control of a waste valve. 

A mixture of rhodium II) acetate (228 mg, 0.516 mmol), the imidazolidinone (1.70 g, 6.15 mmol), and dry chlorobenzene (20 mL) is heated under reflux for 18 h in a flask fitted with a Soxhlet extraction apparatus into which a thimble is placed containing an oven-dried mixture of sodium carbonate and sand (2 1, 5 g). The progress of the ligand-exchange reaction can be monitored by HPLC (p-Bondapak-CN column, methanol). The resulting blue solution is concentrated under reduced pressure, and the residue is purified by column chromatography (reversed phase silica, Bakerbond Cyano 40 mm prep. LC packing, methanol). 

Application of Prep-HPLC to PGS Analysis. Reverse phase liquid chromatography has proven to be well suited for cleanup of plant extracts by prep-HPLC (4, 46, 47, 48). When the mobile phase is initially an aqueous buffer at pH 2.8, all but the highly charged (e.g., zeatin ribotide with 5 AMP used as a representative compound for zeatin ribotide) plant hormones are retained at the head of the column (Fig. 1). Since the PGS are retained, samples can be injected onto the column in a dilute form. In-... 

Isolation of Compounds VI-VIII. Methyl / -D-xylopyranoside (0.4 g) and saligenin (2-hydroxybenzyl alcohol, 0.3 g) were reacted in water (0.25 ml) at 140 °C for 80 minutes. A portion of the mixture (150 mg) was dissolved in 50% methanol in water and separated into fractions by preparative HPLC on a reversed-phase Waters Prep/PAK 500 Cis column with 50% methanol in water as solvent at 0.25 L/min with RI detection. The first (1L) fraction contained three peaks that were further separated on a reversed-phase Bondapak Cis column (4.6 mm x 30 cm). The solvent was a gradient of 30 to 90% methanol in water at 1 ml/min and the compounds were detected by UV at 280 nm. 

---