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 Our Real Life Example
To develop the isocratic method for Metformin and Glyburide, described above, the aqueous normal phase (ANP) of Metformin was defined as our first course of action, as this was the compound that was so polar that it could not be retained on conventional reverse phase columns. Metformin is a highly polar basic compound; therefore we started with the recommended acid isocratic mobile phase. Solvent A: 0.05% v/v H3PO4 (0.5 ml of H3PO4 (85% concentration) to 1 liter with DI Water) Solvent B: Acetonitrile HPLC Grade We began with 20% solvent B, and as we expected the polar base eluted near the Solvent Front (SF), we progressively increased %B, 30 to 40 to 50 to 60 to 70 to 80 up to our definition of infinite retention (20 minutes) to create our Retention Map of Metformin on this column under these conditions. We also expected that this polar base would begin to retain longer as % B increases, probably between 50 and 70 %, and therefore looked for how other non-target peaks behaved and noted when their behavior was ANP or ARP. For Glyburide we expected it to function in ARP, so we started with our mobile phase at 90% solvent B and expected no retention, with SF elution. Progressively we reduced the concentration of acetonitrile. We used these data points to add to our Retention Maps. We examined to see the overlap in initial Retention Map profiles and optimized percentage concentration of solvent B relative to required retention / selectivity noted on the Retention Maps. Click here for final results. 
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