Aqueous reverse phase = ARP
Aqueous normal phase = ANP
Organic normal phase = ONP

Normal Phase: Retention times decrease as most polar solvent % increases.
Reverse Phase: Retention times increase as most polar solvent % increases.

This page shows three chromatographic options for the separation of Metformin & Glyburide with the first and third chromatographs showing the least polar compound eluting first, the second chromatograph showing the most polar compound, (Metformin) eluting first.

By manipulation of the Metformin ANP response and Glyburide ARP response, that is by using the fundamental difference in their retention characteristics, it is possible to easily put either the highly polar or the mid polar peak first and having the second close by or infinitely retained.

The option even exists to make these compounds of vastly different polarity (LogP differences of over 10 to power of 6) to co-elute.

To develop this isocratic method, first the aqueous normal phase separation (ANP) of Metformin was defined, 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 start with the recommended acidic eluent.

A : Solvent = 0.05% v/v H3PO4 (0.5 ml of 85% concentrated H3PO4 to 1 liter  with DW)
B : Solvent = Acetonitrile

Begin with 20% B, expect that polar bases will elute near Solvent Front (SF), then progressively increase % B to 30 to 40 to 50 to 60 to 70 to 80 and so forth and expect that a polar base will begin to retain longer as % B increases. It is probable that the optimal concentration will be between 50 and 70 %, be aware of how other non-target peaks behave and note if their behavior is ANP or ARP.

For a compound that is moderately polar such as Glyburide expect it to separate in ARP order. Therefore start with 90% B and expect no retention with SF elution. Progressively reduce acetonitrile %.

Examine to see overlap in initial Retention Map profiles (simple plots of % B against time) and optimize % of B relative to required retention / selectivity.