Chromatography Forum

Hi

During the analysis of the drug Tizanidine we use a mixture of 80% of a pH 3.0 buffered solution of sodium hexanesulfonate + 20% acetonitrile for the MP (modified USP method). When this is performed the retention time is approximately 10 min for the Tizanidine peak.

However, one of our chemists did by mistake replace the acetonitrile phase with methanol and suddenly the retention time switched to 60 min.

I have never seen these dramatic changes in retention time when replacing acetonitrile with methanol before. Can someone perhaps explain this behaviour?

The column is a C18, 25 x 4.6 cm, 5 um maintained at 50°C. The flow rate is 1 ml /min. HPLC: Waters Alliance.

I find that the lower the organic content, the greater difference there is between using ACN vs MeOH. For instance, if I have a method with 60% ACN and replace it with 60% MeOH, my retention more or less doubles, while it can quadrouple for a method that has 20% or so organic.

In my experience, using pic amplifies this effect.

The change of mobile phase ( CH3CN to MeOH ) for alkyl sulphonates is known to have a very profound effect on basic compound retention.

A whole new table of solvent strength realtionships was once offered up for alkyl sulphonate ion pair chromatography, but I'm not sure if it still applies to modern columns. It's shown in Practical HPLC Method Development by Snyder, Kirkland, Glajch.

Bruce Hamilton

ACN vs MeOH

With ACN the total amount of ion pairing reagent adsorbed on the column is smaller due to the higher hydrophobicity of ACN.

Also I would expect that electrostatic interactions would be stronger in a MeOH:H2O than a ACN:H2O mobile phase...