Chromatography Forum

Hi everyone! I ordered a standard that I want to use for quantitative analysis. The standard comes dissolved in pure methanol, however my sample injections for my unknowns are in 70:30 MeOH:H2O.

Question - when diluting my standard down to build a calibration curve, is it recommended that I calculate what volume of H2O to add for each dilution to create a 70:30 MeOH:H2O solution? Or will there be a negligible difference if I keep everything in methanol?

70/30 is already pretty high, so my guess is that a 100% MeOH diluent won't cause a problem unless you're injecting a large volume. In any case, it's easy to check: try it and if the peak shape is OK, don't worry.
If you really want to avoid 100% MeOH, just diilute with 70/30. The little bit of excess MeOH from the initial solution won't hurt anything.

What Tom stated: injecting from pure methanol, keep the injection volume small if larger injections hurt peak shape, you'll need to try out.

Or make 70/30 solution and just use that to dilute everything, then you won't have to calculate how much to add.

Typically - if possible, feasible - one would rather have the sample extraction solvent match up to that as used for the injected standard. But that's not a necessity; we often extract samples (consumer products, duh!) with methanol or dimethylformamide and then assay using aqueous-ACN mixtures on HPLC, works fine. In general, we inject 5 or 3 microliters so don't have any effects from the strong solvents and a partially aqueous mobile phase. It also depends on how much organic is in your starting mobile phase/isocratic mobile phase.