by
lmh » Wed Nov 22, 2017 2:59 pm
You can estimate the t0 anyway; work out the cross sectional area of the column in mm^2 using pi-r^2, and multiply by length (in mm) to get the volume in uL. Probably only about 60% of this volume is accessible to solvent. All HPLC systems will also have a dead volume of tubing etc. between the autosampler and the detector, which you'll have to guess. Add this to the column volume and divide by flow.
A 250*4.6mm column has a volume accessible to solvent of about 2.5mL, so if you're using a column like this on a system with not too much tubing and 1mL per min flow-rate, then a retention time of 2.5min means the compound isn't retained at all, and any increase in acetonitrile percentage will make no difference. You need to decrease the percentage until you have retention. If you have no idea what percentage is appropriate, one option is to run a gradient from about 3% up to your current percentage and see what happens (I don't recommend 0% because some columns don't like running in 100% aqueous and will "de-wet", temporarily losing the ability to interact with anything, and requiring a long purge in 100% organic to recover).