Q: I am thinking of replacing the column cleaning liquid (and mobile phase) with methanol. What proportion should I use and what flow rate should I set?
Refer to the instruction manual for your column to check whether or not methanol can be used with it. Before delivering solvent with a high proportion of methanol, in order to prevent the precipitation of salt in the mobile phase (e.g., buffer solution), first deliver a volume of methanol and water, mixed with the same ratio as the acetonitrile and water currently used (e.g., when replacing a 4:6 mixture of 10-mM phosphate buffer and acetonitrile, first deliver a 4:6 mixture of water and methanol), equivalent to about five times the column capacity, and then increase the proportion of methanol. For example, if you have been performing analysis with a 60% proportion of acetonitrile, try using an 80% proportion of methanol. If the proportion of methanol is greater than or equal to the proportion of acetonitrile, salt is normally not precipitated since methanol/water has greater salt solubility than acetonitrile/water.
If methanol is used instead of acetonitrile, the pressure may increase. Deliver mobile phase at about half the flow rate used in analysis, and then increase it within the maximum pressure range of the column while observing the effect. Care is required in gradient analysis because the pressure may increase as the proportion of organic solvent increases. To be safe, it is better to set “P.Max” for the pump. (This is the maximum pressure at which delivery is possible. Delivery stops if the pressure exceeds this setting.)