Q: If I switch to methanol, do the retention times change? Also, is it possible to continue using the same column?


Because the elution capacity of a methanol/water mobile phase is smaller than that of an acetonitrile/water mobile phase (except in cases where the proportion of organic solvent is equal or very close to 100%), if acetonitrile is replaced with methanol in the same proportion, the retention times increase. You must first try using a slightly larger proportion of methanol (e.g., if the proportion of acetonitrile in the mobile phase in 60%, use an 80% proportion of methanol), and adjust the proportion so that the retention times are equal to those obtained with acetonitrile/water. Note also that the separation selectivity differs between methanol and acetonitrile and so the elution order may vary with the compound.
In many cases, you can probably use methanol with the same column. In particular, in method development with an ODS column, an acetonitrile/water mobile phase is often experimentally replaced with a methanol/water mobile phase in order to change the separation selectivity. Note the following points when using methanol:

  • Refer to the instruction manual for your column to check whether or not methanol can be used with it.
  • 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. 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.)


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