John W. Dolan

The question went something like this: "I have a method that works well, but I am trying to scale it down so that I can save acetonitrile. The method uses a 150 mm × 4.6 mm, 5-μm particle C18 column operated at 2 mL/min at 30 °C. This is an isocratic method run with 40% acetonitrile and 60% of 0.1% formic acid. My sample is dissolved in mobile phase and I inject 20 μL. My peaks come out at 1.5 and 1.65 min, with an overall cycle time of 4 min. The resolution requirement of R_s > 2.0 is obtained easily. I decided to switch to a smaller diameter column to reduce the acetonitrile consumption. So I switched to a 150 mm × 2.1 mm, 5-μm C18 column that I found on the shelf and dropped the flow rate to 1 mL/min, because the column is about half the diameter of the original one. Now I can barely pass the resolution requirements and sometimes fail. The pressure is higher than before and the peaks come out earlier. This seemed so simple, but I must have done something wrong. Can you help me?"

The Rule of One

First, we need to make sure we have not made a chemistry change in the system. Based upon the question, I'm not sure if the column chemistry is the same between the two columns. There was a time when everyone thought that all C18 columns were created equal, but today, with literally hundreds of C18 columns to choose from, it might be more surprising if two are chemically the same than if they are different. The 2.1-mm column should be from the same brand and line of packing material as the original 4.6-mm column. Because this wasn't mentioned specifically in the question, I want to make sure it is not overlooked. A second way the chemistry of the column can change is if it has been used for other samples. A column that was "found on the shelf" might or might not be new. If it is used, it still might be OK to use, but this decision should be based upon a column log sheet that records column history and column testing. Any column with unknown history should, in my opinion, be filed in the dumpster. Columns are consumable items with finite lifetimes, and it isn't worth the risk of creating problems with a method by using a column with unknown history. Either of these changes, a different manufacturer's C18 material or a used column, can mean a change in the column chemistry and, thus, a possible change in peak spacing — one of the possible reasons the resolution requirements are hard to meet with the new column. For the moment, let's assume that the 2.1-mm column was from the manufacturer and packing type and was new or like-new.