Liquid chromatography problem solving and troubleshooting

Question: After mixing my mobile phase and using it over the course of several weeks, the retention times of my compounds gradually increase, ending up approximately 20–30% longer. Upon preparing a new mobile phase, the retention times almost immediately return to where they originally were. Then, the situation occurs again over the lifetime of the new mobile phase. This cannot be a pump problem, because I rigorously degas my premixed mobile phase (methanol–water, 50:50). Presently, I relate the changes to an internal standard; ideally, I would like to not have to do this. I am stumped as to how to maintain retention times in this real-world chromatographic situation. Answer: Your problem is an interesting one and may be a case where an " ounce of prevention " is required, but you applied a " pound of prevention ". Let me explain. Degassing your mobile phase is a correct " ounce of prevention " to protect your pump from swallowing an air bubble and ruining your chromatography. However, I believe that the increasing retention time results from a situation of overdegassing to the point of causing evaporation and loss of the more volatile solvent. Vigorously degassing over a long period of time can change the ratio of volatile to nonvolatile solvent in the mobile phase mixture. In your situation, because you mix 4 L of mobile phase at one time, degassing over a period of approximately 4 weeks can change the mobile phase composition, because the methanol preferentially evaporates. This would result in a weaker mobile phase and a longer retention time. I would suggest that you degas every morning and once or twice during the day rather than using a continuously active helium sparge, or you might consider purchasing a degassing unit that intermittently (rather than continuously) allows gas flow. This periodic degassing minimizes preferential evaporation of one of the solvents. Coming to this conclusion is a matter of eliminating other possible causes that are improbable because they do not correspond to all of the observations that you made. Because you were experiencing a problem with retention time, only a few other sources could be the cause. The first cause that comes to mind is that your column is changing with time; this could be column fouling with non-eluted sample, a plugged frit, non-equilibration of the column, or overload of the sample injected into the column. However, none of these …