A reader recently sent in this e-mail: “I am trying to find information that I can put in an SOP regarding how long a mobile phase can be used before it must be discarded. I’ve heard 1-2 weeks from some sources, whereas others say a month, and some people say not to worry about it – just use the mobile phase until the bottle is empty. Can you shed some light on this matter?”

It would be nice if there were cut-and-dried answer for questions like this. Unfortunately, the answer always seems to start with “it depends.” Let’s look at some important factors that can influence your decision on how long to use a mobile phase.

   Microbial growth in the mobile phase is a concern when the organic content of the mobile phase is sufficiently low to support such growth. I remember doing a test years ago in our laboratory, where we took a common aqueous mobile phase, such as 25 mM phosphate buffer, and let it stand at room temperature on a window ledge for an extended period of time. After two weeks we began to see an increase in the bacterial count, and I’m sure the solution would have become cloudy if we left it much longer. Based on this, and other experience, we decided to write our standard operating procedure (SOP) so that aqueous solutions could be used only for a week before discarding them. Of course you could limit growth by adding organic solvent to the solution. A general rule of thumb is that once there is 20-30% organic present, microbial growth will be minimized. But those little guys are clever – if there are microbes that can live on oil spills at the bottom of the ocean, they’ll figure out a way to live in my mobile phase. I think the one-week limit is reasonable. But if you are using UHPLC, there can be sufficient growth in 24 hr to cause frit blockage with the 0.2 µm porosity frits in many UHPLC systems. For this reason, many UHPLC workers replace the buffer daily.

HPLC Solutions #65: MP Lifetimes

   Organic solvents, such as 100% acetonitrile or methanol should be no problem in terms of growing bugs, and if they evaporate, you lose volume, but not concentration. We set a one-month limit on the use of such solvents, although this is an arbitrary time limit and probably conservative. If the organic solvent is mixed with something else, there may be selective evaporation of one component that could cause a change in mobile phase composition. I’m sure this is a problem with some methods, but I’ve only seen a clearly assignable cause for this problem a couple of times in my career, so I don’t think the problem is widespread.

   A couple other pointers on mobile phases. First, don’t ever replenish a reservoir – use the solvent and clean or discard the reservoir. If microbial growth or other contamination occurs, adding fresh solvent only contaminates the next batch. Second, you may be able to save time by making a concentrated solution of the buffer, such as a 1 M solution, that you can keep in the refrigerator and then dilute and adjust the pH quickly when it is needed. Finally, if you do inadvertently leave the buffer on the system long enough that you observe a cloudy solution, replace the sinker frit in the reservoir so that it does not contaminate the next batch of solvent. You may need to flush or replace the transfer tubing, too. You can help to avoid this problem by pulling the frits out of the bottles when the system is not in use (protect them from contamination by inserting them in a plastic bag).

   So, although there are no hard-and-fast rules for this game, I recommend replacing the aqueous portion of the mobile phase once a week and the organic portion once a month.

This blog article series is produced in collaboration with John Dolan, best known as one of the world’s foremost HPLC troubleshooting authorities. He is also known for his research with Lloyd Snyder, which resulted in more than 100 technical publications and three books. If you have any questions about this article send them to TechTips@sepscience.com

Published  May 14, 2019