Restek
Home / Resource Hub / ChromaBLOGraphy / It Was in the Water.. Now What

It Was in the Water.. Now What?

2 Sep 2021

Hopefully, you have read my last post on this topic and you have found a reliable source for Type I water to use for your mobile phase. We are not home free quite yet. While we are preparing mobile phase and immediately after, we should be concerned about the following:

  • Particulates and microbes, which can be introduced even after the water is purified. Most of these usually come from the air, but sometimes also from surfaces of the container or mixing utensils.
  • Dissolved gases are always a possibility, but exist in higher concentrations if water or solvent is stored at colder temperatures. The mixing process can also trigger the inclusion of more gases from the air.
  • Leachates from the storage container.
 

So what is the best way to remove particulates and microbes? LC grade solvents are prefiltered and most do not require filtration, but if using water that is purified at your facility, you probably need to filter it. Most importantly, if you weigh out any solid materials and add it to the solution as a buffer or modifier, you will need to filter the solution. Membrane filtration is the most common way to do this. Check out my blog post here that discusses product options from Restek for this: What can I use to filter my mobile phase?

Although some microbes, particularly bacteria, can be removed by filtration, a few may sneak through and could multiply. Here are several things you can do to keep microbes from growing and keep particulates out of your mobile phase, even after you have filtered it:

  • Premix your mobile phase with some or all of the organic solvent you will be using. Depending on the components, sometimes as little as 10% of organic solvent is enough to kill microbes and keep them from growing. This is the most efficient way to prevent or slow down growth of microbes.
  • Keep your mobile phase bottles and all containers covered to keep out dust and microbes from the ambient air. Also, make sure that all glassware and containers are clean before you use them.
  • Some labs store their pre-mixed mobile phase in a refrigerator, but I do not recommend this. Cooling the solution does slow microbial growth, but it often creates other problems. Cooling decreases solubility for some modifiers and buffer components, which could trigger precipitation, depending on the initial concentration. Cooling the mixture also increases the solubility of gases in the solution, creating the need to degas more thoroughly or perhaps a second time. Overall, refrigerating mobile phases probably creates more problems than it solves.
  • Make sure your mobile phases are assigned with appropriate expiration dates and discard the mobile phase in a timely manner. Make sure to clean the container well and refill it with freshly prepared mobile phase- do not “top off” the solution. Many laboratories have SOPs that specify how expiration dates should be assigned to mobile phases, or sometimes this is specified in written methods that they follow. Regardless of other measures taken, this one is probably the most important guideline to remember. Opinions on how long a mobile phase should last vary widely, but in the absence of test data, a reasonable estimate is up to 1 week for highly aqueous solutions (90% or higher) and up to 3 months for organic solutions.
 

Removing dissolved gases is sometimes another challenge with mobile phase solutions. Fortunately, most modern LC systems include degassing modules. This is the most efficient and thorough way to remove gases. If you have an LC system, perhaps an older one that does not have a degassing module, you can purchase degasser modules to fit your system. To be fully integrated for automation, usually you will want to use a degasser that is the same brand as the rest of your LC system. However, there are also some degassers made by independent manufacturers that are compatible with any system. Restek sells the DEGASi PLUS Mobile phase degasser that can be used with any LC system.

Other less efficient (manual) methods of degassing are these:

  • Membrane Microfiltration/Vacuum Aspiration- This is quick, and removes about 60% of the dissolved gases. However, it can be cumbersome and sometimes it is likely to alter solvent ratios due to evaporation. Restek does sell Membrane Microfiltration equipment for this.
  • Helium sparging- This is the most efficient manual method of degassing, since it removes about 80% of dissolved gases. However, it can be costly, due to the shortage of helium gas that is not recovered. Sometimes sparging needs to be repeated daily, so it can be time consuming also. Sometimes continuous helium sparging is even recommended. Restek does sell solvent filters that can be used for helium sparging.
  • Sonication- This is easy, but not efficient enough when used alone. Sometimes sonication is used in combination with vacuum degassing/filtration or helium sparging.
 

Another possible issue with mobile phases is leaching of materials from the mobile phase bottle, storage container, inlet filter or tubing. This concern commonly arises with respect to adducts observed with LCMS or LCMSMS analysis, usually Na+ or K+ adducts.  Organic compounds could also potentially leach into the solution if plastic materials are used inappropriately. This can affect analyses performed with any type of detection. Here are some measures you can take to avoid these issues.

  • Use only borosilicate glass containers, preferably Type 1, Class A or Type 3.3.
  • Never use plastic bottles for storage and make sure any mobile phase caps, inlet filter and tubing are clean, in good condition and fully compatible with the solvents you are using.
  • Never use Parafilm® or other plastic films to cover solvent reservoirs.
  • Avoid the use of detergents or dishwashers to clean mobile phase glassware. After cleaning, follow up with a rinse of (water-miscible) organic solvent followed by DI water.
 

Thank you for reading and I hope you find these tips helpful. Here are some suggestions for additional reading:

Routine LC Maintenance, Simple Steps to Preventing Unexpected Downtime

Simplifying your Membrane Microfiltration Glassware Purchase

Diagnosing and Preventing High Back Pressure in LC Systems

Live Long and Prosper-How to make your HPLC columns last longer

Capping your LC Solvent Bottles (video)

Mobile Phase Preparation for UHPLC: Impact of Filtration Through 0.2-µm Membranes on System Performance (American Biotechnology Laboratory, October 2008)

Filters and Filtration in Liquid Chromatography- What to do (LCGC North America, 02-02-2017, Volume 35)

How Does It Work? Pat II: Mixing and Degassing (John Dolan via Chromatography Online May 31, 2016)

Degassing Mobile Phase Solvents (John Dolan via Chromatography Online, December 5, 2010)

Degassing Mobile Phase Solvents (Shimadzu)

What is Mobile Phase Degassing? (Chromatography Today, August 18, 2014)

Controlling Contamination in LC/MS Systems, Best practices (Waters Chromatography)

Modern Trends and Best Practices in Mobile-Phase Selection in Reversed-Phase Chromatography (LCGC North America, 10-01-2018, Volume 36)