Problems with mobile phase with Buffer:MeOH (60:40)

[adomenech]

Hello,

Recently we are having problems in the lab with a mobile phase which is a mix of Phosphate buffer 10 mM with methanol (60:40).

The problem that we have is that sometimes when the mobile phase is purging, it starts as normally but then the pump does not have any more strength to purge the channel. What we do is discconnect the check valve and connect it again (without taking it out or changing it), and it suddenly has the strength to purge the channel.

Also, it has happend to us during a sequence. It is injecting normally and sudenly the pressure drops and it does not have any more strength to pump the mobile phase.

Before putting the mobile phase in the system, we make sure that the solution is mixed well and that it is cooled down.

This problem only happens when using this mobile phase, if another system is set up, this does not happen.

We do not consider that the mobile phase has a high salt concentration.

Does anyone know where this problem can come from? How could we solve it?

Thank you very much,

[TylerSmith123]

When you all are making your mobile phase is it at all filtered? It sounds like your check-valves are getting stuck or they're running out of time and you need some new ones. I'm not very sure what you mean by strength, but I assume that this simply means that the pump is not meeting running pressure... which can also be a problem with a faulty check valve.

Just to make sure because you all think it's the fault of the mobile phase, are you guys running isocratic? Although your phase does not have a high salt-content, depending on your valve switching and mobile-phase compatibility an issue could arise if this phase is potentially mixed with a high-percentage organic phase. Additionally, when you say "cooled-down" is this implying that the phase is brought down to room-temperature or are we talking even lower? Is it heated to speed up solvation of the salt? Its not unheard of to cool down a mixture and then solubility getting worse, ie resulting in precipitation, but I doubt you guys are going below RT.

Please provide some more details and hopefully we can give you some better answers!

[DR]

Phosphate mobile phases (or at least the aqueous portions) should be filtered, and the final MP should be degassed. Outgassing in the pump or undissolved particulates could be the culprit.

[Multidimensional]

While partially obstructed filters may cause the issue you observed, the specific problems you are dealing with are almost certainly related to your selection of mobile phase. Near equal amounts (~ 60/40) of methanol and water will contain the LARGEST amounts of dissolved gas inside the liquid. This gas must be reduced before use. You must always degas the mobile phase BEFORE introducing it to the pump head or issues such as sticking check valves, cavitation, bubbles etc will result in no flow or intermittent flow. Use of a clean, fully functional vacuum degasser OR helium sparging should be used to prevent these types of basic problems. The HPLC pump is the most important component part of an HPLC system. The pump requires regular maintenance (you should have fully serviced the HPLC pump with new seals, cleaned or new pistons and replaced any inline filters before use) AND a supply of degassed mobile phase to operate properly. Please degas the mobile phase.

[adomenech]

Hello,

Thank you so much for all your replies! They are really helpful.

Regarding the mobile phase, when I say cool down I mean bring to room temperature, we do not refrigerate it. And to dissolve the salt we do not heat it up, it is just that when mixing the aqueous solution with the methanol it heats up and we wait for it to cool down.

We always filter the mobile phases before using them and we normally ultrasonicate them in order to degas them since we do not have a degasser. Maybe that could be the problem, there is still gas in the mobile phase.

From the technical service, we have also been told to check the degaser of our HPLC since maybe that could be the problem as well.

Thank you so much!

[TylerSmith123]

Hi Ado,

Do you guys have a degasser or not? I'm sure that you made a mistake by saying that you do have a degasser, because if you did you wouldn't need to sonicate the solutions. So I would recommend a second method for degassing using a pump to pull a small vacuum on the bottle for degassing. It would help if you had a trap obviously to avoid water reaching your pump but this is a good method for removing excess dissolved gases in your phases.

[DR]

@TylerSmith123 - they have no degasser.

So, I do agree, applying a modest vacuum while sonicating could help your degassing efforts a lot.

If you need to run this method often, it would be worth while to invest in a degasser.

[Multidimensional]

These two statements make no sense at all. Please explain:

"

• we normally ultrasonicate them in order to degas them since we do not have a degasser. Maybe that could be the problem, there is still gas in the mobile phase.
  From the technical service, we have also been told to check the degaser of our HPLC since maybe that could be the problem as well

."

Do you have a degasser? In the first statement you say you do not. In the second it is advised that you check your degasser???? Do you have an inline degasser? Please share with us the exact make. model and part number of the HPLC pump you are using.

IN any case, you need to have and use a properly functioning vacuum degasser OR helium sparging for your mobile phase of Methanol and Water. Using vacuum filters or a sonic bath will not solve the issue as they will only help to liberate gas in the solution while your are vacuuming or sonicating it. Once you stop the vacuum OR remove the bottle from the bath, the gas in the atmosphere will quickly bleen right back into it. This is usually seen as large drift over the first hour as the solution refills with gas. Proper;ly degas the solution to allow the pump to operate normally.