phosphate mobile phase

<p><span>I have some troubles with a phosphate mobile phase. Even if I wash the sistem with water for half an hour and after that with water+ACN (70%+30%) for an hour, I had stucked a tube. Is there anyone who can tell me what can I do not to stuck my tubes, please?</span></p>


  • uhhh... Forgive my ignorance, but what do you mean by you "stucked a tube"?

  • lizh

    Can you tell us the stength of your phosphate buffer. Phosphate is notorious for solubility issues. At a guess I would drop your salt concentration. Try to use the least possible. It is likely precipitating.

    The more details that you provide on the method the easier it would be to help.


  • I mean that probable the phosphate precipitate in the sistem and the pump couldn't get the mobile phase through this. I found the filter which obstruct my normal flow and I sonicated it, and now everything is ok. thank you anyway

  • So, the mobile phase is:

    A = 0.01M Sodium Phosphate+0.88mM Potassium phosphate, pH=8

    B=ACN:MeOH (1:4)

    gradient mobile phase (A predominant)

    Washing mobile phase is: W:MeOH (70:30).

    Somebody told me to use as washing mobile phase first of all Water (just water) and after a while the mixture W:MeOH. What do you think?

    Thank you,


  • A couple comments... I don't recommend rinsing you columns with 100% water. I don't know if the BEH supports are more rugged than regular silica supports, but I don't think it's a good habbit ether way. If you want to pre-rinse your column, I normally rinse the column with 90%H2O, 10%organic for 5-10 minutes (I normally store them in 90-100% organic), and then change over to my mobile phase.

    As far as the filter clogging, I don't know which one you are having an issue with. If your buffer is clogging a filter before it is being mixed with organic solvent, try filtering it through a finer filter, i.e. 0.2 um. If the filter is clogging after organic solvent is mixing with the buffer, pre-mixing some of the buffer with the organic may alleviate the problem. So instead of mobile phase A being 100% buffer, change it to 95% buffer, 5% organic. You will need to make the appropriate changes in your gradient as well.

  • Thankyou for your comments.

    My filter is clogging before buffer is beeing mixed with organic solvent.

    BUT, what do you think if I filter mu buffer through a fine filter and after that I make the mobile phase A 95% buffer+5%organic?

  • I wouldn't bother filtering the buffer before mixing it with the organic. Do all the mixing and then filter the final solution.

  • lizh


    Our chemists generally don't like filtering after pre-mixing the mobile phase, as vacuum filtering can change the composition. Usually they filter the buffer and then mix with organic.

    If you are worried about precipitation, then that says you are already aware your molarity imay be too high and if you are that close to the solubility limit of the phosphate buffer then the concentration is too high. Phosphate buffer mixes should never be murky, they should be clear. Something is not right.

    Also make up fresh buffer in small amounts as required as bugs love to eat phospahte and will cause issues.

    Limiting the salt concentration where solubility issues are suspected for ACQUITY UPLC is always a good idea as it reduces issues such as precipitation in the the small ID lines and filters that will block when precipitation occurs. As a rule of thumb we prefer 10 mM or less and try to avoid salt in the needlewashes. (Caveat - if using partial loop with pressure assist injection mode - PLPA, that may not always be possible as the weak wash and sample diluent are co-injected and can effect the early eluting peaks' chromatography).

    Finally have a good shutdown method to rinse out the buffer thoroughly.


  • dougm

    This is an interesting thread. My comments describe a very conservative set of suggestions.

    Regarding phosphate buffer at pH 7 or 8, almost as soon as you prepare it, bacteria will begin to grow. This is especially true when the buffer is 100% aqueous, as is the case with your mobile phase A. In general, if you NEED to use phosphate buffer at pH 7 or 8, you should add some organic modifier to the mobile phase container (5-10%) and adjust your gradient accordingly. Prepare only enough mobile phase that you will use in 24-48 hr, and discard unused (aqueous) mobile phase every 24-48 hr. Yes, this is conservative.

    Regarding filtration, I agree with Liz. I do not recommend filtering mobile phase that contains organic modifier. I have seen several cases where the filter used was NOT compatible with the organic modifier (ACN) and the dissolved filter material plugged the inline filter and/or column.

    My suggestion: 1) dissolve your phosphate salt in as little water as possible; 2) filter this through a 0.2 um filter; 3) add fresh, 18.2 Mohm-cm water (0.2 um filtered by water system) and adjust pH; 4) add organic modifier; 5) discard every 24-48 hr; 6) thoroughly rinse your mobile phase container with fresh 18.2 Mohm-cm water; 7) repeat from 1).

    Do not use soap to clean your glassware. Dedicate all glassware, graduated cylinder(s) and filtration apparatus to mobile phase preparation/storage ONLY. Use it for nothing else. Ensure your water system is functional and serviced regularly. Calibrate your pH meter. Do not use bottled water unless you have to.

    Other suggestions about avoiding phosphate salt precipitation at high ACN concentration should be heeded. Want to check this? Prepare a 100 mL solution that contains a mixture of your mobile phases A & B at the maximum %B. Does the solution remain clear? It must.

    Good luck,


  • Yeah.. I admit that this wasn't the best advice. I was thinking about resolving a potential precipitation issue and lost site of good technique. The way I typically prepare my mobile phase is how Doug suggested.

  • JeanL

    Here are a few suggestions if you must use phosphate buffer.

    1. Use the ammonium salt. It is more soluble than the sodium or the potassium salts.
    2. Use the lowest concentration that will maintain your pH. If the pH is 3 or lower, just use phosphoric acid.
    3. If you get a clog, try flushing with warm HPLC grade water containing 1% acetic acid.
    4. Avoid having the buffer contact 100% organic. It will not be soluble.
  • thank you for your suggestions, but there is something that I can't understand in poin 4. Can you be more explicitely, please?

  • JeanL


    Phosphate buffers are not soluble in 100% organic solvent. There are places in the ACQUITY Binary Solvent Manager, like the solvent select valves and the mixing tee where two different mobile phases share a common flow path. For example, if you have been pumping phosphate buffer mobile phases and immediately switch to 100% methanol or acetonitrile, without an intermediate flush with water, there will be a point where the buffer and the organic will come together. At that point precipitation will start and something down stream will clog.

    I hope this helps. JeanL

  • dougm

    Valentina, I believe JeanL is describing the solubility issues associated with phosphate salt-based buffers mixing with high ACN concentrations. To obtain an understanding of this, take 1 mL of your phosphate salt buffer (e.g., sodium phosphate, ammonium phosphate, etc) and place it into a 10 mL graduated cylinder. Add ACN, 1 mL at a time. Shake after each addition. Note what happens after you add more than ~8 mL ACN. Phosphate salt buffers precipitate in high ACN concentrations. Isocratically one must take care when pre-mixing phosphate salt/ACN mobile phases to ensure that the mixture remains clear. In a gradient, pay attention to the maximum %B (B = ACN).

    I hope this helps.


  • I think that he was talking about ths, thinking of what you said to me in a message that you have sent me (mixing a little bit of phosphate with a lot of organic).

    yes, it helps, thank you

  • yes, it helps, thank you.

    ? that means that I will never can switch immediately from phosphate mobile phase to 100% organic. so I should switch it gradual to a maximum permitted organic %, but never switch to a little bit of phosphate + a lot of organic couse I'm gonna clog it. this mean that I should change my gradient method so I will never have more than 80% organic?

  • JeanL

    I was able to find an article in a Google search that may help you prevent phosphate problems. At 80% organic you may be at the point of precipitation. It will depend on your phosphate salt, the pH and the ambient temperature. ACQUITY UPLC systems have narrower tubing, finer filters, and simlar particles in the columns. Therefore, samll amounts of precipitate will cause more problems than in HPLC systems. See the attached for the solubility properties.


  • Miami

    Thanks for the tip. It is really helpful. If my mobile phase condition will be phosphate buffer at pH 2.6 and 25% of Acetonitrile/75% buffer isocratic, can I use the same care for ACQUITY UPLC system? I assume the premixed mobile phase is better than that of mixed online for isocratic condition.

    Thank you very much!

  • for more information about buffers solubility in aqueous-organic elunents read de article that JeanL sent me a few time ago. it's a very good article