Carryover issue on UPLC

<p>Dear All,</p><p style="height: 8pt"/><p>Have you met carryover issue on UPLC system? Currently we are trying to develop an UPLC method for carbamazepine, and the carryover issue happened! The strong solvent and the weak solvent we used for the injection system are 100% acetonitrile and 10% methanol individually, the wash volumes for both solvents are 200 uL. We have tried to change the strong solvent to 100% methanol and to increase the wash volume to 600 uL but useless. We have met this similar issues on the other drug substances before. Since we could not overcome this issue on UPLC system, we back to HPLC system without any problems.</p><p style="height: 8pt"/><p>Do you have any good suggestions to resolve this problem? Thanks a lot!</p>


  • Dear Shihhao,

    I have been running applications on the ACQUITY UPLC since late 2003 and I will try to assist you with this problem. Could you please provide me with more detail?

    1. please describe your sample and sample matrix.
    2. chromatographic conditions
      1. column,
      2. chromatographic solvents
      3. gradient
      4. flow rate
      5. wash solvents & volumes
      6. injection volumes
      7. mode of detection
    3. system description: do you know which needle you have installed? Is it still the needle which came with the systems?
    4. Sample manager serial number (this can be found in the console or on the front panel of the sample manager when you open the door ).

    my email address is:

    Any information you choose to share concerning the applicaiton you are running is considered confidential and will not be shared with others without your consent.

  • lizh

    I have alerted our chemists to your question, but carryover could be due to many causes, in general the critical differences at the root is solubility and this is more troublesome when the system volumes of typical HPLC direct injectors and the loop injector used with ACQUITY are so large. Additionally, the additional transfer step in a loop means that sample is left behind and must be washed out. Carryover can be attributed to solubility issue, where the compound would rather stick to something in preference to solvating in the sample diluent. This happens as you cannot always tune the diluent to all sample components. So when you transfer from a vial, to a loop and then make an injection in ACQUITY UPLC there are opportunities for solubility losses within the UPLC Autosampler. As the volumes are so small solubility is more problematic. In a direct injector the needle path is of a higher volume, usually x100's of uL and is part of the pathway of the system mobile phase and if solubility loss occurs it is not left in the Autosampler because the mobile phase washes it away "directly" with much the larger volumes. Therefore, we will be asking you some questions about injection method and wash conditions to get to the root of the issue. I attach a little tips and tricks presentation of things to consider.

  • Try running with partial loop only instead of partial loop with overfil and see if the problem goes away. I have run carbamazapine without problems, (in plasma) I'm sure with the infomation that Patty requested she will solve your problem.


  • Dear Brian,

    Very thank you for your suggestion! We have tried running with partial loop injection and the carryover issue disappeared, but it caused a huge system peak at about 0.5 min.


  • Shihhao,

    As a quick side not to Liz and Brian's comments.....It is always recommended that you use 3 times more weak wash than strong. This probably won't effect your "carryover", but may address peak shape issues that may arise in the future.

    Good Luck!


  • One of the major differences between ACQUITY Sample Manager and HPLC systems is the material the needle is made out of. Most HPLC systems have stainless steel needles. The ACQUITY system standard needle is PEEK. Hydrophobic compounds may stick to PEEK. There is an optional stainless steel needle available (art number 205000362) to make the needle more like the HPLC system.

    When you choose wash solvents, the strong wash can be any solution that dissolves the compounds. Altering the pH for some compounds can help. If you want to increase the wash solvent volume, the strong wash will have the most effect. However, you must then flush with 600uL of weak wash which should be similar to the initial chromatographic conditions. The 600uL is required to remove the strong wash solvent.

  • lizh

    It appears that you may need to adjust your wash conditions, now that you are using Partial Loop with Pressure Assist mode. One of our best applications chemists is going to send you some illustrations and suggestions to help.

    However, as the Product Manager I so want to underline a point, whenever you state a required wash volume, and then along comes an application where that volume is not optimal. It is always a balance of enough wash to manage carryover, against lengthening the cycle time. Often a small volume of an aggressive solvent is better if the primary concern is speed. However, the solvent strength may have to be managed as with Partial Loop mode with pressure assist matching weak wash is important, but is less important for PLNO mode.

    The UPLC Sample Manager has two wash modes weak wash and strong wash and weak wash alone. Whenever any volume is in the strong wash is entered both are used. For those that are interested I have attached the exact sequence of events and rationale behind the volumes. This can help when making the best choices for a particular application.

  • Dear Shihhao,

    I ran your assay on one of the ACQUITY systems in my laboratory, I did not detect any carryover using either the PA or PLUNO injection protocols. I made a minor change to the weak wash solvent to insure that the carbamazepine would be fully soluble in it and I used the "default" wash mix most often employed in our Marketing Laboratories (25% Acetonitrile, 25% MEOH, 25% IPA, 25% water). I also adjusted the wash ration of weak to strong to 3:1. It is likely that the VDD (volume detection device) on your system has been contaminated, possibly as a result of borderline solubility of the carbamazepine at the original conditions. The VDD should be changed if you are not able to eliminate the carryover using the slightly modified settings contained in the report which I am sending to you under separate email.

    Please let me know if you are successful.

    FYI - I was asked to detail the wash solvents used.

    weak wash: 50%methanol & 50% water (in this case the same organic concentration as the sample matrix): 600uL

    strong wash: 25% Acetonitrile, 25% Methanol, 25% isoproply, 25% water: 200uL

    We often include some isoproply alcohol in our strong wash solvent (depending upon solubility wiht other components); IPA has great wetting properties


    Message was edited by: Patty - Systems Marketing Laboratory

  • RD


    Can you post what you used for weak wash (I'm assuming the recipe you noted was for strong wash) ?

    This may be beneficial for others.

    Thanks & nice work,


  • Greetings,

    Just a couple of, hopefully, useful pieces of information.

    First, during the wash sequence the strong wash is, actually, diluted by the weak wash. In all candor, this was not fully apreciated until we began to experience carry over of anthracene during the development of the fluoresence detector Qualification tests. During the strong wash segment of the wash sequence, weak wash solvent is forced up through the bottom of the wash block while strong wash is passed through the needle. The internal diameter of the needle is washed with 100% strong wash, but the external surface of the needle is washed with 50% weak wash and 50% strong wash. However, there is a remedy to this situation. The Partial Loop with Needle Overfill (PLNO) injection mode is designed such that neither of the wash solvents are coinjected with the sample. Only mobile phase is in the sample loop when sample is introduced prior to injection. This means that both the strong and weak wash solvents can be at whatever strength is necessary to clean the system. In the case of the fluoresence Qualification, it was necessary to make both the strong and the weak wash solvents 100% acetonitrile. It must be stressed that this strategy will only work with the PLNO and Full Loop injection modes. The pressure assisted Partial Loop injection mode does coinject weak wash solvent.

    Second, the system peak that Shihhao mentions after switching to pressure assisted Partial Loop injection mode is almost certainly an air peak. The pressure assisted Partial Loop injection mode coinjects the post sample air gap. Neither the PLNO nor Full Loop injection modes inject either of the air gaps.


  • Dear Shihhao, I had the same carry over problems with amphetamins. Addition of 0.5% formic acid to the strong wash solution solved the problem. Could be worth trying since CBS seems to be a basic compund similar to the amphetamines. Rgds M

  • lizh

    I thought it was worth adding to this excellant advice. Adding Formic Acid and acidifying the needlewash does help for acidic conditions, in the same way adding base for basic conditions.