Acquity UPLC area values to achieve good precision and accuracy

<p> Transferring a validated HPLC method to UPLC, I am not acchieving the same precision, linearity nor accuracy. The concentration of my samples is 0.15 mg/mL and the UPLC injection volume is 1 µL. within this conditions, the peak area is about 2450000. Is it too low? Would it be better to try higher injection volumes, i.e. 2 µL? I'm using a BEH C18 1.7 µm 2.1x100 mm.</p><p>Thank you for your attention.</p><p></p><p>Susana</p>

Best Answer

  • rune
    Answer ✓

    Hi Susana

    Using the PLUNO (Partial Loop Using Needle Overfill) injection mode, your injection volume should stay inside the 10-75% of loop size specification that we state for good recovery and precision. When you injected 1ul on the 15 ul loop you were outside of this range, but injecting 2 ul you are inside the range ( 1.5 - 11ul), which is probably why you see the improvement.

    best regards



  • Ashok

    What type of injection you are making ..Full loop or PLUNO, make sure you have correct loop installed, since you are injecting low injection volume the loop plays an important role also it depends the injection type. I would double the injection volume and use PLUNO for the injection. 

  • lizh


    The last reply askig for data was good and I would also like to know if this is UV, what is the AU.



  • SPS-KP

    Dear Ashok,

    Thank you for your answer.

    The loop installed is a 15 µL loop. I do not know which kind of loop is "PLUNO". Anyway, I repeated  the tests using an injection volume of 2 µL instead of 1 µL and the results improved significatively:

    Recovery values (accuracy) ranging from 97.7% - 102.7% (Vinj 1 µL) were reduced to 99.0% - 100.5% and the highest relative standard deviation (precision) has been reduced from 1.5% to 0.7%. This is consider acceptable for us.

    Using an injection volume of 2 µL of 0.15 mg/mL solutions, the area values are about 4800000 µV.s and the peak heigh is about 1.20AU.